AU2003266472B2 - Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism - Google Patents

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TO BE CONMPLETEED BY AP-PLICANT Name of .pplicant: Actual -Inventor(s): Addrcss frrService: Invention Tithe; PONT Dr NH-MOLRS AND COMIPNNY and GENECOR TNTFRhNAIIONAL. INC".

Lia A=n Laff'end, Vasanflha Nagarajari, Ctuidcs Edwi Naka- Mum CALLINAN LAWRA[E, 7111 High Strant, KeCw. V ictori a 3!O0l. Austndlia RTOCON VERSION OF A FBR-MENTABTT CARBON SO01.)RXQL TO I,3-PROP\N EDIOL B Y A SITN GLE IC RE ORGAN I SM The fbi I ~I ng smrexnenr tk a Cull1 descripti on of t h is invention, inic Iludi ng the best nctliW EuCo performing it known to is:- ETOCONVERSION OF A FERMVENTABLE CARBON SOURCE TO I ,3-PROPANEDIOL BY A SINGLE3 WtvCROORUANLM ThiS invetio comprises a proes for fte bioconversion of a fennentabe ca~rbon souice to 1,3-propanediol by a sinzgle midcroorganism.

1,3-Propanediol is a moriom& having ptntial utility in the production of polyester fibers and ie mnanufactur of polyurtbmnes and cyclic compounds.

A variety of cbcrncal rout"t to l,3-propanediol ace known. For aunpla ethylene oxide may be convented to 1,3-propanediol over a catalyst in the presence of phosphine, waftr 1 carbon monoxide, hydrogen and an me", by the catalytic solution phase hydraon of acrlein fofllowd by reduction. or [nurn hydrocarbons such as glycerl, reacted in the pircscnce of carbon monoxide and hydroge over catalysts having amoms from group Yin of the periodic table.

Although k is possible £o gener=t l,3-prcqmicdiol by these methds, thecy r expenive ad geneae waste surea cotaining enviomental pollutants.

It as been known for -over a century that i,3 -prcpancdioi can bc prouced from the fxennauion of glycerol. Bacteria stwains able to produce 1 1 3-propanedial have been foid, ft~r cxamplr,, in the groups Cifrobacer, Clostridiwn* -&wrobaeer, lyobaazer, Kiebsietta, Lactobacllus. and Pdlobactar. In ec~h ease studied, glycerol ih convented to 1,3-propan-ediol in a two? step, cnzyme catalyzed cnctwn scqt'cnc. I lii; &st step, a dehydratas earayte thec conversion of glycerol to 3-hydrozypropijonaldehyde (3 -HIP) and water, Equation 1. In Itic secondi step, 3.1W is reduced to l,3-pwopanediol by a NAD 4 +-ILnkd oxidoicduc~e 1 Equation 2. The 1,3-pwup~redi is not metabolized further and, Glycerol 3-HP H 2 0 (Equationp 1) 3-HP NADM 4 -4 1,3-Propmnediol NAD+ (Equation 2) ancam mi high coenration in the media. The overall reaction consumes a reducing equivalent in the bunr of a cofactor, xeduced ft-ntcocnamje adenine dinucleotide (NADJI), which is oxidized to nicotinamide adenin clinudleatide

(NAD

4

I.

The production or 1 3-prepanediol fr-om glycerol is generally perfonnd under anaerobic onitions usig glycerol as the sole ca~bon source ani the absence of vrhcr exogcnvi~s xedieing equivalent acceptors tinder thee conditions. in strains of Cttrobacter, Clonridiwn, and Kkebsddlla, a. parallel Ilkpathway for glycerol opera=e which firt involves oxidtonl Of glycerol todihydroxyattfl (DHA) by a NAIY- (oriNADr-) linked glycerol dehydrogena. Equation 3. The DHA, following phospholltion to dihydrxyacetone phosphat (DRAF) by a DHA kinase (Equation 4).

Glycerol +.NAD+ i DI+ NAJ)H H+ (Equmiom 3) DR-A An' DIM ADP (Equaion 4) becomes available for biosynthesis and for supporting ATP generation via e~g., glycolysis. in contrastto the 1,3-propanedial pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADII.

Wn febskella pncuwoixae and Ciirbactr freundif, the genes encoding the functiopally linked activities of glycel dehydzatsse (dkafl), ,3-propane-diol oxidameductasr, (dhall, glyol dehydrogease (dhaD), and diydroxyacxtone kinase (dhaKL) a= encxmpassed by the dha regulon The dha regulons from Cirrobaccer and Kiebsitila have been epressed in Eschericzia colt and have been shown to convert glycrol to i,3-propanediol.

Biological prcresses for the. preparation of glycerol ame known. The, ovenvhehnixig majority of glycerol producers are yeasts but so=e bacteria, other fungi and. algae am also knlown. Both bacteria and yeass produce. glycerol by convertig glucose or othecr carbohydrates through. the fructose-I 1 6-bispbosphate pathway in glyclysis or die Enibden Meyerhof Parns pathway, whereas, certan algae convrt dissolved carbon dioxide or bicarbonate in the chioroplasis into the 3-carbon intermrdiaes of ihc Calvin vycle. In a series of stcps. the 3-carbon intermediate 1 phvsphoglyceric- aci-d, is convented to glycenuldehyde 3-phosphat which can be readly intrionvexted to its keto isomer dihydroxyacetone phosphate, and ultimatly to glycerl. Although bi-ological methods of both glycerol and 1 1 3-propanedfiol production ame known, it has never beau demonstrated that the entire proces can be accomplished by a single organism.

Neither the chemidcal nor biological nuethod described above for the produaction of 1 1 3-propanediol is wvell suited for inustrial scle production since the chernical processes air enrgy intensive and the biologicM processel requtfe ihe expenive staring material 1 glycerol A mthod reqiring low enegy input and an inexpcrnsive starting nmerial is needeIt A more desirable proces would iunoporate a microorganism that would haLve the ability to convrt basic carbon souarces such as carbohyd-rates or EUgaIS to the desired 1 ,3-propmneiol endproduact.

Although a single organism cownvrion of fernnetitable carbon source otheir than glyceroel or dihydryaeeoae to 1,3-piupanediol would be- dcsirabl, it has been documented that thre are signiiat difficulties to ovezeame. in suach in endeavor. For example. Gonschalk ot al. (EP 373 2301 teacdi ht the pmwth-r4> most swrains useful for the production of I ,3-proparnediol 1 including Ciirobacter frewidli, Ctauirdium asuobinytcwn, Closrtdum baayliwnm, -and flebsielta pncumoniae, isds&ZuIbed by the presce of a hydrogen donor such as frucose or glucose. Strains of LWcrbadilar brevis and Lacwabaciflss buchnern wich produce 1 ,3-propaneol in co-fcrmn~taiions of glycerol and fruictose or glucose, do not grow when glycerol is provided as the sole carbo ourcc and, although it has been ishown that nag cells can metaboliz g~ucose or frutose, they do not produce 1,3-prepanediol. (VaigafDA Cunha etal..J. BacreloL 174, 1013 (1992)), Sindladly, it has been shown that a stain of Ilyobacfer potytropus, which produces 1 ,3-pxupancdioL what glycerol and acetat ae provided, will not produce l,3-pramnediol from cadhon substrates other than glyceol, including fructose and glucose. (~eib et Arch. Mtcrobiot. 140, 139 rzuly Tong et al. (Appi. Biiochem. Dknech. 34, 149 (1992)) has taught that recombinant &cherlchia coil trasformed wih the dA rgulnn enoding glyceol dehydraae does not produce I 1 3-propanediol fro either glucose or xylose in the absence of exogenous glycerol.

Attempts to improave the yield of 1,3-propanedfiol fom gLycerol have been 21) zeported whctu co-substaies capable of providing reducing equvalents, typically fcmnnentable sugai, ame included in the procs. Imprenms in yield have been claimed for resting cells of CO~bacrerfreundii and Kiebsiella pneuwonae DSM 4270 cofennenting glycerol and glucose (Ciottschl ela.r ur~ n rnDn et al- DE 3 734 764); but not for growing cells of Kkbsila pneu4wnioe ATCC 25955 cofermnenting glyceirol and glucose, which produced no 1,3-propanediol Tong, MDLE. Thesis, University of Wisconsin-Madison (1992)). Increased yields have been reported for thic cofermentarion of glycerol and glucose or fructose by a rcmbinant Eschediclda coil: hewever, no t3-propanediot. is produced in the absence of glycerol (Tong et at., supra.). In thee systems, single, organism use the carbohydrate as a source of gcperadng NADH whil providing enrgyj and carbon for cell maintenmne or growth- The disclosures suggest that sugmn do no enter the carbon strAn that produces 1,3-propmnediol. In no case is 1,3-propanediol produced in the asnce of an exogenous source of gly-cerol. Thus the weight of literature clearly suggests that thte production of 1 .3-propanediol firom a carbohydrae source by at single organism is not possible.

The problem to be solved by the pmeset invention is the biological prodcdon of 1.3 -pnw-anedlol by a single organism from an nexpen.=ve. catbon 13/09 2007 THU 14: 51 FAX +61 3 9859 1588 CALLINAN LAWRIE 0008/015 0 0 0 substrate such as glucose or other sugars. The biological production of 1,3-propanediol requires glycerol as a substrate for a two step sequential reaction in cn which a dehydratase enzyme (typically a coenzyme B 1 2 -dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADI-I- (or NADPH) dependent oxidoreductase.

C The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process which utilizes this reaction sequence for the \production of 1,3-propanediol. Furthermore, in order to make the process CnI economically viable, a less expensive feedstock than glycerol or dihydroxyacetone is O 10 needed. Glucose and other carbohydrates are suitable substrates, but, as discussed 0 above, are known to interfere with 1,3-propanediol production. As a result no single organism has been shown to convert glucose to 1,3-propanediol- Applicants have solved the stated problem and the present invention provides for bioconverting a fermentable carbon source directly to 1,3-propanediol using a single organism. Glucose is used as a model substrate and the bioconversion is applicable to any existing microorganism. Microorganisms harboring the gene for a dehydratase are able to convert glucose and other sugars through the glycerol degradation pathway to 1,3-propanediol with good yields and selectivities.

Furthermore, the present invention may be generally applied to include any carbon substrate that is readily converted to 1) glycerol, 2) dihydroxyacetone, or 3) C 3 compounds at the oxidation state of glycerol glycerol 3-phosphate) or 4) C 3 compounds at the oxidation state of dihydroxyacetone dihydroxyacetone phosphate or glyceraldehyde 3-phosphate).

SUMMARY OF THE INVENTION The present invention provides a bioconversion process to produce 1,3-propanediol comprising contacting, under suitable conditions, glycerol or dihydroxyacetone with a single recombinant microorganism expressing an exogenous glycerol dehydratase enzyme from Klebsiella or Citrobacter, the microorganism selected from the group consisting of members of the genera Aspergillus. Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansemula. Debaryomyces, Mucor, Torulopsis, Methobacter, Bacillus, Streptomyces and Pseudomonas.

The present invention further comprises the product of the above process.

The present invention further comprises a cosmid comprising a DNA fragment of about 35 kb isolated from Klebsiellapneumoniae wherein said fragment encodes an active glycerol dehydratase enzyme having the restriction digest in Figure 1, columns I and 2.

1309/O7.at13772.spOip ,2 4 COMS ID No: ARCS-160826 Received by IP Australia: Time 14:50 Date 2007-09-13 The prcsctil invention further comprises a trant'Tumned microorganism comprsing a host microorganism and the above cosmid.

The present invention also comprises a transformed microorganism comprising a host microorgani sm anid a first DlkA fragment isolated from .lkbsiella plumnoniae, the first DNA fragment encoding an active glycerol dehydratase enzyme having the restriction enlzy-re digest in Figure 1, colunns I and 2, end at least one second 1)2A fragment isolated fromi Ke hsic tn pneumoniae, the second DNA fragment encoding an active fu nctional prote6in 01 her than a glycerol dehyd-alase en-Yme.

The pi-esont inIveT1 iOn futer encompasses a Reo mbinant eUca-YOte iicroorganism comprising a host cclI selected (iom the group consisting of yeast tilarentaus Fugri and expressing a glycool dehydratase euzyme- Recombinant mi roorgan ism s embodying the invetliiin are set forth in the BRIEF DESCRTPTTON OF TIHE BIOLO GICAL DFPOSITS- BRIEF DESCRPTION OF THE FTGL1RES Figure 1 show s reslmition diges ts (EaoR I, BamTR 1, EoR V and Not1) of cosmid. pkP I, pKPZ and pKP4 labld as columns 1, 2 and 4 respectively, and separatiun on a 0.8% agarose gel elecirophoresis. Kiulecular size markers were loaded on the lanes in the end. Coluns&1e1d es numberF lAMd 2 reprec vosrnids containing a glycerol dehydratase enzme. Figurt 2 shows a pardal physicul map of pKPI iand the position of the genes hazed on DNA sequence. The genes were identified based an Comparison of deduced op=n reading frmic witb he ir(nbahk d=t base using the Tfa progttn provided by a sequence analysis software of the University of Wisconsin (Geneics Computer Group, Veuisan 7, April, 1991, 575 Scicnae Drve. Madison, WI 53711].

EOU t E1ENEU M The n-nsfornemd F- cecli DHqx corLuiirdn-g cov~in& pKPI cunamLgL a pan ion of the Klebsiella genarrc encoding the ycem1 dehyduatase enzyme was deposited en IS April 1995 with te ATCC inder dhe of the Budqpest Treaty and was designatd ATCC 6979, The tranfocued F. coil 1, CQnUAin4~m c c-nid pKP4 containing a portion of dr-e KkbsieLW genone enoding a dial dehydratas enzyme was deposited on 18 Apri1 1995 with the ATCC under the terms of die BudapestTreaty and was designated A7CC 69790. *The Pseudomona aerugiwara strain PAO 2845 :pDT9 tromfociied wit a plasmid ccaraiin S the dM8o operon was depositd on 11 Apri 1996 with the ATCC =ider the tems of the Budapest Trety and was designd ATCC 55760. The flchta panoris stain MS'4-2.8 1, nusfonnd with non-replimsive plasinids contiing expqvssion cassette for die dlAaB 1 B, dhaB3ffa and taT genes,.i deposited on 11 April 1M9 with the, AW'? under the terms of the B3udapws Treaty and was d=sigated ATCC 74M63. The Sacdwaromre cereisiO, &=lin pMCKIIIOJ17(IM)#A, trnsformed with a plannid contiing the dhzB 1. &0a2.

dimE3 3, and daT operon. was depositd befeR' the filng of the ins=n itmrationa pplication, on May 9, 1996, with the ATOC under the trm of the Budapes Treaty and was designated ATCC 74370. Thie Rrepwimyces t1dcns s=An SIII4Z7 =rasformd with a plsmid containing the dWufl, uflua2. dbaD3.

and daT operon was deposited betfe the filing of the instnt istematonal application, on May 9 1 1996, with ftcATCC undr the fte= of the Budapest Tnsry and was designated. ATCC 9:3052. The Bacillus &heWnormis strain BG IBSIhM (Clone trunsfunrd with a plasmid conrningtea dhaB31, dhzB 2 and dhali3 opron, was deposited befoe the filin Of the instant inftenaional application, on May 9. 1996, with the ATCC under the trumsof the BudapeSt Treaty an was designated ATCC 9805 1. The Bacitos sbtdlis strain 902864/pM27 (Clone WI), =mfonnA with a piasmid contaning the dhafl E, dbhaf2, dhafl3 end dhaT operon was dnposited before rhm filing of the instan snfemational application, on M-ay 9, 199b, with the ATC Ivd% ch. teAni vt4zkj Budapcst Treaity and was designard ATCC 98050). The AspnrgiijUaniger uriW- TGR40-13, transformed with a plasinid containing the dhoB 1, dhzM2, dhrnB 3 arid dhaT operon, was depoited before the filig of the instat iternatonal applicatn *n May 9, 1996, with doe ATCC under the trms of fts Budapest Treaty and was design=e ATCC 74369. WACCE zvfe to the American Type Cultur Collection irnernati-Onal deposicoy linked at 12301 Parklawn Drive, Rockvlie 1 MD 20852 U.SA. The designatfions refer to the accesionnumber of the deposited material.

ApplicaMt have provided forty-six sequence in confonnity with "Rules for the Standard Represenation of Nucicodde and Amino Acid Sequcnces in Patent Applications"(Annexe I and fl to che Decisionk of the President -of the FPO, published in Strpptenicm No. 2 to 07 MW, 12/1992) and with 37 C.FR. 1S21-1.925 mid Appendices A &ME B Reqairemeamt faf Application Disclosrn~s Conring Nuceoides An4'or Amidn Acid Sequences").

The preent invention provids a metho for a b5Riooical proucion of l,3-propanediol from a fertmentablecarbon source In asinge organism. 7T method incorporates azicrorgmnism connig a dehydnase enzyme which is contactd with a carbon substt wad I 3 Tpmdiol is isolated fiom the growth maedia. The single organis may be a wild type organism or may be a g-enetic-ally altered organism harboring a gene encoding a dchydrazas enzyme The present method provides a rapid, inexenive ad environmentaly iraponsible souve of 1,-propanediol mawnor usehil in te prodution of polyeswn and other polymers.

As used herin the folowig term may be usecd for ireireration of the claim and specificati As used herein. the cenn '"nuclek acid" refers to a jarge molcule which can be sine-stided ordoubl-smnded, composed of maonx (nuclevride2) containinga sugar, phosphate and either a purine orpyrimidne. A "nucleic acid fi~="is a fraction of a given nuclec acid molecule. In higher plant.

deoyribonucleic acid (DNA) is the gentic material while fibouucleic acid (RNA) is involved in the tranfr of thte informLtion in DNA into proteins. A flegeomelr is thec entire body of genetic material contained in each cell of an organism The term nucleotide sequence" refers wo a plymer of DNA or RNA which can be single- or douable-tmded, optionally containing synthetic,. nonnatural or altred nucleodde bas capabl of incorporation into DNA or RNA polymers As used hemrin, "esseniially similar 'deit DNA sequeniccs thai may involve base changes t" de not cause a changt in ihe encoded am-ino adcicur whichb invyolve base changes whiich may alter one or mnore amino acids, but do not Waffc the funcvionial prpades of the protein encoded by the DNA 3cqucncc. It is therefore undcntooed thamthde invnton encozpaase more tan the specific exeznpluzy sequences. Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changcs tha: do not sub stantialy affec the. funa propertie of the, zeuttin protein molecule are pio ontemplated. For exmnple, alteration in the geesequence which eflect the degency of the genet code, or which result in fth production of a chrniL ,ctq eival cnt af, L.:naC acid at a g sitc, z 'zc "r torthe amtino acid aisnine, a hydrophobic anmio ak rna ye subriinu,'6 a codon encodintg another less hydrophobic residue, such a glycine, or a more hydrojJhObk residue., such as vulkne. Imnane, -or isoleunme Si-iflarly, changes which resul in 5ubstitution of one nee atively charged residue- fdr another, such as asparcic aci-d for glutanil acid, cr one positively charged rcsidue for another, sucli as lysine for arginine, can also be expected to produce a biologically equiv~alent product. Nucleoti-de changs which result in alteration of the N-tcnninAl and C-tenn ia portions of the protein molecule would also not he, expvctcd to altr the actvity of the protein. bn some abs, it may in, fa= be de~sirable to make mutants of fth sequence in aider to study the effecut of alteration on the biological activity of the protein- Each of the proposed modifcations is well Mthin the, routine skill in the art, aw is deterznination of reterntion of biol ogical activ ity in die encoded product. Mormover. the skilled. artisan recognize that "essentially siznilsf" sequenes encompassed by this invcntion are also defined by their ability to hybridize under strinigent conditons IX SSC, 0. 1% 31)3, 65 0 C),withi the sequenc exemplified heein.

i 4 Ce-nePI refers to a nucleic acid fragment that exresses a speotific protin, including regulatory sequnce preceding non-codig) and following nomcoding) the coding region. -Native" or

M

wild-type"gene refers to die gene as foond in natue with its own regulatory sequence.

The term "genedically altered or genetically altered. mkcroorgnish 1 refers to any microorganism, muicablc for use in the present invention, which has undergone 2n alteration of the native gentic machrrTy of the m~i=rznzsm.

Micaorgmism may be genetically eltered by underoing trarnfnn 01 by Vectors comprising heterologouz nucleic acid fragmts, rnutAgmfesis with murgenizing agents UIV light, ethaneslonic acid) or any other mthod whereby stable vlahezon of the cell genomv occur.

The tern 'cansmucf refers to a plasruid- virus, autonomously replicatinr sequence, genuin Iintegrating sequpence. phage or nucleotide sequenoe. inear narcircular, of a single- or doubie-straded DNA or RNA, derived [toni any msoc, in which 2- numuber of nucleotid sequences have been jined or recwnbined into a unique construction which is capabl of innvdudng a promotr fragment and DNA sequence for a selected gene product along with appropriate 3'untrunslated sequence into a cell- The termL "vwransfnition" or UtransfcctiwV' refers to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired Se=e may be Lntegrated into chrornosomal DNA or introduced as extrachromosomal zeplicadingscquences. The term "n-an~scnnna refers to the product -of a innfon naton The term 'genercally altcrd' ref=r to the process of changing hereditary material by transformation or mutation- The term 'cxpression' refers to thie vrndpticif and transLation to gene, product from a gene codin for the sequence of the gene product.

The tarm !IPIaandM or Yver" pr "cosrald" aw used herein refers to an extra chrornosomal element often cantying genes which are not part of the central netabolism of the cell, and usually in the fann of circular double-stranded

DNA

molecules.

The, term "dehyciratase enzyme" will refer to any enzyuie that is capabke of isa meriting au conventing a glycerol molecule- to the product 3-hydroxyprapionaldehyde- For the, purposes of the present invention the debydratase enzymes include a glycerol dehydrase and a dial dehydratase having preferm d substratetq -of glycerl =nd 1,2-propanediol, respectively.

The term "carboni surb=='x or 'cabon suiiO rnto,= mny carbon source capable of bcing mnetabolized by a rnieroorpanism wherein Mhe substrate contains at Iet one cadbon giom, provided that the carbon substrate is other than glycerol or- dihydroxyacne Constrction of Rjembinant OQpniML: Recombinant organisms ontaining the necesary gene that wi encode the cozyrnatic pathway forthe conversion of a carbon subsrae to 1.3-propanediol =ay be constaicted using techniques well kniown in the arc, In the present invcnt ion genes encoding dehydratase enzyme were isoLated from a native host su-ch as Klcbsidlla =nd used to t-nnsfonn the E. coil host strains 0115m ECL7D7 and Me-thods of obtaining desired genes from a bacteria genarne ar co~ntio and well own in the an of molec.iLur biology. For example, if the sequce of the genn is known, suitable genosnic libraries may be created by resfflcban eadonuclease digestion and may be screened with probes compireninary ui the desired gene sequence. Once fte sequence is isolated, the DNA may be atilfe~d using stadani pnncer directed arnpi ificanion methods such as polyxnerase ch~ain reaction 4,6583,202)10o obtain wnoun's of D)NA 5uitbc for UInraformadion Using appropria vectors.

4 kjtesatively 1 *o=njd libraies may be crated where large siegments of genomric DNA (35 A5kb) may be packaged into vectors and use-d to ta~onn appropriat hosts. CoAnid vectors are unique in being able to aconvno daze Ingse quantities of DNA. Generally coarnid vectors have at leant One copy of the Cos DNA sequence which is needed for packaging and subsequent cire~duization of the foreign DNA- In addIftion to the cos seLqutn3:-c tcs ve!ciors also contain an origi of repli cation such as Co'l M I dn. Z. r ss:.2cc mr~cr su ch 3a azcnc resistant o ampilin or neornycin. Medliodsof using coswi vciowrs for the nansfonnarion of suiitable bacterial host amt well dtscribed in Suxnbrok, 1, W Molcular Qnnw A Labortory Manual, Second Fclhion (1989) Cold Spring Haeoor Laboratory Pxnss. bic in incowporarcd by re ference.

Typically to clone cosans, foreign DNA is hislted and ligated, using the appropriate. resoiction endonucleases, adjacent to the co's region of the c*Smid vctor. Cosii vccwn containing the linearizd foreign DNA is then reacted with a DNA packagin vehiicle such as bacteriophage During the packaging proces the- cos sites are cleaved and the foeign D)NA is packaged int the head portion of the bacerial viral particle. These particles are d=e used to transfict suitable host crils; such as E_ colt- Once injected into the cell, the foreign DNA cirularizs under the influence of the cos sticky ends. In this mannr large segnwnt of foreign DNA can be introducd ad eaprrsed in recomb inant host Odts Cosmaid vectors and couid transfonnadun mnethodst were used within the contezt of the pe-an invention to clonc larg segments of genornic DNA frim bacterial genera known to posses genes capable of processing glycemio to 1,3-prapaniediul. Specifically, genonmic DNA from K. pnnanonwre was isolated by methods well known in the an and digested with the rescionv enzyme SaWfA for insertion into a coanid vector Stiperos and package using Gippackl packaging extracts, Followinig ostructiont of the vectorE. coli XLTI-Blue hM cell were tranfozred wit cosmid DNA, Transfornmnts wer screened for the ability to convert glycerol to 1,3 -propanediol by growing thec cells in the presence of glycerol and anayzing the media for l.3-Frpnmdol formazion.

Two of the l,3-propanediol positive ranfounanrs wero unalyzed and the cosmid wcrnanrdpKPI and IpKP2. DNA seuencing revealed atnii homoclogy to the glycerol clehydrarase gene from C. freundii, demonstraing thffi these transfontnants contained DNA encoding the glyol dehydratase gem. -z Other 1.3 -propanedial positive tfLfamt wer anayzed and the cosmid were named pEI'4 and pHI'S. DNA equencing revealed tha these osd catried DNA enoding a dial. deliydratase gene- Altho-ugh the instant invention utilizes the isolad genes from within a Kieb.*cia cosrid, alterae sources of dehydratase genes include, but arc not limted wo, Cftrobaaer, Clom-iia and Salnonetla.

Ottr gemes diat wrnl positively effct the production of 1 1 3-propancdiol may be expresd in suiLabkr hvsts, For czaxnpk it may be higly desirabl to over-express certain enzynmes in the glycerol degradaion p athw ay uidior other paxhways at levels far highr than currently found in wild type cells. This may be accowplished by the secriree cloing of the genes encoding those einzymes into minniicpy plasnilds -or placing those genes under a strong inducible Or constitutive proatr. Methods for averc=prcssing desired proteins arm ommon adwell known in the art of miolecular biology and exampldes maiy be found in Sambrook, wp~ra. Furthencire specific deicton of cerzan genes by umhods known to those skilled in the art will positively affect the production of 1,3-propanediol.

Exaznplez of such ruediods ran bie found in Methods in Euzyxuol ogy, Volume 217, R- Wu editor-, Academic Press:Smn Diego ([993).

mutanTs: In addition to th4 cells exemplified it is coutcmptd That the present method will be able to make use of cellsq having sigle or multiple muatations specifically designed to enhance the production of t,3-propanediol. CellUs tha normraly divtzt a carbon feed stack into nori-produotive pathways, or that exhibtt sigificmnt casbohite repression could be mtated to avoid these phenotypic deficienieis. For exmmnple, many wild type -cells ar subject to catabolite repress ion from glucos an by-products in the media ad it is contempLated that mutant strains of these wild type oigaisnis, capable of l,3-propanediol production th1at are resisram to glucose rpresion, would be particularly usmfi in the present invention.

Methods of creating muvants ame common and well known in the art. For exampl, wild type cells may be -exposed to a variety of agents such a radiation or chmical mutagens and then screened for the desired phenotype When creating mutations thraugh radiation either ukyaviolet (LTV) or ioruzing radiation may be used. Suitable short wavo TJV wavelengis for genetic Mutations Will faD.

within the, rane of 200 urn to 3 00 rim. where 254 nm is pitfeneod. UV radiation in this wavelcnrth principaly causes changes within nucleic acid svqucncc fr-om guanidine and cylosim to adem-inand tOymidine. since atls have DNA- repair mechanism that would repair most UV induced mucataous agents such as caffeine and other inhibits may be added Io inrterpt the repair prcess and naxxmuz the number of effective mutations. Long wave UV muatoio us 4 iht in the 300 um to400 tmn rngc c ata possible but are generally nut as effective as the alin wave LTV ligt mnIe= used in conjuncion with various acivatvo such as psoralen dyes that interact with the DNA.

Mutagaesiz with chemical agents is also effective for genr aing mutatus and cormonly ilsed .substuwts include chemicab that affci:t nourpliting T)NA such as HNO 2 and NH 2

OH

1 as well as agents that affect replicaig DNA such as acridine dycs, notable [cri c;?ising 1 J23Ircslff fItl l~ 5pcciflcict~ fur cureing mutants using radiation or rhernical agents air wewi docuiern1cFd in thr an. See for example Thomas Brcwk in Biotechnology: AlT1ksmk of Inqbsbiia MigN~i Second Edition (1989) Sinaucr Asocimcrs, Inc., Sunderland MA., or Deshpandc, Mukund V.,AppL Biochem. Biotechnol. 36, 227, (1992). hercin inccrparued by referrnc.

After mutagenesis has occurred, mutants having die desired phenotype may be selected by a variety of methods. Random screning is mort cormon where the mrucagenied cells am selected far the ability to produce the desired prduct orutrediaze Altemartively, selective isolation of mutants can be perfotned by growing a muragenized ppulaadon on selective media where crlly resistant colonies can develop- Miethods of mutant selection are highly developed and wll known in the art of indllsrria] microbiology. See Brock, Sura.- DeManci~ha e aL, Food Chu. 14, 313, (1984).

Mumftions andlt~sfomatigns in the 1,34m==diol roductim Wbwhwaa Rrrentative enyme pathway. The production of 1,3-popsnediol from glucose can be accomplished by the following series of steps. This series is represntave af a number of pathways known to those skillEd in the aM Glucose is convened in a series of steps by enzymes of the glyolytic pathway to dhydroxyactone phosphate (DHAP) and 3-phosphoglyccraldehyd& (3-PU).

GiyceroL is then farmd by either hydrolysis of DHAP to dihydxoxyactone (DHA) followed by rndution, or reduction of DHAP to glycrol 3-phosphate (03P) followed by hydrjysis. The hydrolysis step can be catalyzed by any number of celular phosphatases which are Imow to be a-specific with respect to their substates or the ativity can be introduced into the host by recombinatiout The reducion step cai be catalyzed by a NATY (or NADP-) linked host enzyme or the activity can be introduced into the host by recombination It is notable that the dha regulon contain a glycerol dehydragenasc (E.C I 1-6) 'which cuxalyztj the reversible reaction of Equation 3.- Glyce-rol 3-HP 1' 1420 (Equation 1) 3-HP -F NADH H+ -4 I ,3-P rndiol NAD+ (Equation 2) Glycol NADF [iRA NAIDH H (Eqation 3) 'Glycerl is converted to l 1 3-propanecliol via dhe intermediate 3-hydroxypropionalfthye (3-HP) as has beeni desaried in detail abovte inerwmediat 3-HP is produced from glycerol 1 Equafion 1, by a dehydratase enzyme which cart be encodc-d by the host or can introduced into the host by reoobinauion. This dchydratse can be glycerol dchydrarac (EQC 4,2,1.30), dial dc-hydmatasec (E-C 4. 2- 1.28) or any other e nzyme able to catalyze this fransfcnzinan Glycerol dehydratac, but not dial dehydratase, is encoded by 1hz dhw regulon.

1,3-Propanediol is piroduced frm 3-1HP, Equattion 2, by NATY'- (or NADr+) [fiked host enzyme -or the activity cart introduced into the host by recorbbidon.

This final reaction in thle production of 1 3-prapanedial can be catayzed by I ,3-pmanediol dehydtogenasc 1. .JIXQI-V) or other alcohol dehydrogenases.

Mutation& an~ transforintions that affeCt carbon chaneiig., A variety of mutant orani comprising vauiauiom in the 1 ,3-propanediol production pahway will be useful. in t present invention. Par cxample the introduction of a niasephosphate isornarase mutation (ipi-) into the microorganism of the present iniverition izs ani example of the use of a mutation to improve the perfonnance by cmtxboil chaineling. 'Me mutation can be directed Toward aL structural genec so as to impair or improve the actvity of ;an enzymratic activity or ma be direted toward a regulatory gene so as to modulate the expression ILevel of an enzymatic activity.

Alternatively, transformaions and muitation cani be combinied so as to control parricuhir enzyme -activities for thie enhancement of l,3-propanedio1 production. Thus it is within the scope of the present invention to anticipate modifications of a whole cell catalysl which k-ad to an increazed production of 1,3 -prop anediol Fe~nrion media in the prcsent invcntkn must contain suitable carbon substrates. Suitable substrates may include but are not limrited to moosaccharides such as glucose and fructose, ohigosacchanides such us latose or sucrose, pclysaccharides such as starch or celu lose or mijxtue thereof and unpurified mrnizus ftrm renewable. feedstocks such as cheese whey purmate-, cornsteep liquor. sugar beet mnolasses, and harley malt. Addhionaliy the, carbon ub mate may A"s be one-carb-onsubstrates such as carbon dioxide, or methaol for which metabolic cionversion into key hyiochnicaL intermediates has been d=mntnte4 GlyccrvZ production horn single carbonk sources (egnechanoI:.

formaldehyde oyr frmiate) his been reported in merhylouophic yeasts Yanuda et al.. Agric. SWL Chem-. 53(2) 541 -543, (1989)) and in bacteria (I lnter cal., B&,clwmirtry. 24, 4148-4155, (1985))- Thewe organism= casaiilate singl carbon comnfds, ranging in oxidation state frm methane to foanate, and produce glycerol. The pathway of carbon ass inilaxion can be diroush rihdlosr monophosphae 1 thro &mrine, or thrugh wyaiue-momophOqpter (Uols chalk, Pngi MtabSism, Second Edition, Springer-Vediag: New York (19S6)), The ribulose monophosphac pathway involves the condentsation Of fonnate with rib? 5j-hosp t on -s'agar Owl r hccmnrr frtose- :and seine pathway essimnilates the one-cartn ciompound io th-. glycolytic pathway via tnethiylcnacrrahydrofol ate.

I1n addition to one aLnd two carton =suhsat erehylottophkc organisnu ame also known to utilize a rnirnba- or other carbon containing compounds'such as meb-thylarninte, glucosarnine and a variety of amino ac-ids for metabolic activity.

For czw-nple 1 methylotrophtc yeast ame known to utiize the carbon from mnetbylsmine to form rwhaloc or glycerol (Bellion ct Al, Mkcrob. GToWT/ CI Com~pd., (Jut Syup., 7th (1993).415-32. flior~st' MuhrCEiT Collin; Kelly.

Don P. Publisher loercept, Antdoycr. LTK). Similarly, various species of Camlida will metabJizc slan-ine or aIsle acid (Sulter et Arch. Microblo.

(1990), 153(5), 485-9t- Hcnce c is, contemnplated thjat the source of' carbon udllzd in the prcsentA invention may encompass a wide varicty of carbon containing substrates and will nly be imnited by the choice- of organism., Although it is contemnplated thzt all of the ab~ove mentionied carbon substrates and mixunes therof are suitable in the present inventioP, preferrd carbon substrates are glucose, fructose, sucrose or methanolin addition to a appropriat carbon sourcr; fermientation meiCa must contain suitable mnincutis, salts, of~ators, buffers and other comfpofletlW known to those. gklled in the arc, suitable foir the growth of the cititures and promotion of fth enzyninfk pathway nrcessary fey i,.3-prpaniediot production. Particular uttention is given to Co(JI) sats and/or v itamirk IB 1 2 or pivC,=oIS thereof.

Typically cells are grown at 30C1C ip appropriate mnedaL Preferred growth nvclia in the present invention are common cornnrercialy prepared media such as Luria Rertani (LEB) broth. S abouraud Dextrose (S D) broth or Yec't medium (YM) ixoth. 0the defined or synthetic gmwth mcdi ma~y also be used and the appropriate medium for growth of the particular microrganism will be known'by someone skilled in the art of microbiology or fennrmentation science. The use oagents kown to modulate catabolite repression directly or indirectly, cyclic adenasine 2':3'maonophasphate, may also be incorporated into the maction media.

Simarly, the use of agents knowv to modulate enzymatic activities methyl viologen) that lead to enhancement of 1,3-propanediol production may be used in conjunction with or as an alternative to genetic manipulatios.

Suitable pH ranges for the fermentation are between pH 5.0 to pH where pH 6.0 to pH 8.0 is preferred as the initial condition.

Reactions may be performed under acrobic or nacrobic conditions where anacrobic or microacrobic conditions are preferred.

Barch and Continuus Fertnentations: The present process employs a batch method of fermentation. A classical butch femenitation is a closed system where the composition of the media is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the media is inoculated with the desired organism or organisms and fermentation is permitted to occur adding nothing to the system. Typically, however, a "batch" fermentation is hatch with respect to the addition of carbon source and attempts are often made at contolling factors such as pH and oxygen caoncentration In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stoppedt Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

A variation on die standard batch system is the Fed-Batch system.

Fed-Batch fementation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in inercrnents as the fermentation progresses. Fed-Batch systems are useful when carabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Ped-Batch systems is difficlt and is therefore estimated on the basis of the changes ofmeasurable factors such as pH, dissolved aoxygen and the partial pressure of waste gases such as C0 2 Batch and Fed-Batch fenrmentations are common and well known in the art and examples may he found in Brock, supra.

Although the present invention is performed in batch mode it is contemnplated hax the. method would be adaptable to contiuous fennentaiwmethods. Continuous fermentation is an opcn sy stem where a defind fermnewnion media is added ccntiuoudsy to a biomcctor- "n an equal amount of ondidoncrd media is renxved simultaneouly for processing. Continuous fermentati-on generally meain the cultures a a cot:en high deasity where cells amc prunsrily in log phase growth.

C~ntkwUUS fermenation alows fur the modulation of one fator or any number of factor that affect vceil growub or end prodwtr concentation. For exvnpc 1 one m-ethod will mainuain a limiting nutrient suchi as the cabori source or nitregrn level 21 2 fixe d rare and allow all Oth -Pa, en.CSVrmlrtL ethier iwccnn. number o f factors affecting girowth c m- bs altered continuous ly while thc czll concentration, mneasured by medi&a turbidity, is kmpt onstant.

Continuous sysn= striv o rn aintaini ste ady state growth conditions mnd thus the cell less due to media being drawn off must be balanced against the cell growth rate in the fennuentation- Menthods of modulating nutrients and growth factor for contintioti farenmo processe as well as techniques for maikuizig fth raw of product formaton arm well known in the an of industrial miaoboogy and a variety 4of mthods are detailed by Brock, supra.

It is comvernpiaed dthde present invention may be practiced using eiter batch, fed-bath or continuous processes and ihat any known mode Qof fermntation would be suitable. Additionally, it is ontemplated ta cells may be ed on a substrate as, whole cel) catalysts and subjcted to ferentation conditions for l$3-pwopanedfiot prduion.

Identiiatio and purlfion of 1.3=propm&iL Methods for the purification of 43 -prupanediol from fermentation miedia are known i the ext. For example propanedials can be obtained from cell media by subjecting the reacztion rnbnure to extraction with an organic advent, distilation and colum cltomarogrsphy 5,356,8l2) A particularly good organic sovent for this prouess is cyclohenane 5,0QB,473) 1$3-Propmnediol may be identified dircty try subrmiti the media to high preu~re liquid chrumacogmphy (HPLC) analysis. Preferred in the preseo invention is a method where fememntaition media is analyzed on an analytical ion imchange column using a mobile phase of (LOIN salfuri-c acid in vn isocratic fashiou.

Cels suitable in fth present invention comprise those that huarbr a dehydratase myme. It is contemplair-d that suitable cells may be either prukaryotic e- etukarytoic, arnd will be limited only by their ability to express ai7 active dchydrarase enzymne. Particuarly useful in the present invention will bd:, CcllS rhn we readily adaptable co large-scale fermentation metods. Suchl organinn re well known in the a of industrial biaprocesing, examples of which may be found in Recmhrninmwt Microbes for induscrial and Agricultual Applkcaxicn&'v Murooka et al., eds., Mn!W Dekker. Inc., New Yaztc New York (1994), and include fennencatve bactcria as well as yeast and flaumn-tous fitagi Typically the enzyme will be eithe a glycerol dehydramase or a1 dioT dehydramae having a substrat specificity for ecither glycerol r I 2-pwopancdIal, respetlvely- Dehydratase enzymnes ame capable of converting glycrol to hydroxyprupionaldehyde (3-EPA) which is then convened to 1.3 -propanedil. Cells containing tbis pathway masy icude mutated or recombinant organisms beonging to the genera Cimrobacter 1 Enierobacrrr, Ciostridiwrn, Kieb.Tielia, Sam one (Ia, and Lacrobadillus. Microorgaisms known by persons skiled in the art to produce glycerol by fcrmentation 1 cg., Aspergiu, Saccharomycej, Zygosaccharornvces-, Pichia, Kheyveromyccs 7 Candida, Hansenula, DunalieUa, Debv-ryomyccs, Macor, Tory (opsis, and Metylobacsuria, my be the hairs for a recombin ant derhydratas enzymne. Other cells suitable as hosts in the present invtntion include Bacillus, E-sche ridzia Pseudemnrnas and Strepromycer.- While -not wishing to be bound by theory, it is beieved that orgaisns beongin to the above mentioned groups, exist in nature that ame su itable, for ffie present invention.

On the basis of applicants' experiental work it is contemliated that a widr- variety of cells may be used in the preant invnti on. Applicants have dcmonstrated for "~ample that cells varying widecly in genetic and pheioric composition tic able to bioconve-t a suitable carbon substrate toa 1,3-p'upnediol.

Cells exremplified include, a K. pJ'wwnoniae mutant stainl constitutive for the dhta genes. ecombinant E. cai ustrns omprising elements of the Kkebsiella gename conain genes encoding either glycerol or diol dehydratase, and recombhpant E. coli strains also traxifcred with elements of the Alebsiella genornes and harboring a mutation in the gene encoding thew triosephosphace isornerae emyine.

Although E colih transformnnts. containing the, d-ha regulon from Klebsiefla pneumonia were able to convert glycerol to 1,3-propanediol. even in the Presence of glucrose or xylose (Tong cc App!. Maoch em. Slouch. 34, 149 (1992)) no I,3-propanediol was detected by these organisms in ft presence of glucose alone.

In dimei contrast to rts disclosure, applianus have discovered that tbree stns of E, coi, containing either o~f two independently isolated cosids comprising the dhta regulon frorn Klebsfrfla pneomnia, produced l.3-propmnediolfiurn a feed of glucose with no cxogenously added glycciol preent. E. coil stain ECL7Q7.

containng cosinid vctors PY-I of pEYp-2 comprising thle t- pnwnon rat d4 regulon., showed detetble though modes production or 1.3 -prapanediol fzatu glcose in the absErisc of cxogca usly added glycerol, (Rnamplc Recombinant E. cfl strains constiucted from an alternte host organism DHlict also containing coarnd vectors pKP-1I or pKF-Z, wer found to be. mor efwivc chin the EcL7Oy7 recorbinans i producing 1 .3-propanediol torn glucose widler the appropriate conditions. (ExampI* Mos effective in producing 1,3-propanediol fromn gicose under the conditions of Example 4 were the recombinant E. col strain AA200 containing cosmd vcaars pl'- 1 or pKP-2, Example 2. E. coi mtain AA200 contains a defective ti-iosephosphate isomeras enzyme, (tpt-) A strain of A-A20I-pKl'I 1,lctd for furl: f rudy fi Kim a pool of independcnt is olatecs from the vr--onatiofl reacziln, converted glu0co se to 1,3-prapanediol in a two zitage reacion. In the, fnls stage, the strain AAZOO-pK.P 1-5 was grown to high cell density in the absence of glucose and giver"'! In the second S12ge. the grown crib, suspended in a medium containing glucose, but no glycerol, convened glucose to 1 .3-propieicol with Nighb converioni and selec dvfly, Exfample 5. Although differing hrnmumochenticaily' chrornatograpfhkcllY. Knd genetically, fth coenzynie I 12 -depudent e=nze glycerol dehydratast (E.GC. 4.2.1 30) and dtiol dehyd-ratase 4.2.1.28) caralyzc the conversin of glycerol to 34tydrurypropionsldehYdc. Glycerol dehydranssc but not dial dehydratase, is enompassed by the dha regulon. K. pnewnoniae ATCC 8724, contzainig a dial dehydiatase but not a glycerol dehydratase convert glycerol to I *3-propanedial (Forage et al-, Bacterial., 149, 413, (1982)). Recombinant E. coi mansi EGL707 and AAZO containing cosmid vector pKP4 encoding genea for 2L dial dehydratase. converted glucose to 1, 3-propanediol, Ewanple 2 and Example 4, K. pneumoniae 8CL2106, pitpared by mutagenesis from a narrmly ocauirrzg strin (Ruch er al., Bacweriot. 124, 348 (1975)), exibits constitutive expression of the diw regulon (Ruch i supnz; Johnson et al., J. Bacteriot N0 164,479 (1985)). A strain derived hromn pneumrniae ATCC 25955 dislaying the sane phecnotype, has been skimaly pmepard (Forage et al., J- BacterioL 149.

413 (1932)), Expression of te Kkc~reflc dha scnctuial genes is. in pait.

controlle by a represso (product of dha M? (Sprenger et al., 1. Gunx Microbial.

135, 12:55 (1989))- Applicant have sho" wn tEt CU 106, which is constitutive forthe dha structura genes, produced 1,,3-propancdiO![ from a feed of glucoue in the absenc of exogenouisly added glycerol, Example 6. TIs is in contrat to wil-d type K. pneumoniae ATOC 25955 which dlid not produc detectable levels of 1 *3-propanedol under the samne condians, Example 6.

Thr- cxprossion of the dbha structural genes in ECL2106 is further controlle by estabolice expression (Sprenger et ul.4-J Cmi Microbial, 135. L-255 (1989)). Eliniation of cawabolite repression can be achieved by placing the ncccssauy su-rurzal genes under the control of afternate prniotors as has been demonstrated for 1 ,3-propaziediol oxidorcdnctuxe (dM7) ft-r C. freundit and dial dehydrarase from K ox-vioca ATCJC 8724 (DanWe ei I BacteriaL. 177,2151 (1995) and Tobimassu cc DIaBl. Chem- 270.7142 By eliniinatig emtabolite repression from FCUI) D6 in this mannerr an improvement iw cite production of 1.3 -propanediol firom glu~ose, in the absenc of an exogenous scarce of glycerol is achieved. An even farther inproverner is obtained by appmroiiatc carbon channcling as is described, by example. with the rpimutatian.

.As the dha regulonsof Cirrobacter and if/ebsie tha Tp. arc stikingly sirnilir, one of skill i the art willf appreciate that teachings that involve the production of l,3-propanedioI from gliteose in the absence of mn exogenous source of glycerol for K/ebsic lua sp- applies to Cirroiwcrer sp. as well.- Furrhennocr. As the metabolism of glycerol by C. b7w5!icunn is comparable to that of K pneurnoniac [Meng el al., Riotec/uwi. and Biceng. 44. 902 (1994)), teachings will extend to Ciosidia sp. Ls well.

MIMILLU

(IEhUTFALM=rODS rrocedures for phosplioxylutions. Lgarions ant transformations ame well knowrj in the afl- Techniques suiiable for use i the rollowhlg examnpe-s may be founid in Sambrook, 1. ct al-, Mnlevdla Qounrf A% Tjjg~re Mmjnj Seocd Edition, Cold Spring Harbor L.ab-oratory Prss (1989).

Materias anid methods suitable for the maintenance and growth of bacterial cultures s=t well known in the art. Tedmique suitable for use in t following examples may be found in Manual of Methods for Genera PaW&rilog (Phi~pp (lebardt, R- E. Murray. Ralph N- Costilow, Eugene W, Nester, Willis A. Wood. Noel R, KMeg end G, Drigs Phillips. eds), American Society fox Microbiology, Wa~hingiorl, DC, (1994) oir Thomas D_ Br-ock in Bictechnolorv: A TextL2okof Industial Microbipoou, Second Edition (1989) Sinauer Associates. Inc- Sunclexand, MA. reagents and materials used for the growth and maintenance of bacteral cells were obtained fromn Aldrich Chemicals (Milwaakve, WI), DEFCO Laboratories (Detroit, MI), GID3CC/BRL (Gaihcrburg, NM), or Sigma Chemnical Company (St. Louis. MO) unless othrwisc specified, Trhe uteaning of abbrcvialians is as follows: "hi" =mn hourAS)," I =wan minute(s), Hsecl m ascond(s), &'rmeans day(s), 'ThU mneans rndlliiters, T m L' means liters 50amp is 50 pgtznL ampicillim and LSB-S Oamp is [aria-B entani broth containing 50 gxnb azpiciliin.

Within the tables te following abreviaians =m used. "CooPh is conversion 1 'Se12" is selectivity based on catonY4 and Thd 1 is not detectd.

Glycerol debydraiase activity in Cell free' erracts Was decennine usingS l.2-propantediol as subst. The assay based on the reaction of midrehydos wit methylbenzc-2-thiazolofle hydraone, has b=c desezibed by Forage and Foster (BiO~~L B3fop4h'.r. Arcia. 569, 24 9 (197 Thc activity uof 1l,3 -prvpanicdal or-idoreduarasc 1 sometimnes referred to at l3-prupswnedicl dchydrogcnawc, was, deftennined int soluicn ori slab ge)2s using l, 9 3-prorpaxiediol and LNAID* ag substrtez as has also been described- Johnson and Lin,.J. Racitriol, 169, 2050 (1927).

The converion of glycero to 1,3-pwopanediol was monitomd by BLL Analyses were performed using stanidard techniques and riaeih available to one of skil in rt =n of chromatograpl y- Oner s it able- method uilized a Watcrs Maxima 820 HJ'LC system using IIV (21.0 rm) and RI detection. Samples w=r injectd onto a Shadex SH-- 10 ll column (8 mmr 2L 300 mrat purchasd fromn Wrens, Millard 1 MA) equipped with a Shodex SH-lO0IIP piecolulmn (6 MM x run). tccrature canorilld at 5(r'C, using 0,0; N F-27S534 as mobile phase at a flaw rate of 0.5 muL/min. When quantitative analysis was desired, samples were prepared with a known amount of iriniethylacetic acid as external standard.

T'ypically 1 the retention times of glycerol (RI detection). 1,3 -prorpaedioI (PI detection) 1 and erinmethylacetic acid (UV! and RI detction) weie 20.67 min, 26.0S mmi, and 35,03 min, respectively.

Production -of l,3-prupancdiul was continued by GO/MS. Analyses were performed using standlard techniques and mastejials available to one of sk i dwsh an of CC/MS. One suitable method utiized a Hewlett Packard 589(1 Series IT eas chromnatograph coupled to a Hewlett Packard 5971 Series mass selective detector (Ml) and a HP-ENNO Wax colunm (30 mi length, 0.25 rom 0.25 micron film2 thickness). The retention tinem and massspectnn of l,3-propanediol generatd were comed to that of authentic 1,3-prop anediol (rnlc 57, 58).

An Altrnative method for GCINMS involved derivatizatiun of the sanuplt To 1 .0 rnL of samnple cultur supenat) was added 30 oL- of conlcentrated 'Ny) perchiori acid. Alteir jniig, the, sample was frozen and lyaphilirsi A 1: 1 mixture of bis(nirnethylsflylft-riflu uacetanxide;pyridine (30 UL) was added to thin lYaPhilizcd matrial, mixed vigorously and placed ait 65 0 3C for ontr- The Sample was clariid of insollubla material by cencrifuigation. The x=sulrng liquid partitioned into two phases, the uipper of which was used for analysis, The smmple was cbnnnaograpWc ons- DR-5 colun (48 mn, 0 Z5 zmnf1.0., 0.25 umn film tbkkncss; from J&W Scientific) and the retention rime and mass specnnt Of the 1,-propanedial derivative obtained from eultuxr supernatant were con-,aird to that obtained from authentic standards The mass spectra of IMS-derivatiueci 1.3 -propaneclicl contuinis the charateristic ions of 205, 177, 130 and 115 AMU COnstrnrdienof K. ogeumoniae cosrid LiI)ries ,tC pnieumoniae (ATCC 2-595 5) was grown in 100 ruL L11 mnedium for 8 hi at 37CC with aeration. JPaccedia (25 ruL per rube) were ctntifugcd at 3,0Q rpm for 15 min in a DuPont Sorvali ULC 2.13 ccntrifuge at roamn temperaturc. The bacter ia were pelkted ad supemarant was deranted. The bamneril me2 pellet -was frozen at -20VC, The chromosornal MNA was isolatcd ai outlined below with special care taken to avoid sheAring of DNA (Le 7 vontexing was avroided). One tube of bactria was reuspended in 2.5 mL -of .50 mM Tris -I UaM BDTA mid 500 ptL of lysozyrne (3I mghmL) was added. The pellt was gently resuspended and the suspenion was incubated at 371C for 15 rmin. Sodium dodecyl sulfate was added to hring the- final concenn-aiion to Th1is resulted in thie solution bcomring clear, Prow man K (50 -ug/raL) was added and the suspension was incubatred at 55C1C for 2 h- The tube w removed and transferred to an icc bath and sodium chloride was ad-ded to yield a 0.,4 M final concentration- Two volumes of ethanol w=r added to the liquid. A glass tube was Iiserred to the interface and the DNA was gently spooled, DNA was dipped into a tube containing 70% eral- Alter drying in vno 7 v the DNA was mmsspended in 500 ul of water and the concentration of DNA was detennined sptmophotomnecricsly- A diluted aliquot of DNA wats run on a 0.5% agscose gel to determLine the intact natuit of DNA.- The chromosoinal DNA was partially digested with Sau3A as outlined by Sanibrook et al., .wprcr DNA (2 ug) was digested with 2 unit5 of Sau3A (Promega, Madison, WI) at room tcmpcrani-v in 200 p.L of total vollume. At 0, and 20 min, samples (50 pLi were reved. and transferred to tube containing umol of EDTA. These tubes were incubated at 70MC for 10 mini. An aliquot (2 p.L) was withdrawn and analyzed on a 0.5% aga-tuse gel clectrophainsis to determine the level of digestion and the rest of the sample (4 8 PL) was stored at Thie gel was stained 'with echidium bronidoe and visualized mider UV to de~termine the partial dijgcstionj of die duxonosomal DNA. A deaease in fth size -of the chromosomal DNA with inucae in time was observed showing flal the decreasc in the size of the chromosomal DNA is due to the actionl of 5EU3A,- DNA was extracted from rest of the sample by standard protocol methods (Samabrok c at., yupra).

A cosmid iihary of partially -digestd DNA from K, pnewnoniae was prepared using Supecwos oaid veact kit end Gigapacki packaging extr=&t using reagents purchased from Stnrtagene (La Jolla, CA). The in-structions provided by the mvanuatrer were followed. The packagd K. pnewnoniae contained 4 x 10$ to 1L0 x lo5 phage itr as detcnnined by transfretiflg E. coi XLI-Blue Mt Cc-srnid DNA wxs iso l ated frmn 6 of the E. -ccl trnsfcnnafls and found tu omDain ~'sinsert f DNA (25 to Oonir aid tsnsonnaionof-E, rofi host ctlls with cusinid MliA for-tht t~~~Olp 3voatdo Synthetic 5Sl2 medium was used in thec s crening of bacterial trmnskrmant for the ability to make l,3-propandt S 12 medium containz: rnN amnoniuwm sulfate, 50 mM potassiurm phosphate buffer, pH- 7,0, 2 mM MgC 2 0,7 MM CaC1 2 50 FIM MhC! 21 I IIM FeC43 I j tM ZzCi, 1.7 pJNI CuSQ 4 Wi CoO 2 2.4 WM Na7 2 MoO 4 and 2 PM thiamine hydrochloride.- Medium A used for growth and ferentation consisted of: 10 rnM vrnmonim sulfa-te; 50 mM MOPS/K014 buffers pH 7-5, 5 mM" potassiuml phosphate buffer, pH 7-5; 2 mM MgCI7; (K7 miM CaC 2 50 jIM Ma 2 I P.M FcC1 3 1 pM ThI; 1.72 WM CuSO 4 2.53 gM CoO 2 2.42 pM Na 2 MoO 4 2 pLM thiamine hydrochloride; 0.0 1% yeast extrac; 0.01 casamino acids; 0.8 pWg/nb vitamixn B 12 and 50) Medium A was supplenmted with eher 0.2% glycerol pr 0U% glyccrul plus 0D.2% D-glucose as required.

IKlebslila pneumoniae ECL21O6 (Ruch yr al., Bacterial., 124. 348 (1975)), also known in the literature as K. aerogcflei or Aerobacler aeragciies, was obtained from E. C C. Uni (Harvard Mcdicai School, Cambridge, MA) and was mibnaid as a laboraory culture Alebsiella pneumoniac AWCC 25955 was purchased from American Type Culture Collection (Roekville, NW).

E. cofl DH5a was purchaed from GibcoilRL and was unsfonnedd with the cosnid DNA isol4r~d from Alebiella pneumwniaec ATCC 25955 containing a gene. coding for either a glycerol or dial delydratasc enzyme. Comd containing the glycw-ol dehydraas were identWiid as pKPI and pXP2 adcarnid containing tbc dial dehydzaas enyme Were identifid as pXp4.

Trnformed DH a cells w jndic a DH nw pxp1 D!4Sc-pKP2. and DHfba-pKP4coil RCL7O7 (Sprengr et al., j. Cr. Aherobiol. 135, 1255 (1999)) was obtamned ftvni E. C. C. Lin (Har-vad Medical School. Cambridge, MA) and was siniflarly lnnisformned with cosmid DNqA from K/c bsIdIa2 pncIumoniac. These wransfonnanw were identifieclas ECL7O7-pXP1I and ECL70Y7-pKP2 comafing the giycerfol dehydralas gene and ECL7O7-pKP4 containing the diaul debvdrmase gene.

E. coi AA200 conianinfg a matiton in the rpi genie (Andcnan et al., .1.

Gen Microbiot, 62, 3 29 (19 70)) was purchased firm the E. cali (jenetic Stock Cente-r, Yale University (New Haven, C-T) and was transformed with Kkbsitua cosmid DNA to give the, recombin=aorganisms AA200-pKPI and AA200-plcn 1 containing -the glycrl dehydnutase gene, and AA20*pKP4. conitaining the diol dehydratase gene- Six transf&nnatcn plates containing approximatly 1,000 caornes of E. co/t XL1 -Blue MR cransfeezd with K_ pncwnmoniae DNA were washecd with 5 niL ILB mnedium and centifuged. The bacteria were peDered and resuspended in niL LB mnedium glycol. An aliquot (50 pL) was inoculatrnd intoa 15 nL rtbe containfing S12 syruheuic mnedium with 0-2% glycerol +4W0 'g pe mL of vitrrnjj 12 MW 0% yeast cxtnt 50a'np. The tube was flled with the medium to tbe top and wrapped withl parafii aLnd incubated at 30 0 C. A sliht turbidity was observcd after 48 b, Aliquots, suialyzcd for product distribution as described above. at 78 h and 13 2 hi, were positive for L,3-pmpa~nediOl, th~e Later time points ontaining incnased amownts of 1,-propanedol.

The bacteria, testin positive for I,3 -propanedi pducdcn, wer, SeriLl~y dilute-d and plaed onto LB-S()amp plates in order to isolatec, single colaries. Forty eight single colonies wer isolaed and checked again for the production of l,3propanediol Cosmid DNA was isolated from 6 independent dlues and tranformed into S coi stmai DH5-a, The- tansfonnmnts were again checeked for the production of .1 3-propanediol. Two rrasfvrnmns wre chaactrizd fulrthr and designiated as flH5ct-pKpJI and DHSC-pKp2.

A 12.1 kb EcOM-Sall fragmnent from pKPI1, subcloned into pM8131 (181 Biosystem, New Haven, CN), was sequenced a-d termed pI-KZS-26 (SEQ ID No: Sequencig revealed the Loci of the relevant open reading francs of the dhae Operon enIcoding glycerol -dehydratase and gene necessary for regulation.

Rgefening to SEQ ID NO,, 1. a flhgint of the ope-n readig frame for 4/urN en~codingdihydxuxyamceekanae is found at basez L-3919- the open readin 6 frame dhnaD encoding giy-ccrol dcblydrogenase is found at bates 983-2107; the open readinS frame dhaR encodiru the repressor is found at bases 2209-413-4; thr open readig ftamn dhaT encoding 1 31mopndL niorceductas is found at bases 5017-61890; the open rcading ftrame dhahl eacodkig the alpha subunit glycerol dc-hydratms is found at bases 7D44-8711; fth open reading frame dhaB2 encoding the beta subunit glycerol dehyckarse is found at bases 8724-9308; the open readin frame dM8B3 encoding die gam=a suibmit gl-ycol dehydrazase Us found in bases 9311 -9736; and the open reading framei dhaoX, encoding a protein of uniknown fwiioni ih f cnd at 97zf9-1 152 Single colonies ol E. col )0-1 -Blue NM tnansfvec? jracdcsr DNA ftrm K. pheumoniac were ino--culate4 into niicrotitct wcibs containing 200 uL of S15 jmcli (aniionium sulfate, 10 mM; potassium phosphale buffrr, pH TO, 1 rnM; MOPS/WOH buffer, pH 7.0, 50 ryM; MZ,g~ 2 mM; CaCl 2 0.7 rnM; MnCI 2 50 uM: EtCI I ttM lnCL, I tiM; CuSO0 4 1372 UM; CoCI 2 2.53 uM; Na 2 MoO 4 .4 uM:. and thiamine hydrocbluridC 2 idA) 0,2% glycerol 400 ng/mL of vitantin B 1 2 U.0I1% ye ast emtact .50 ug/mL ampicillin. In Addition to the miactiter wells, -a master plate containin L13-50 amp was also inoctdaced -After 96 100 uL was. withdrawn and centifuged in a Rainin microfoge tube containing a 0.2 micmo nylon mcmbrane fiR. Bacteria wee retained -and the filtrate was procssed forHPLCanysiA. Positive clones demnon~strating 103-propanediol produ ction were identified a Rer screning aprximately 240 colonies. Three positive dones were identifiedt to of which had grown -on LB -5 0 amp and one of which had not. A single, colony, isolated from one of the, two positive clones grown on LB -50 amp and verified for the prodluction of 1,3-ropanediol, was designatd as pKP4. Cosiid DNA was isolated from E. coi strins! containing pENI and E. coii strain DH-5a was trrnfornnd An independent trsmfonnmnt, designated as DHStt-pKP4, wasq veilted for the production of 1 ,3-pzopsnediol.

WCL707 E. coi strain E0L707 was transonned with cosnid K. pncwnmoniae DNA cotrespondirig to pKT I. p1ZP 2 p1(1 4 and the Suiperos vector alone and tine-d ECL70D7-pKP 1, 2CL707-pKP2, ECL7O7-pKF4 and BCL7O7-sc. respectively.

BCL707 is defective in gid, and pisb which ecde the, ATP-dependent glycrol kinas; NAD-linkcd glycerol dehyckogenase. and enzyme 11 for dihydroxyaceton of the ptiosphoenpolpyriivaoe dependent jphospltotrwsft-ras e stem respectively.

Twenty Single C01lomcz Of each eoumi-d iransfonnation and five of the Supercos vector alone (ngative conrl~t-mnfomution, isolatled from plates, were trnsferred to aznaste LB-5Danw Plate. Thes isolates were atso tested for thecir ability to convert glycerol to 1,3-papm:-ffiol in order to dotrmne if they contained dehydrazase acivity, The nisfonmants were tinfed with a sterile Coodtick to rnicrotitcr plates cantaining 200 piE of Medium A supplemented with either 0.2% glycerut or 02% glycerol plus 027% D-glucose.

After incnbadon for 45 hr at 30 0 C, the contents of the- mierociter plate wells were filtered throught an 0.45 p. naylon Mwte and chronaiographed by HPLC. T1he results of these tests are given in Table I- Tabikl Convcrsion of glycerol to 123-propanediol by tranformed ECL7O7: numnber of positivei listnbro izol ars tcaied 2jmIOaui Glyce jvrol plUS ClucqSe ECL7O7-pK-PI 19)20 1 2 E0L707-pKP2 18f2o 20/20 ECL7O7-pKP4 0120 20/210 UCLO7sc0/5 E. COiL strain AA200 was unsfonned with casznid K. pneumoniae DNA orresponding to pRY 1, pKI'2. pKP4 and the Supuexs vctor alone arid named AA200-pKPI, AA200-]pKP2, AA200-pXP4, and AA200-sc. resjicnivcly. Strain AA200 is defealtve in triosephosphare isomerase.(pl Twenty single coloniies of each costai cranfomiadlon and five of the empty vector transformation were isolated and tested for their ablity to convert glycerol to 1 3-propanediol as deribed for caft strain S-CL7(It The results of these tests are given in Table Z Tatk2 Conversion of glycerol to 1,3 -propanediot by transformed AAZO: N wmber of positive isoltes/umbr of isolates tested £ThnMWOlcrol Glycera PlUS.Glulcose AA200-pKPl 17/20 17/20 AA200-pKP2 17/20 17/20 AAMO-pKVI W20 16/20 £4200-se 0/5 Conyea 'n of D-9 lucose 19 1.3-4 pz-pnediol Lty F. COlt £Iehydraae activitY Glass serum baffles, filed to upmify withI media (ca. 14 mL of Meditim A as defined in Ex ample 1 Suppleiesled with 10 [LghfmLkanamycin and 0.2% D-glucose. plus or minus 0.5- 1.0 caM cyclic adenosine 2':3 rc~hosfrlac (cAMP)). were innoculated with selecred single colony jaglakcs of E. cofl ztrnin AA200 couaning the K.pncmnnae dlvi regulon connids p]KPlI orpVKP2, the K, pneumoniae pdu operon pKP4, or the Supecmos vectur alone. In order to avoid conn ct with glycerol. the irrnocul adon wa pefrmcd ficr.' e ithcr r agmr-2 3ht, of LE -5 Oamp or fi-r a Liquid cuictire Qf the sa rncx Li The xrAtiii5 wren neubatcd for ca. 72 hr at J0'C whie shaking)4 ac 250 rpm. G rowth was determd by the change in absorbance at CO0 nm what the initia ODW was -0.020 AU. The cxtcnt of glucose depleion and product distribadton wmi detenn.ineciby 1-PLC Single colony isolates are identifir-d by a nUni--td suffix AA200-pKP1I-m, Cumulative results axr presented iTu'lc 3 and Table 4- Conversion of 0.2% P-glucose, to I proanediol by nJfgfllcda Lo twlstraip AAZOQ:, wihout c&MP I l3-ppsne- -T,-usformanC OD. n. diol] (rilv) Coi.% e AA200-pKPJ-3 0.06 0.517 1 AA200-pKPl-5 0.115 nd 0 ORY.07 ad 0D.076 0.2 14 0.116 rid 27 0 0.205 0.3 17 8 AA200-pKPZ-10 0.08 0.2 13 7 AA200-pKPZ-l4 0. j17 0.5 17 14 H0,129 0,7 19 AA200-pXP2-20 0.094 rid 1t 0 AA200k-pKP4-4 OL, M. CA8z 2 AAIOO-PKP4-l9 0.197 0.2 34 3 AAZO-ffU'4-20 0,20m OA 38 1 AA200-sc-4 0,-097 ind 22 0 0-176 n'S 46 0 Table Conrversion of 0.2% fl-glucose to 1,3-propanediul by turaned R, coi stran AA2,00: w~t cAMP [1,3-propane- Transfomiant ODrr diol] (m M) Cmn Set- AA.20-pKPJ-3 0.102 M. 19 12 AA200-pKPI-5 0.0&9 1L5 2-1 37 '0236 1,4 24 28 0.07 Q.8 15 23 AA-200-pEPI-20 0.153 nd 40 0 0.185 0,9 27 16 AA200-pKP2-10 13.098 0-2 13 7 AA200-pECP2-14 0.213 2.0 1627 1,0-155 0.6 2-5 12 AA.2O-p.P2-20 0.198 1.2 40 14 AA200-pKP4-4 0.218 0.1 31 2 AA200-pKP4-19 0.2.23 0.2 37 3 AA200-pKP4-20 0,221 10.2 35 3 AA200-sc-1 0.111 nd 23 0 0.199 cad 49 0 0-122 ad25 0 'The idetntity of 1.-propanedicl was vcrffied by GCAMS as described in the GENERAL METHODS.

Lonveton of D--I vuccse to -L3Vro~anedolji hx :ljofirrin DH{S0;. ranfcnucd wFjth Klebsiellioypneurnonioe, DNA containingTstcydras ativitv E coli auain DH5 a, containig fte K, pneumoniae dha reg-lon cosinids pKP1I or pKP2. were teste-d for their abiity to convert V-glucose lo l,3-propanedio1 as described in Examrple 2. The results a=c preented i Convcrs ion of 0.2% P-glucose to 1 1 3-prcpancdicl by trnsfoce E. cc U strain DH~rt plus and minusr cAMP Transfonnhn DH1cz-pKXl DH5a-pKP1 DH.Sa-pKP W% 0.630 0.774 0554 0.6919 [1.3-pwpane- 0.5 0.6 016 0,7 Con.

100 100 100 1on 2 3 3 3 (k01ver~ion of D-!1u1CoFC tQ I±EpvarvIo b .7Ts071f ,2l 1T~nst2-1rKd with KlebsTiei~o~*e~roiq N containing, dehvdntas activiXY E. cobi strai ECt7O7, containing the Et pnewncniac dha xeg-uori cosrnjdz pKP I or pKP2 the IC pnewn antae pdu cperom pKP4. or thc Superco s ve ctor alone, were tested for their ability to convert P-glucose to 1,3-propPanediol a~s descibed in Example 2. In each casr, onversion was qumnitantve. Theso tiwwts amepresented in TAb~ 6- Cuaversion of V-glucose to 1,3-r by cnnskfomed E. co h Str ain EC wit-.m~d VrjrhoutcAMt [1 .3-propane- OD~n 0 dioll m (without _CA.~ Transformarn ECZL707-pKPI E-CL707-pKPI -3 ECL7O7-pKP 1-7 ECL7OI-pXF1-10 ECL.707-pKPl-1 S ECL7O7-pKP2- ECL7O7-pEY2-5 ECL707-pKP2-9 E-CL7O7-pKP2- 15 E-CL707-pEP2- 19 E-CL7O7-pKP4-8 ECL7O7-pKP4-9 M-07 0.619 iD.582 0.593 0.584 01588 0.630 0.542 0.589 0.577 0.499 0.544 panediol L707: [1.3-propane- ODmm diod) (MM).

0.422 0.1 0.522 D.2 0.408 0.1 0.433 0.1 O-409 0M 0516 0.2 0A8& 0.1 DM85 0.1 0.504 0.1 0,361 cO.1 0,354 rid E-CL7O7-pKl'-10 0.35 nd 0,265 cal RCL77pKP4I14 0.512 ind 0.319 c0.1 13CL70Y7-pKP4-17 (k545 rd 0.338 <0.1I JECL7O7-sc- 1 0-9 d 0.385 ad EXAZELE TWONI !a=c conversoi- Of D-Eticosv, tammeltoi by LEawii co1i AA2zQQ-p1 Baffled flubs (250 rnL) contaiing 50 rnL LB-amnp medlium were inoculated with single cQoonies of AA200-pKY1.5. The cells were grown. in duplibee overnight at 30 or 37rCwithi sakig (250) rpm).

(3rovai culturc were Ttun 10 mlinutcsr lO0000 rpm. 492) anid resuspended in production medium without glucose (10 rM (NH 4 2 50 4 -5 rmMN POtassim phosphaie buffer, pH 7.5; 50 niM MOPS, pHf 7,5; 0 .01 yeaw ext ract; 0.01l% cas aminn acids; 0.3 -pg/niL vitaruih B, 1 2; and 50 pg/inLL ampicillin) containing either trace rmrads A. (0.08 pM CoO 2 0.'06 pM CuO 2 7 piM FeSO 4 2 gMI

E

3 B0 4 0.2)pM MnC42 0.1 pM Na 2 MoO 4 1 0.08 pM N0C 2 0.3 1M ZoSO 4 and 0.03 mM thiamine) fir race metals 1B, (0.7 mM CaCJ 2 253 LM CvCI 2 1.72 d CuSO 4 1 -0 P.M FeCI 3 2 niM M90 2 0.05 rnM MnQ 2 2.4 2 p.M Na 2 MaO0 4 JIM 7nCI 2 and 0-03 jimM thiamine). The cells were spun a second thuc, ncaaspended in 50 atL fretsh production me-dium containing 1)-glucose arnd dispensed into 60 itt serum bankis whi-ch were capped and sealed with buty! rubber septa. The bankls were shaken (250 rpm) and samples wididrawn with a syringe through the seprri mid filteid trough a 0,2 p fiter befor anaysis.

Resuhs; are shown in Table 7 mid Table 8; rusidual glucose was measured by enzymatic analysis (B3iochemnistry Analyzer, Ye~low Springs hiniruents Co., Inc,) and 1,3-propancdi ol was analyzed by HPLC.

Tablez Conversion of 0.2% D-glucose, re I .3-proparediol by EsYcherichi call AAL20-pKPI1-5.

D~ugjicgtC rections w EzvxCfo-n a Time [Glucose] [1,3-propauc- Con- Sed.

Experimmt~ (days) (MiM) diol] (mIM) M(0b #1 15 0.1 2.3 99 #1 4 0.1 23 9.9 #2 1 2,8 23 75 14 02 4 031 2.4 99 11 a~he reactions mubnirca, cuntaining um=t metals A, wer incubated at 37 0 1C.

Convemion of 1% D-glucose W0 tm [glucose] [1,3-propance- Call- Sri_ (days) (inM) diojf (mM) 1 39 5,6 26 2 35 8.3 34 23 3 33 8-4 38 1 rl'he reactions rnixptuts.$ containing u-=c metals. B. welt incubated at 30 0

C

bA th t, end of the icac-ion, the prescnce of 1 ,-propnedict was confuied by CC/MS and 13 C-NMR (3W(1 MIIz).

Glass serum bottles, flildwto capacity (ca 14mrnL) with mcdiaL wer lightly innoculated b-cm a LB agar plate containing K- pneumorce ECLZ 106 or K. pflnfeurfnit ATCm 25 955. The miedia contained 50 mM glucose. 3 ruM (NMt) 2 7S0 4 .0.9 MM GAO 2 4pVM CoI-, 0.06 pM CuC3 2 7 p1M FtSO 4 2 2PM

H

3 B 04, -0.1 xM M0S4, -0.2 pM MnQ 2 0,1L pM Na 2 MvO 4 0.08 JIM NiC:Zr 0.3 pM ZnSO 4 0.1 mg/mE, PL-cysteinc. 10 p.M etlhylcnedia nhxetetrazcttic acid, 0-8 kg/inL vitam1in 5 12 pOtassiurriphosphate as indicated in Table 9, and eliher mM IEVES or 50 mM MOPS buffezrpH 7.5. Thy reaaions were incubated for 47 hr at 30'C whil shaking at 250 EPL OtbWXiSe. the reaCtion weS pc-rfonned as dr-scribe -d in Example 2. The results =r given in Table 9.

Conversion of P-glucose to 1 1 3-propar'edicl by KlebsiEl.

pncunwiae ECL2106 but not by Strain Buffer Pi [GluCOSe] [1,3-EkopaflC- (tm) dioll (mM 2105 MOPS 53) 11.4 012 2106 MOPS 2.5 13.9 0.2 2106 MOPS 1.3 14.8 0.1 2106 MOPS 0.6 15.8 0O1 2106 EEPES 5.0 21.1 0'1 2106 HIEPES 25 23.4 0.1 2106 =EPS 1.3 2654 0.1 2106 HEPES 0.6 27.5 0.1 7-5955 mops 5-0 4.4 nd 25955 MOPS 2.5 5.4 rid 25955 MOPS 1. 2.8 nd 25955 MOPS 0.6 738 nd 25955 REPES 5.0 7T0 nd 25955 HEPES 1.5 13.5 rid 25955 HEMPS 1.3 10.2 Rd 25955 24EPES9 0.6 17.8 nd Produ rion ofi .3-trcpanediol by rcornbinsn Pidxia rasroris ConStrocli-on of nrrai puijxsg exprEsion jasmid 'The 0.9 kb EcoRlJXbalI fragment in pIfUN-D (Phllps Petroleum, Batrtesvifle, OK) Was replaced by the 0-9 kb ]EocRlI/Xmal fragment from (Inviirogen, San Diego. CA) to generate the plasmid pHMt-D4B2 which contins t following elements:. YAOX, P. pasroris methanol inducible alcohol oxidase I (AOXI promoter, AOXJ ternz, P. panoris AOX I transcriptional twieriation region;RI1S4 P. pasworis histidinol debydrogenase-cncoding gene for selection itt hirY4 hosts; kran, sequence derived frwm transposon TnVOS encoding amin-oglycoside 3'-phosphatmnferasc, conferring kanenycin, neomycitt and G418S resistance in a wide variety of hosts, and useful ax an indicator of c-assette.

copy mnmber; J'AOXJ F. paswari,5 scquc-ncc downstreanm frcom AOXI, used ini c-onjunction with SAOXJ for site-directed ved±or integration; ofi, pDRS22 origini of DNA replication allowing pfasnid manipulations in E- cofi; and amp, P~-laase gene frmn pBR322 conferring resistance to ampicilli. An additions]l fe-ature of pMlL-fl4 2 is that mnitple exresion cassetles (5 AOXI gene AOXI rerm) can easily be placed onto one piasmnid by nxbcloning cassettes on B3gl2/Xba I fragments into BaanHIt'Ca I s its.

C~nscrjtin of niasmid for co-exnression of dhaill and dhafi The open reading frames for dhaBl aid dM8E2 wer inplified from costnid pK?1I by PCR using p~ixners (SEQ ID NO: 2 withi SEQ ID NO.:3 and SEQ ID Nk4 with SEQ ID) NO:,5 for dihaBi and dMR2 respectively) incorporating EcoRI sircs at the5'ends (10 mM Tris]pHF8.7,50 n-M KCI, 1.5nM MgCa2, 0.LXI1% gelatin, 200 jiM dATP, 200 pM tJC T, 200 paM d=TP 200 pM MTTP, 1 pM each prinior. 1- 10 iig tagct DNA, 25 uniits/xL Amplitaf DNA palymerese Perkin Elmer Cetus, Norwalk Cr). PCP. pnrnexor were 1 nqm; at 94"C. I trim at 551C,2I 1in at 72 0 C, 35 cycles. The products were nibeloned into t EcoR I sit-t of pH[L-D4B2 to generate the expression plasruids pNETl9 and PNIfP2 conrtiing:dhja I! and 4habZ2 respectively. TUc daR I expresion cassette on a Bgl2/XbalI frurnent from PMTPI9 was subelaned finto the HhnffIXbalI site of pMP2O to generate pMPZ1. Plasid pMP21 coc mn expression cassettes far bath d/raB2 and dhatE, MrK the 1154 selectable mrarke of $asznid for c-epirssion of dhaBi and dhaT The open rcading franie for dhaT ma dhcaB3 were amplified by PCR horn cosmidp" I using primers (SEQ ID NO:6 with SEQ ID ND;7 and SEQ ID It) NO:89 with SEQ II) NO: 9 for dhaT and dM8B3, respetivdly) incorporating EcoR I sires at the 5' end.; The products were subcloncd into ti-c EcoR I site of p1ID42 to generate the expression plasnilds pMPl17 and pMTJH containing dhaTI and d/aa3, r rcccivcly- The dlwT expresion cassett on t7BgZXb41I fragment from pMPI 7 was sube loned into tht 13 amI1XbalI s it of pP.W 9 to rreeriu- r)MP22 vhich cornains expression casseues for both dhaTl and dhaB.

The 4.1 kF EoRi frament ciontaining SUC2 was deleted from pFLK2O (Phillips Petroleum, B artleaville, OK) to generte pMP2. S0C2 encodes for an inverizse which may be used a second selectable mnarker in Pichia, The 4M kb Hind3 fragmnent contmining lacZ was deleted form pMF2 to generate pMP3. The 0&4 kLb Hind.3 fragment containing A OXI term from pMfL-04 was sitcloned into the Khind site of pMI3 to generate pMPIU.

The 2IC kb Pgl~tXbaI fra~mcnt irkpMPJO0 was rcplaccd with the 5.0 kUi Bgl~balI fragment comnix g the dha83 and dhaT expression cassette from pMP22 to generate, pMP23. The 5A4 Lb PstiIBgl2 fragmnt concainin SUC2 from pRK2O was suboloned into the Psil/Dgt2 skte of pSP73 (Prmegi, NxMo, WI) to generate pPi la. Plasmkid pM~l [a was cut with EcoRt, filled with T4 DNA polyrner=s and religated wo geeate pMfl lb. 7te 1.1 kb PSt/Bgl fragmnt in pMPIO was irpland with the 5.4 kb BgI2/Pstl fraumet. containing £UC;?frpoM p Ib to generate pMPJ12.

The. 1-0 k, 5rAl/B g12 fragment in pMP23 was replaced wvith fthe5.2 kb ScalllgI2 fragmnt containing SUC2 hrn pMFlZ to generate pMfl4. Plisnid pMP24- cotains expre-ssioni cassettes for both dhaT and dhraB3. and the SUC2 selectable miarker.

Tflgflnjlrolntf 1! paivtods with dhaBljdhaf2 ;jirssion pVaszni pMP21 R pantoris strinh (3TS 115(his4) (Phiffips Petroletum, Bhrttesville 1 OK) was tranfomined with 1-2 tg of Bg!2-linearized plasmid pbM using the spbcrolast tumfoaaion. method dc dbed by Cregg et aL, (Riot CelL, 210115. 3376, Cells were r eertd on plate without histidinte for 3-4 days at 3oVC.

urnsfOmasur arise After integration of plsmnid DNA into dhedm o mz Ranformants were parced onto a YPD Dacta yeat exuaa*, 2% pepwne 2% glucose) umster plae.

Scring of P. paste-s flnSfonns ft dhaB/ and-dzafi2 Chromosomal DNA was pieparrd horn his." tranfonnanms descrbed above and subjected to PCR analysis withi primer specific for dhafll and d/w2 High copy number suram were selected from ltrasfonnants ontinin both ethOS) and dM122 by growth mn YPD mdia supplementd with inetaing levels ofG0418 (Sipma, St. Louis, MO) up Co 2000 ±g/mL. Resistance. to a high level of G413 suggest& significant daplication of expressivn cAssttes.

S--cond ar tnns~maton ofP.Vpasrs with dihafl3ldhoTxpssiM yaismid pIAP24 Transfonmts with resimtace to a high level of[G419 as described above wee r-transfonned wit plasnid pMP24 un in g t pheroplsst iransformasan method- Cells were ffirs regenerted on non-selective plates for 2 days at 30 0

C.

after which top agar containing the regenerated cells was scraed from the plate, and vorte~ed zeiyiely ini 20 niL w-ater. After passing though 4 folds of cheesecldc, the cells were pelleted by cenudlugatian and resuspended ink 10 rob wratr. Alignots of 2030 u.L were plated onto sucrose plates znd incubated for 2 days at30 0 C. ALI umasformunts arise after integraion of phLqsrid DNA iWO the chromosome. Transformmnts appear as large colonies in a baokgraund oa siaa olonies, and re-quirr isolaton- After 24 hi growth widh shaking at 30'C ini Msu media (pcx L, 13.4 g yeast niagcn base w/6 amino acis, l0 g sucrose, GAg biotinl), tralfounzs were steaked4 onto Msu plates (IMEu media plus! 15 g/L agar) and grorwn for 2 days at 30uC Large isoated colonies wereparched oto a YPI) master plate.

Scrceri varswris douNblersnfwinants for dhraB d4a12.s/haB.3-and dhaT anrd their-carreUpadinezn a Vjyiir Charomosomnal DNA was prepared from suc* double iransformanta describe-d above and subjected to PCR analysis with primers spcific for dhaR1.

dbafl2, dUzD3q and dhaT. Thus, the presentce of aDl four ope-n reading framnes was confiMned The presence of active glkcerol dehydratase (dhaR) and ,3-propmnediol oxido-reductase (dhafl was demonstrted using in vitro enzyme assays.

Additionally, western blot analysis confincd protein expression from all four Open reading framnes -Cell frete extracts for thse prnrein chuactedzadis were prepared as follows5, Double trmnfornnats ontinirng diwiBi, d/ta2, ethail. and dhwT were ruwn aer-obicaLly with shaking at 30'C in MGY (Per L. 13A g }S-ct nitrogen base wca amno A6&,s OA4 mg biotin, 10 rnL glyctbol) for 2 days..The c*lls we=e peilerd by ccnrifugatiui 1 reuspended in MM (Per L, 13.4 g yeast nitrogen base w/o amino acids, 0-4mrg biotin45 mL mcthanok) and incubated as above. After appmrpziatly 24 h, the cells went harvested, resuspended in buffer (Q.A M nijcene/KOU buffer, pH 8.2, 50 mM KCI. and 2% 1,2-propanediol), mechanically disrpted (using a glass rod while vortex ing in dhe precnc of glass beads), and certrifugect One =&rin that showed positive far the presence of all four open 'ading frame s (dhtzl, dA0l2, dhaBS3. and dM7') and thecir conesponding activities WM dc: igaatcd1 MP4 2.-81 and was scccied for furthe-r study.- LrF iyf ppk in o 1-:V~ne~i:P1usiz mnmbnan Pi 4i pavtri P. pasrorr NIS P4 I2b I (ATICC 74363) were grown in a B iosta-tE knncntcr (B Braua)Riotch. Inc.) in 1.5 L rninirnnl mediumn containing 2.5 KH 2

PO

4 2.1 g/L (NThE} 2 S0 4 10 g/L glycerol. 23 gIL MgSO 4 -7H 2 O. 0.18 gIL CaLS0 4 2F1: 2 0, and 0.29 mfl- PTMI. FThiI is a .;tu)Ck fl l dl S4tdL: 01', CO,117&fa fln, 24 MM CuSO 4 4.8 ruM YI. 18 mM MnSO 4 0.8 mM N2 2 Mo0l 4 0.3 rnM H 3 B0a,.

2.1 ruM CoCI 2 70 rmM ZnSO0 4 26 mM 142304, 234 mM Fy30 4 and 0U mnM biatin. The fennenter was contr-olled at pH 5.0 with addon of 2 M NU 4 0H4, 30'C, and 30% dissolved oxygen tcnsion through agitation control. A cultur of P. pasworis MSP428 grown in YM broth at 30CC was uwed as an inoculum,; rub of the culture was used to inoculate the fermnter.

When glycrol was shown tn b3e depiecd (24 hi after inoculation), induction of the AOX promoters was initiated by the addition of a mecthanol feat, The teed contained I liter of miethano, 5 rub PTNII and 5 rub of a stock biatir' sohulon pxtpard as 0.2 g/L in warer. The mnethno solutio" was addted maunAlly to maintain an average concentration of 0.5% as dewmnined by JTPLC anaysis.

FiftymtL ot cells 0Mw 20 ALT) were removed ta the reactor eftai 15 h ouf induction.

The50rn ceflsuspenwio wspdieedd respmded !a 1 L nitrogen sparged base mediumn (6.7'g/L yeast nicroge base, 3 .0 giL yeast extract, g/L K 2 HP0 4 1.0O gJLKII 2 PO4, 3 .0 g/b(N4H 4 2 S0 4 O ttraed to pH 712 and filter sterlized). Coepzyme R 12 prepmrd as a stock solution at 2 mg/mb in nitrgen sparged waer, was added to t cclisusperion to give a final concentration of 20 pig/mL. Thre media stock solution were prepared in b=s medium containing 1% glucose, 0.5 5 laccose and 0-5% glycrol (wft). and glycerol A stock solution of chloroquinc (1.06 g/SO rn1L, pH- 7.2) wvas "ls prepared, Two rut., of ine edia s lock solutions and 1 zuL mlixture of chioroquin and waver VD give the final concentratons ined in Table 10 were plac ed in t0 Mt crimp sealed scrrn baffles an4 sparged witi nitrogen before adding 1 miL of cells with caenzymne B 12 mixture. The seu boffles wver inicubated at 300C with sAkig. SampleS.jaken finmediately after the addition of cells and after 24 h incubation were analyzed by HPLC. The rmsu am shown in Table- In vivo production of l,3-propanedio1 us*n rvcoinbinant Pichia uwouis chioroqo mne 1'3 -propmnedial rcaaion inediumal trda) (mnM) glub 0 0.04 2 gin 2.5 0.2 3 glui 5.0 0A1 4 lu 10.0 0.1 giulvgy 0 0.2 6 9glu/gly 2,5 0A4 7 glufgiy 5.0 0.4 8 gtn/gly 10-01.2 9 gly 0 0 2 gly 2,5 0.3 11 Fly 5.0 0.3 12 gly 10-0 1.4c lkc than l10l of echl substrae was9 used in 24kh unless noted.

bN 0 glucose remained after 24 b, flThe presence of l 1 -prvpanediol was confimed by GC/MS as described in GENERAL METHODS.

of a Pilcdo gawrIs inublvjnansfonnantlor production oflI vrc'panedioi frvM P- UJucvse P paxtoris MSP42.81 were grown in a B3ios=at feretr (BI Draun Biatech, Inc.) ij 1.5 L mninimal medium contairing 8,5 g/L KH 2

PO

4 2- gL

(N"H

4 2 S OA, 10 g/L glucose, 2.3 gIL MgS0j7Fl 2 O, 0.18& gIL CaSQ 4 2H 2 O, and 0.29 milL ITMLZ Otherwise, feunencation and idutfon conditions were identical to those described in Example 7. Fifty m-L. of cells were removed ricni the reactor after 15 hi of indutmo The, cell snspcnuion was handled as described in Example 7. wit the exception that a modified basc medium (6.7 g/L yeasjt nitrogen base, 1.0 g/L K11 2 P0 4 I gILK9{P) 4 ,3 8 g/L(N114) 2 SC)ttittltedtopH7.2ad fiter stulzd) was uscd. The three media stock solin ons writ prepared in ii& modified lbase medium as well. All othe inolutions were the same. Reaction miuxe~s were pxtpard as described, And inaibatcd al 30'C with $lwking.

Samples taken immdately after the adcdon of celm and aft=r 75 hours incubation 'were analyzed by HPLC.' In a revacin Containing glucose as the carbon source and 5 mM chloroquine, 0.17 MM l,3-propancdiol wai prouced.

f~glasi cosmction for the =nfm.rrnaiion and t=re sion of dhaB nd daT i saccbaronycs cerevisiae Cuinn-ction of V r ljl nrscensioni p]iasmids Two types af cqnncsion phsrnid wc'r creazed, thusc that cOuld iaiegmatc byr ircvubination into chromosoames, and ihose that could exist as rcplicating episusia element For each typ of general expression plasmid aL ycast promoter was prtscnt1 Rnd Sep2ated fromn a yeas uwranszption terminator by fnipnents of DNA cotnairiing rcog'iidor sites foi oe -or nJi&Mt xcsL~Uictncl~l~tS2 Each type of gener aepresion plsmd also contained the goe for 0-Iactamsc for selection in E. cati on media contaiing imnpicillin, an origin of replicatin far pLssmid inaininece in E. coil. and either a 2 maicron origin of replication for episorna elemens or sequences homologous to chose found in S. ccrevisiae chromosomes for recombinaton and integration -of intm.oduced DNA ino chroomes. The selectble ItotitiorWu rmkers used for yeast "n preent on 11ie- cx.wprsion pasmids wrc one of the following: ff15 geie encoding inidazoleglycerolphospamm dehydwaase. URA3 gene encoding orotidchn 5'-phosphate decazboxyiase, f(Pl I ene enoding N-(S-phosphmibosyl>mnfitranilae isornzse and LEU2 encoding ji-isprcpylnmlatme dehydrogenase.

The ye=s promoter used wcmt ADH I or GALI and the transcription terminator ADHW1, CYCI or AOXI; the lattr ftrm Pic/ria pastern.

Plascnid pOADOK (Clomtech, Palo Alto, CA) was digested with HindMl and the single-strand ends convered to iBoll ends by lgafilan with HindlJ-Xmnl and EcoRI-XmnI adaptors (New England Bliabs, Beverly, MA).

Selection forpawicls with correct EcoRL ends was achieved by ligatioxn toa kmamrycin resistance gene on an LeaF! fragnmt from plasrid pUC4K (Phannacia Biotech, Uppsala), uransfortnaiion itoa E. coil train DHSCX and seletion on LB3 plates containing 25 pg/nLkmnycin. Mme -resulting plasrnd (pGAD$KAN7) was d igestcd with Sna~l and HEaR! aid a 1. 8 kb fragmeuttwithi the ADH I promoter was isolated. PLasmM pGBT'9 (CLunte cli, Palo Alto, CA) was digested with SnaBi and EcoFi. und the 1-5 kt ADIII/QAL4 flglefltc zeplaced bpy the 1.8 kli AU-H I promnoter fragment istead hrorn pGAD/KANZ2 dyi with iSnaDI and Eco~jl The resulting vect ox (pMCK1 1) is an replicating pjaunji4in yeast with ADHI Promoter and tennfnator and a TR1 marker.

?lwnid pGADGH wus digested with SnaDI and Hindin and a [.8kb fragment containing the ADHI promoter isolated. This fragment was liated inao the voer PRS40S (-Siratigene. La Jolla, CA) previously digested with Sm.! and l-iiidM. Positive clones were identified by insezional-inactivadon of the plasrid-encoded krcZ alpha peptid and the presence -of the ADIII promotar fragment. The resulting pkumnid %pMCK4) contained an ADH I promoter and a LFU2 maiker.

The -0.2 kbNael-EcouI fragment frcrn pGflT9 containg the ADI temiinator Wv&93 fig ated to EcoRI-I-indfl d igested p1(3403 (Stratagee, La Jolm.

CA) to yiel-d the &kb JJ1yRflidJRVNS,. The -2.0 kb SnaDX-BcoRi[ fragmnent from pO3AD/KAN2 containig the ADHI prmo~ter was ligated to Smul-Ec oR! digested pRVNS5 to yield the 6.8 kb plasmid pRYN 6 with the ADH1 IPromoter and terminator and a unfique EaoR] cloning site in bectween.

The Cf4 kb HindMl fradgmentt from pOADOR comaing an additions! Xjnnl site was deleted and the vcctor was religated to ykcld the 7.0 kb vector pGAD-D3. Vector pGAD-D3 was. diges ted with Xznnl and th A kb fragment omna= tg the ADIII promoter and terminator and an intervening HiaM~ cloning sire was purified. The, pRS4O4 vector (Straragene, La Jolla, CA) was digested with Pvull and the larger I.S kb fragmnent with TRP I was purifid and ligated to the XmnI promoter and ternator fragmnent horn pGAD-D3 to give plasznifd pRYNI- The open reading frames for dhaT, dhailA, and dharB) we= amplifEcd hrm p11K13-26 (SEQ rD NO. 1) by PCR using primers (SEQ ID NO:6 With SEQ ED NO:7, SEQ ID NO:A withISEQ [DlNO.9. and SEQ ID NO:Z with SEQ ID NO:3 fobr S/ta, dhtrSj, and dha~i, respctively) irtcopomdfng coR I sites at the ends -(10 raM TnsL5pH 8 5 0 mM KC1, 1.5 MM M9C0 2 01002% gelatin.

20041 d ATPD, 200 IaM d=T, 200 jrM dGTP. 200 pM dTP. 1 pM each primer, 1-10 rig target DNA, 25 uinirnL Airpiuq DNA polymerase <Perkin Elmerx Cerus, Norwalk PCR pnn~ern were I main Rt 94 0 C, I main at 55*C, I rmin aT 721C. 35 cycles- 7T produmt were suboloned into the EvoRi site of pHIL-D4 (PhIllps Petroleumn, B artlesvile, OK) to generate the pl asnidsb pMPL13, pMP 14, and pWflS containting S/tar, dhzB3, and dhl/tB respectively.

CogtruC-ripn -of plasrnids for exresson -of ancri The replicating plasraid pGADj'KA142 was digested wit EcoRi to remove the kwnrrqcin rest tuna fngmcnt, dephosphorylated, and ligated co the dilaT EcOki fragment horn phP13, Tin rcsddtng plasmid (pMCKI3) haO divaTcorrectly orientd for tramnriin humm the ADH1I promoter and cnun4t, L-EU2 marker.

Phpsmid pIWYG was digeted wih EcoRI and ligated to the divaT ECORI frupnent foMM pMPJ3. The rezulfhag plid (pRVN6T had dharonecdy orientated for tranprion from thec ADILI promoter vend contained a ML3 Mnarker.

C&orction of plaanids for exprwion of VdhJ The replicating plasnid pGADCGl was digested with Hfindlll dephosphorylaed, and ligaterd to ft dkaBI 1-irzdlf fragment fr-om pMPIS.- The re ul inZ plasinid (pMCKI 10) had dieRI coemc[y orivi jr fcG r LnSCrpTt~n ftm the ADH I promotcr and contained a LEU-2 market..

Cons~k cuctisgn of PJaUrndsio .rt ~reso ri oL'JaR2 The replicatinig plasruid pMCK I11 was digcsted with EoPJ 1 dephosohory1inted, and Ii gated to thec dhtaB2 E m~R1 frag-nent from pPW2O. The resulting plasmid (pMCKI17) had dh~aB2 iMcdrefy oriented fOr LLansciipl f1rom the ADH I promoter and contained a TRP I marker.

t'lasmid pRS403 was digeste with Sinal and ligated to a SnaWI/Ntir divaf2 frament from pMCKX17- The resulting plam id (pMCK2I1) had dluz2 correctly crimtetd for trariptio fromn chc ADH I promnoter- and contaied a 1153 marker.

Cpnstnxction pf p1 asnid forxag sso fd8 Tat rcpliC4Luflg pl asrmid pYES2 (Tnvitrogc-n, S an Dicgo, CA) was diges(e.k' with EcoRi, dephospharylated, and ligatd to the dM5E3 Eco~l fagment from IpMI14. The reulting plasrnid (pMCKl) had dM8B3 c retly oriented for transcription from the GALl promoter and corgained a URA3 marker- 7te rrplicating plasmid pGAfAL/AN2 was digested with LicaFJ, dephasphorylared, and ligatcd th dk4W3 EcoRi fragment tram pMF 14, The xesulting9 plamoid (pMCK 15) had dhafi3 correctly oriented for transor pton from the ADH I promoter and contained -a LEUZ market.

Plasruid pRS4QA was digsted with Pat and HindH and ligated toD the ?stlfcoRV dhaB3 fragment from pMCXI5. The resulting phasnid (pMCK2O) had dhall3 correctly orientated for trnscription from the ADHI pruffloter and contained a TRPL marker.

Transotjjsai tfS. cerevriae with diva exptssion p]iasid S. cerrvisae swa4in YPH1499 (sira3-SZ lys 2 -S0l adel -101 rrp-d16 hi3-del2010 1eM2-ddl) (Stratagene., LaJo~a 7 CA) was tranformd withi 1-2 g±g of plwnid DNA using a LiCllpolyethylene glycol protocol published by Sntagne (Catalog #217406). Alternativelyt transformalion was ach-ieved using a Frozen-Ei Yeast Transformation Kit (Caulog#T201l) fromn ZymoRecrh- (Orange, CA). Colonies weic grown on Supplemented Minimal Medium (SMM yeas nitrogen base without amino acids. 2% glucose) for 3-4 days AxC 29C with one or more of the following ujdtioUs adeone= sulfte (20 zxjL), Irn=![ rngfL), Lt-owophan (20 mg/L), L-hisriin (20 rnzJLX L-leucine (30 mgG4) L-Iysine (30 mg/L). Colonies w=r streaked on Aeective plates and use-d to inoculate liquid media. flepending on the vector u-sed, colonies arose eithe after integration of plasmid DNA or from replication of an episom. Innadition to transformations with sintgle plasmid "yps, co-transformations with two or more plasmids were caried out.

Eziprss ion of direR activil yn W-usorels Eyfj Strai n YP11499 transformed with plasmids pMCKl, pMCKIO0 and pMCKl 7 was grown on S upplanented Minial Medium containing 0.67% yeast nitrogen base without amidno acids, 2%7c galactoset, 2% rAffinose, 20mzgfL adenine suLfae 7 30 mg/L L-lysine and 20 mg/L bisddine- Cells were homogenized and ~excras assayed for SAA activity. A specific activity of 0.-021 units per mug was Obtained- Constrttcdon of sllernate rpictin g and integration plani~sdor the zranformaa i f5 jev~iiq A general purpoDse expression plasrnid. is constructed by isolating a SnaBl[EcoRI API promoter fragment from pFlAt)/CAN2 2md ligating tis fragmnt into the vector pRS46 (Stratagene La Jolla CA) previously digested wit HincU and EcoRE, Positive clones amre itified. by 'senional-inactiation of the plasmidd-encoded lacZ alpha peptide and the preence of the ADI-11 promoter fragment. The resulting plasmid {pMCS3) is digested with EcoRi and Sinai 2nd ligated to th-i U}2 kb ADH I vemilator fragment released from plasmid pGET9 by digestion with EcoPI and Neal. The resulting plasmid contains both AiDfl promoter and tenninarr sequence and a URA3 marker- CaQnstfiQon of p1 4SHIid fg' fZpI'rs 19n Of 4& The vector pMCKS is digested with EcoRI arnd dephosphoryiated. The dhaT gene- is excised a~s an EcoRi fragment fom plasruid pPI 3 and ligated to pMCK. *Me resulting plasntd (pMCK7) has dhaTcorrectly orienated for tnnscription fromn he ADH I promoter and ontains a tTRA3 mrker.

The integration vector pltS404 is digestd with Kpnl a" Sna. The dhaT 9c=e with flanking promoter and terminator is excised as a KpnIVSact frgment fromn plasmnid pMCK7 and ligated to pRS4O4. The resulting plasmid has ShuT coffectly orientated for tranScription from the AD I promoiter and conAS ft.

ThPI. uakerdsfrexsiu AhaB The vector pMCKS is digested wit RooU, and dephosphorylated. The dMA)I gne -is excised as an Ecodi fragmevt from plasm~id pMPIS and liated to The resmnjg plwnid (pMCKS) has dWIn) correctly orientated fot transcription from the ADHI promoter and cotains a LIRA3 mnarker.

The ntegratioo vctor pRS 4 03 is digeted with CIO! and MIRT. The ShOE gene with flankig promoter and renninator is eidsed as a ClaJ/AaI fragment from plhsnid pMCKS and ligated to pRS4O3. The rvsuJdng plasmdd has dhaBJ corre ctly orientated for tnanscdiption from thc- ADH I pro muoer and conmain a. h{S3 marker.

The rplkcating plasrnid pYES2 is digestd with Hind--lanid SrmBl, and the GALl promoter elemernt is replagcd by liation withA Sna~i and Hindli di g"Ysed API-U1 rmoter fr-arenr from pC-iADGII1. A dhoBl l!ndlfl and XbaI fragmaent fromn pMIPI9 is Llgaed to those sitesm in the modified, APIDI proornte version of pYESZ. The reulting plasmid has dhaBI coneetty oriented for transcriptioni from thie ADH I promoter and contains aUIRA3 ninilcr- The vector pMCK4 is digested with H-indli and dcphosphozylacd. The dhatUI gene Ls excse d as amjnW loI fragment frun plasznid pMPI15 and ligated to pMCK4- The resulting plasrnid has dM8) corretly orient ated for tmascription Vra the ADHI promer and contains a LEU2 marker.

Constuction of plaaids Ioxxnre~zion of dM~jai The vectors pRS404, pRS4O5 arnd 2 RS406 ue digested with Sinai. The dAR82 gene wi-th flanking promoter arnd tuniaor is excised as a SuaBI/Nc fragment from plasmid pNICKI7 and ligated to c~h of the integration vectors.

The resultig plasnids have dM22 correctly orientaed for transrptio frxn the ADH I promoter and conain either fth LEM2 TRPLY or tlkA3 markers.

EXUIMLUJI

Smigg .crvsd o h rmor=m corivcsion -of D-rzlucoe to 1.3-prnanedkd1 Ssnnn -of S. cerygrte for dha ryenrj Quromosonil DNA from Ura+. His'r orP be1 t~rmnnnts, constructed as described in Exmnples 9 and 10, is analyzed by PCR wi-ing pdiniers specii for each gene, as described for Pichia parworis (SIBQ ID NO,:2-9).

Proifpciio of 1- 3 -pnpan;.iol from D)-g ucoscjy S_ cerena rnsomdtJ Tranzfonnarus containig d/wt. dhavl. dhaB2 and dhcB3, costrucied as described in Examples 9 and W1. are grown aerobically or anaerbicialy with shaking at 29 0 C inSMM supplemented with 20ingJL adevinesulate, 30 m9AL L-lysinc, 1 mg/AL vitaminl B i2- Growth continues until statioarvy phase is reachedi and the presence of I.3-propanedio1 is detmined by RVLC. Tranfoirnat S. cereviae pMCX1/1O/117(HA4)#A was. deposited and designated ATCC RXAMPE flL £dirnM -poaei2from D-gltu s!AX Costidhann zsrcuripnumn ATCC 603 ndr a hya1pgen nmosphe Genera! zvw~J vonditiorns for Ckn-igm pst eurianj Closrridium pastc-urianumn ATCC 6013 was g-rown in 60 mL Crimp~ sealed bottles containing 10 mL of mnedium, unless nofted The crimped hovdes: containing the mediuim were aseptically sparged with nktrogen prjir .o ininoculaton. Basal tncdiim (Medium adjusted to pllI 7.2, containd the foliowing comnenwts Lan g/L: KH 2 F0 4 1.4; Nul- 2 P0 4 0.69; NH 4 CI r 1.8-2.5; KG,. 0.50; MgSO 4 ,7H 2 0 1 0.5 0; Cad1 2 0.02; Na, 1.0; yeas cysteirie 410a, .0.50, sodium bicabonate 1 2.5; p-amino brcnzoi-c acid, GANJ8; ~0biotin, 0.000040; sodium citrae*2H 2 0, 0.10; PeSO 4 .7HzO, 0.050; CClr76HzO, 0O0OUON MnCIy24U 2 O, 0.0010; ZnCI 2 0-OD050; Na 2 MoO 4 -21- 2 0, 0.0025; NCi 2 6H 2 O, 0.010; and CuSO 4 -5H 2 0. 0,050; to which rjubon components were added as indicated Uclow- ALI incuhat ions were performed at 30'C with shaig at 250 rpm A 10 niL batch of Medium A supplemented with 5% glucose was inacutnccl with I niL of a frazern stock of Clostridium pastesrianunij ATOC 6013 which Q uitaiitd qpprxiniarey15% (YWv) glycerol, in duplicate. After 96 hi, mL o the growing cell suspeinioas was passed into 10 niL of fresh medium and growth was continued. After 24 h, die atmospher in the newly innocnlated vials wam pressurized to 30 psi with hydrogen ga and incubat ion was ontinued for a fuflther 96 Ii. Theaqueous pahase was sampled at the beginnin and end 9 f the final 96 h far analysis by 1IFLC as described in the GENERAL MHODS.

The resuilts ame *own in Table 11I.

Convcnsion of D-&zaose to 1,3-propane~diol by Clostridium past: uriaLim ATCC 6013 u ndeir a hv-drurM" anonhem Time Glucose Glycro! l2-Prupanc.

h) Repliate m) md dial n'1a) o 114 nd 2.7 96 A 47 ud 3.4 o B 119 0.1 96 B 59 0.1 'The pnesence of 1.3-propanediol was conllrnxd by (COMS as described in the GENERAL MflODS.

Frvd'lcti 60n of 1.3, -napandio fror mggv vio on ii,=[WutlI- All cells were pxrw' according to the protocol in Examnple 12 -A 10 niL. bautch of Mcdiunt. A (dcscri'brkl in E--anwle 12) suppLemented with 5% glucose was inopiloed with 1 ml -of a frozen s tock of Ciasetdiwm pasteutdantn ATCC 6013 which contained approximately 15% (Y/v) glyctrol, in duplicate. After 96 hi, 0.5 ml of dho growing cal suspensions was passed inTo 10 mL- o f frosh metdium and gwrowth was, cutttiziued- After 2-4 h, methyl viologen ([,$-dinethy-4'-bipyridhtllm dich~ode) was added to che newly innoculabed vias to a Final concontration of I iM and ncnbation -was conti~ned for a faither 96 h. The aqueous phase was sampled at th-i beginniing and end of the fina 96 hi far analysis by I-PLC as described i5 the GENERAL IS METi-HODS. The results ameshown in Table 12.

Convemsion of fl-glucose to 1 ,3-propmncdiol by CkosldiwnpitWraWmr A=C 60 13 in the prsence of methyl vioo&e Tim GUCSC Glycerol 1,3 -PropanjeoA 113 2 96 A 28 1.8 3A O B 87 0.3 2-1 96 B 40 3.2 4A4 'The preence of l$3-propancdiol was confirmed by GO/MS as described in the G3ENERALMETHODS.

EIP~dgoLZ- Mcliim A supplemented with I% (vWv) glycerol I %h (wfv) glucose waz inocnlatcd fromn a frozen stck of Clostri ium past eurinnuni ATCC 6013.which contained approximtely 15% Cv/v) glycerol, at a ratio af I 0.2 wL &ozc mtock pci 20 =iL medium. Mt 48 N 10 niL f the cel lupens xwms added to 90 niL of fresh mnedium anid growth was continue-d for 24 h, The 100 mL cell suspension was chifld on ice and fth celLs collcctcd by cnurifugation under anaerbic onit ions. The cells were washed 3x in anaerabic buffer (50 mM phosphate buiffer, pHi 7.2+ -0.5 g/L cysteine-HC1, previously gassed with N 2 and antaclaved under N 2 and resuspended in anaerobic buffer to a volume of S niL. In duplic eperirents, one niL of this cell suspension was inoculated into 10 tnL of Medium A s xppienmed with I% glucose and 0 mM.

1 inM, 5 mM. or 10 mM mnethyl viologen and incubated for 240 hi. The queoFus phase was sampled a% the beginning mrid cnd of the final 2403 b for analys Ls by RPLC as described i the GENERAL METHIODS- TIhe results are shown in Table 13- OaN 1 13 Conversion of D-gr]ucosc- to I ,3-propancdlol by Closrtridiurn pcrneurianurn, ATCC 6013 in die presenQe of !cthvl Yiot e (MY) MV Time Glucose Glycero 1 ,3-Propane- (mrM) Regplicate (MM) dial (mMk4)a UD 0 A 38 nd rid 240 Ab 40 Rid ud 0 B3 nd rid nd 240 Bb rid nil Id I QA 40 lid nd 240 Ab rid 4 1 0 1D 45 rid rid 240) Bb d 5 2 0 A 37 rid rid 240 A nd 4 2 0 D3 38 rd nd 240 1B nil 3 1 0 A 40 rid oi 240 A nil 2 0 B 43 nd aid 240 B lid 3 'The preence of 1,3-propanedial was cofirmed by GC/MS as descibed in the GENERAL MhrHODS.

bfly 120 Ki, glucose was depleted and additional glucose, I1% Final corixttatofl 1 was added.

Ev.=Efl=n3. ClOfTridifm parmranium ATCC 6013 was inittiJY rnaintaird in tinlycollate medifumn Wifcol&) and tansferred to Medhink-.

supplemented with 0.4% glucose for all subsequent smudacs- After severs! tansferdo ugh the JItterrmedum, =n inocudumn was prepaied by gowing a 1 xnit aquot of stock culmur in 10 niL the descibed mediu ovenigbt A ser of nenun bottles, conining methyl vivkgpen at the concetttii Jindicatd in Table 14 ini fre-sh mediumn bottles weEr inoculated with 1 rnL of tie oveniig cultre and again incubatd far the times indicatrd in Table 14. Botles we= peioicly sampled for glucose utilization and anLtyzed for the presence of l,3prupanediol and glucose by 1*PLC as describe-rd in the GENERAL M]ETHODS. Table 14 suirnadzcs tiv ai,.alytical results- Produ ction of 1 .3-rwrdiol fromn gluco se Lwy Clostridiumi r?9twranium A= 6013 methyl vzolkgen thme Fcs rpancdinl 1 0 0 22-50 1 0 5 0 0 2 0.1 0 250 2 0.1 5 00 31.0 0 25.3. 0 3 10 5 10.0 2.4 LO 9 0 2.4 'The idet of 1,3-pyopanedial was veri~td by CC/OMS as described in the GENERAL MrrODS.

Censaucrion of gsneal Purpse I&prsion Plasrnids- for Usein The exprSSin vectr p-TacJO The E. coli expiession vector, pTaclQ contains the lazlq gene (Farnaugh, Nature 2 74, 56573 (1978)) and tat promoter (Anmanin cc Gene 25, 167 (1983)) inserted into the EcoRi of pBR322 (Sutcliffe et inl. CcddSpring Had'. Syn'V.

Quanr. Viol, 43, 77 (1979)). A mnultile cloning site and terminat sequence (SEQ IC) NO:l10) replaces the pBR322 sequence- from EcoFi to Sphi.

Sybc onins! be-glycerol dehydratase rzezgs Cd/to1. 2. 3) The opcn reading frame- for dhaB3 gene was amplfied from pH 11 2 8 26 by PCR using primer (SEQ ID) N&4 1 and 42) incrprating an ECORt site at fth end and a Xbal site at the Y en~d. The product was subclonrd into p~inMUS29 (New England ioilab. Inc., Beverly, MA) to generate the plasznid pDHA33 containing dMI?3.

The region ccntaining the entire coding rg ion for the four genes of Ate dhaB operon fOnt ipH128 -26 was cloned into pj~luescripW KS (Stratagene.

La Jola, CA) using the restriction enzymes -pnif and EcoRi to create the plusrni pM7, The dhaRX Senw was remorve4 by digesting the plasrnid pM7, which contains ehaB1 .2,34A). with Apal and XtbaI (deletin pat of d/raBS and aU of dJhaBX). The restinkg 5S -9b framnt was pmiffir4 and Ligated wid the 325-bp ApaI-XbaI fragment from plasmid pDHAB3 (restoring the dhafl3 gene) to create pMI 1, wlhich Contains dhazflfl..2.3).

tihc open rea-ding fr-ame for the dIhaD) gene wats amp]fWe d from pHil 2 8 2 6 by PCR using primnem (SEQ ID NO:lI I and SEQ ID NO,,12) incorporating a Hindli site and a onsensus RUS ribosame binding site at thk end and aL XbsI site at the 3' eid. -Me product was suticloned into pLitanu.-28 (New England Bialsh, Inc,) to generate the plasmid pDTl containig dM81l.

A NoxI-XbaI fragment from pM I I contaig part of the dha81 gene, the dha82 gene and the dM8ft3 gene was iserted into pD)Tl to cr-eate the dM8 expression plmsnid. pIWL2 The JidTI-Xbal fragment containing the dhA(1,2,4) genies from pflfl was inserted into pTacIQ to create pD1'3 SubdJjan theic.-rpndo dvhydneng on gene (dhaT The KpnI-SacI fz-grncnt of pF{K28-26. contaidn the complete 1.3-puropanediol dehydrogenase (d/ATJ gene, was subcloncd into pliltiescripi KS+caradng*plasmidpAlll. Thle dhaT gene vas amplfied by FOR froam pAlM1 sternlate DNA using synthetic, primers (SEQ ID) NO: 15 with SEQ II) NO.) 4) incorporain an Xbal site at the end and a Bamnfi site at the 3' nd Trhe product was sivbconed into pCR-Script (Stratagent) at the Srfl site to generate the piasrnids pAJI4 and pAl-S containing dhaT. The plasmid pAFI4 conans1 tile dhaT gene in the correct orientation for expression from the lac promoter in pa{-Scfipt and pAHlS contains the dhaT gene in the apposite orientaitio The Xba-RanI ramnt hrorn pAH4 containing the, a/taT gene was insered into pfacIQ to generate plsnid pAHS. Thr, Eindff-BariJJ fragment fmm pAILS contakung the, RRS arnd dhaT gene was intd into p33luezcr-iptIt KS+ to create pAHll I -The HindMl-Sa firagment from piAH& containing the RiB S, dhaT gene and trnninator was inserted lawo pBlucscxiptl SK+ to cmratc pAN 12.

An expression cassette for the dhaB (1 .24) and dhaT was assembled frm the individuzl dM80 2,3) and dhaT subclones descrnibed above using standard molectd or biology methods, The SpI-Kprd fragment from pAiB voncainin. the EBS, djhwT genc and terminator was inscrted into the XbaI-LIpnI s hcz 'Of P=f to Create pAl{ZL The Sinul-EcuRi fragmet between the dhaJ3 and ShaT gene o I pAH23 was removed to create pAH26. The Spel-Nod fragmnt cofltaindinta S &coRL sie fi= pDfl was used to replac t Spel-Noti fragmen of pAUZE to genrate pAI-27.

An exprion cassette fordhAW and 4haB(1 2,3J was assembled frcm the individual dhaB(l 2,JJ and dhaT sduoc described previously us*n Standard mokoula biology methods. A SpekSacl fragmemt containing t dhafi(1,243) genes from pDT3 was irnsencid into yAH II at the Spl-acJ sites t o =rate pAHZA- Productipn 4 1 .3-propanedid by Recombinant Sfreptornve lividans ;5t0nZ1~U&? Jmrmc. PV!Mf Two 'vmrions of the glucose isornerse promoter fioym the vcao-r pCOs 12'1 (SEQ ID) Fa15) or pCOL12llew (SEQ ID NQ:1 t6) werz amplified by PCR uSing primners (SEQ ED NO: 17 and 18) iucoqporading Spel and BcoRI siteu at the S1 end an a Hind]]] site at the 3' end The products we~re subcloned into p~iirnus29 (New Englandfliolabs, Inc., Bevcdy, MA) to gene-rate d-e plasmids pDll and The 41 kb expir nsion cassette far dhasr) 0LI mid cl/tT from pAH27 (Example 14) was insened into pDfl -or pflT using the restricton enzymes WinMcI and S aII to create pDTIJ I nd pDTI 2.rcspecttveWy Consinicion of a plamid forso-exuiessian of dhafirl 2.2) and dha Ti The 4.3 kb exression casette for dhaB(1,2.3) and dhaT was removed from pD1771 or pDT 12 by digestion withL Ecafi and Sail. The virctor p13 4 8 8 101 was digested with the rcstrictioan emme BcoPJ and Xhai. The expres ion cassette and vector were ligated along with a Sul-Xba Linker (SEQ ID NO, 19 and to create pDT 13 and pJIT14, respectively.pI 14 88 -Il}I consists opication origi fromn p0J101 from Sfreptrnnyces Hvidans (Kendall and Cohen, 1 Bacteriol. 170. 463-4 ((1988)) and pUIJS fro0m E. coli Nonndax ct at. Gene. 26, 101 The sequence ar derived S follows: bases 1-2096 ame hum p11101 (1-20S6), and bass 7688-8437 are frm pUlli1 (8080-H 30). Bases 2087-3 368 ame from the thostrpon resistance gene from S- azur-cis (rh-omnpsn et. al-, Gene. 20, 51 (1982)). B3ases 3369-76S7 are from pUC 18 containing thre cryrhromycin rcsistmce genr from S. erydu-eus- (Thornpson et aL., supr) insemtd at the 14n1 sit, iransnio uin-af 5epromv c IiuyIdans with 4fWh(/ 23 and _dhaT The plwnids pDTI 3 or pDT1 4 w=r t&anfrmed into Srptamyces tvdans =12 using standsxd protaoplast tramformarion techniques (Hopwood et 21., Geiiemiuainof ISternm tq cs The John Limes Foundation (1985)).

The wrmfuzwnu were selected on plamte-s contaiing 50 pgknuL dhiosnrpton incubated at 3m1C. Spares firorn the trsafionuts were repinted to obtain pun adtue& Detoction ofETX=,wA ulehvdratas afRftyj The Streptomycrs trarmfonnsmts were grown in 25 niL of TSB (tryptone souy broth. Dhfco. Veun it. Nff) plus 1% glucose, 2% glycerol. I ingtL vitamin BI12, pg/mL thiosnrptona at 3T fox 3 days. The cells were harvested by cenrtnfugati-on snd resuspended in I niL of 100 suM Tdis buffer. pH- 7-4. The cells were broken iusing a French Press (20,000 psi) Arid the- cell extract was assayed for glycerol -dchydraxae as described in GENERAL MIETHOJ)S, Coll rxtract frm S. Uividans TK23 transformed with ether pDTIS3 (Clone E8)r pDl4 (Clawe #2) contained glycerol dr-hydratase with a specific activity of 0. 1 U/mg.

Production of 13-proaned1fol in rcrnbintrrepwrnvcer (Mvdans S. ltvidans T1S23/pDTlA4 ((lane 42) (ATCC als-o iclendified as S. lividans strain SL 1412). inoculazed fromn aTISA plate, was grown in 25 niL of TS (Tryptone-S oy Broth, Difco 1 Detroit, bU) plus 1% glucose, 2% glycerol, I zng/L vitamin 1 i2, 50 higlinL dostrepton int a 25 niL flask. The shake -flask was incubated al 30VC with vigorous shaking for three days, after which 3 nigA 123-pranedioi was detected by G]C-MS mnalyrsi (ThS derivative) in die supematmnr its described in GENERAL METHODS.

Produtopn -of I .3-rpponediol frmVhdumxn using r' cWgb jmrir Srromvces lividAns Growth for damonstation of 1,3-prapanedaol production by Hividarw TIK23 containing pDTI3 or pDTl 4 proceeds aerombically at 30 0 C in shake-flask outirs (edlenrnetyer flasks, liquid volume 1/10Oth of total volume).

Cultures in rich. media shak-flat a=t started by inoculation ftrm twodays oldUTA-plates (trypticase 25oy agar, EBL L 1043), Rich media ame either TSB (nypficase soy broth; BBIL #11768), Liquid Broth (which contains per liter: 16 g nryptone, 10 K yeas extract, and 5 g NaCJ). mecdium B (ThE supplemnted with per L- 10, S glucose 7 2 niL Modifed Balch's Tra_-c-lment Solution in which NTA is irplacead by citric acid, 2-0 g NafQ.3 4.0 g K 2

HPO

4 I M9 vitamin B 12 final PH or medium C (zmium B. atxpH 6,4- Thecompositon of ModfficdBaldhs Tracce-Elcmcnt Solution can be found in.

Uthd u-tedadN0a~fBetilI Greztart et ml.. eds. p,.58 American Socicty for Micrubiology. Washiton. DC (1994))- Cuitta in nuinirnal media sbake-fl asks an naned by inoculation tkvm iwo-days old iquid 'ME cuhumrs, using n 1130 mocuiwn. Minimal media am either. NM22 (which onatains per liter: 12.0g. glucose, 113 g K 2

HPCJ

4 1.1)g (N1- 4 2 S 4i 0.2 g Difto yeas awuact, 04 1 MaCI, 2 Ig vktmin B 1

L

2 Old 10 nML Modified Balch's Tzmce-Biermt Solution modified as dbo ye final pH 6.7 MiCIU); medium D (mediumn MM3 22 supplemented with 2 g Na 2

CO

3 IL, rmualpH ar rnediarn E (rncdiumn D, final piU 6A). Media D ankd C and thc nanffN,),l ined~ ar-c flher-stenlizcd the ath-ii media =t autoclave-d.

The s hakc-flasks ait incubated a i 300C wic h vi gorous sh&king far two days, afte which they ame samnpled far HPLC analyski of the vupcmnatant- Glucose- is added, the culture is incubated for 1 h under aerobic conditions, after whkch the cultu= is tran [enred to2 Z in volume glass tubes (wich arc neadly filed to thep top), Thes tites ame subsequemy ncubated under anaeroblic conditions at 30'C2 After incubating for 2-5 days, 1,3-prcpanediol in the supernatant is detected by IIPLC as descxibed in GENERAL METHODS.

EXAMPEL17 Constrution of Genera Purpose Pgasrid s. PI1.sMids for the Ceexprssinof edha&J-3k and d0&q In Bacillu and Proiucticn cf The replicative high cop numbey shuttle vector pYSO2 Ls used to coexpress dhaB(I-3) and ct/aT in Bacilus. pVSO2 was concced by cloning a~n EcoRI/BamHlI fragment carrying an alkagiuc seria protease from BacWfls lenrur fused to fth B. subtilix apr prmoter into pBSIV. pBSl9 is a- derivative of pBS4Z (B and and HennerDNA 3, 17 (19864)) in which the EQoRVM auuW fragmm bus ben replaced by the EcoR/ind~rllpolyliter from pUC 19 (Boebringer Mannheirn). To facilitate sequencing andcllCR reactions, a 45 bp synthetic linkr (SE1Q ID N0:2t) was introduced by PCR bctwccn the end of die proteae gene and the imcriptivnal tenninator.

The replicative lowy copy mzmnba simile vector pSS 15--B is use to coexpress dha8(f/-3J and dhaT in Bacillus. PLasmjd pSSIS.-B was consaructed hy digesting Pluasnid plP 13 (H-ai=ia et al., Mot. Cen, Genet. 209, 33 5 (1987)) with infflIISal (sites present in potylinker), fillig the ends with T4 DNA Polyfmrae and rcligatng to generate pSS 13- A 2kb EcoRPI33m~lfrapient f~rm pVSOZ was inserted into the PScoRJJBamifl site of piwsnid pSSl3 to cruuc Plasrnids forlhoOver-pxrssian of "a(1I-3) and dhaT cassettes In order to cre-ate aBa-cias consensus zibosome binding she at the 5' end of dkaT, an RoPl/Xba linker obtained by anneling Synthetic primem (SEQ ID) NO:22 with SEQ ID NO:23) was inserted inca the Eco~/baI site of' pAH23 to Crate pM 17. A HfndIfl/glJE liker, using synthetic primrs (SEQ NO:24 with SEQ ID NO:25) was added at the HindlI'bglR site of plasmid pMl7 to intoduce a Sall site at the 51 en of ShaD) to create pM2O. The 0.3 kb Mu/Kpn! fragment fTrn plasmid pM2O was replaced with the 0. 3kb MbiiIKpil fromi plasinid pA4 to introducc, a Hindu sixe tu czr=te, pM2I.

A SIJ-Xbal linker (SEQ ID NO:26 and 27) was insened into pARS which was digested with the restrction enzymes, Sall-XbaL to create pDTI5. The linker destroys the XbaI size and changes the reading fumre so that the dM7' gene is tS fused to the, open reading frmet of protcsze coding sequence of plasmnids pSS and pVSZ. Thet 1 Lb Sail-Midl fragment from pDTI5 wa5 then inscrtcd into pAJ24, replacing the eisting Sail-Mid fi-agmcnt to create pOT 17.

A Sall-Xbal linker (SEQ IID NO:28 and 29) was inseed into pAILS5 which -was digeted with the restriction enzymes Sall-.Xbul, to caze pDT16. The linker de-stoysxthe Xbisheuand changes thc reading iamneso that the daT gene is 'fused to the ope reading fram of poly-His coding sequence of pUSH I (S-Chon and S&human, Gene 147, 91 (1994)) The 1 kb Sail-Mifl fragment haom pDfl6 was then inserted into pAI-24 replacing the existing Sail-Midl fragm-ent to create POT'S8.

Fhlaind pDT4 (containing diwB (1 .Sfl was constracted by incroducing the: Z.7kb EcoRL'Xbal fragmenit tin pDfl2 into pUC) S (HovhriugcZ Mannhekn) digested with EcoRI/Xbal.

Flasmids for the over-Zprcsion of dbaaianddorUf-3J ca 5er~s in BRaci/ius pDT1 7 wvas digested with S ad, cads were filled with T4 DNA poly-merase, and the DNA was flested with Sail to release the fragment conting dhzaT and dimuB. The fragment was then ligated to p5515-B3 digcstecd with. Hinxil (ends blunicd with T4 DNA polymerase) and Sail which created pM2UT Plasinids for ft~ ov r-exprssiof -of dha Bfl)-31in80c11u usins! o I ac-~beo idcbesgg A 2.7 kb BglIFHind]Jl fragnt contaiing dhaB(I-3) 6or plasmid pDT4 was cloned into the HiJIdII/aTIJJ site int the polylikr of pUSHI to ceate pM2&, The dha Igcncw a:5 fused to the opn nxcading frame of Pol)]y-I Lis coding scnqlenice of ]pUJSH!.

Tr=&rmiqno Jamis nsari The plasmids pM26 and pMZ7 w=r t Rndonrled into F- Uceheniformnis B03307 by nuxrz.Wflnftiof (MeCuen and~horn.LJ Bacferial. 107.

63&-645 (197 and selected on 10 ug/niL kmnimycin and 30 iugbn chloranphiclcol, icspectivtly, Thec sein plnsmi~ds w=r transformed into P, lichean ils strui'IBGIS 1u8sing standard prDoplast ransfonnatir'f tecbniques (Pngai cc al., Microbioogy, 140,.305 (19W4)) and selected as above. B5. subdli.s strain B028 64 was nnsfornicd with the lAmid pM27 by natural transformaIdOn- Transfoirmznts containing plamlids were se-lected -on LA plees containing 10 wghnl_ chloraxn7rencol.

PNasniid pMZ6 wx; Aso iranzfonned into B, sm~briis stai 1162 (Sairo ct a1., fot Cot- Gerwr., 170, 117 (t97 and trainsfornrmnu containing plasnids 'we= selcted on .A pacs conwiniing 10 ugh-nL erythro'nf tnd 20 ug/mL kan amvta2 All tratsfoflt wttcm gown at Detection f yiyverol dehvdrat oii B. lichenfiis strain flG188 trafnsfrd with p?' 42 (CIOne 8) was grown in 25 iL of LB (Difco} plus glucose and I1Q ugu knamycin at 3-0 0

C

overnight. The. cells were hrveited by cetnuifugatiof and vrsended= in I rnL_ of 0. 1 M Tykine/(KOH briffcr, pH S.2, 5-0 rn4 Q K1 I M ffdioriiDtol and! 200 uLM phcnyhnethylstllfoflyl fuoride- Cel1 c7nract was obtained by breaking The cells ini the FzrnltPes (20,0)00 PSI) and analysis for glycerol dcliydratas t w;as performed as dc~scrbed in G2NERAI- METHODS. A specfic wtivhy of 0.036 U/mg was obtained. FHc specific activity o[ 1.3 -propuAneiol dehydrogenase, measured as dcscribed in GENERAL MJ3THODS, was 0.2 U/rug lichenifonnky swain 30G18 8 transformed with pM26 (Clone #8) (Al-CC 9-01 was grown in a shuke flask containing 25 mb of LB3 (Difco plus 1% gLucoe and 10 ag/nt kanamycin at 30cC overnight with vigorous !buklfg.

after which 1 mb was used to irioculatc 25 niL of LE plus Il9G rlucose. I*1 glycerol, '0.33 tkg/mL vitamin B mi ad 10 ugfinL kanarnycin in a 250 niL flask Shakc flasks wver incubated at 30CIC with vigorous shaking an-d after 9 hi of growth 300 ug/L 1.3-propanediol was detected by GC1WA ('VM dvirfr~lon) as descrd in GENERAL METODS.

B. subsiffs strain 11G 2864 transformed with pM27 (clonc 1) (ATCC 28010) was grown in pL shake fl1ask conairnig 25'mL af LB3 plus I% glucose. I% glycerol, 033 ugruL vitaminB~12' and 10 uS/niL chloraRnpqheuical in 9L250ntb11-flask. Shake flask were incubated at 30VC with vigoou shaking)Zja after 43 11 ofgrcwdi, 1,3-prvpanediol was detectecd.

Ppmuiti gf 3 -hcdror=yPc~~tnpdehvde bysjreminanh Bacilu Bacillus feunernaciozu were carried out in 15.5 LtotW volume Biolafitre fermenret. working volume iailly 7 H=,rs increaing to 9-5 liters duin the rin, Aerobic conditions were insud by aeration with air at a raft of 7 liters/minute, at an impellerf speed of 650 rpm and a back-presure of 0.8 bar (arobic. conditions amy defined by the %A Dissolved Oxygen (100% DO defined at ambient prsmurt measttd. with installed Do-probes; a minmalvalne of DO was considered arobic). The pH was maintzined at 6J70 by automiativ addidi Of 10% H2O or ZS% NH 4 OW- Temnperaur was maintaned at 3092C.

The1r following compounds were batclie4 into thle tank and vteriLize~d at 121'Cfor 3lniines2 grmnper liter) 6 NaU 2 F'0 4 1H 2 0, 10 K 2 H-F0 4 1.5 NaGj, 10 (NI-1) 2

S

4 0.2 FcCI 3 1.5 tryptuoc, 65 yeast extract, 10 ink of B3al-ch t s modified trace-element s olution (tbdslfor Oencg i and Molecuh{U ~4~ZiI~fl P. Orhardc et ds) p. 158, America Society for Microbiolo)gy, Washington. DC (1994)) and 2 MAZU DF204 (a cusomn-made antifoam). After steriliation, 350 gramn of the 50% glucose feed was added, together with kmnarnycin and chiloamphenic-ol (both up to a final concentraion of 10 mg/lter).

0.6 litrr of a 24 hou= old Bacillus licheniformis B3GtS3afpM26 (dlon #8) shnkefl ask, growing in LBGlI 10 g U)ypwne, 5 g yeast extract, 5 g NaG, 10 g glucose), was used to inoculate the fcnnenrer. The culture was then allowed to grow and exhaust the glucose; a pil rise over 6.60 triggered the glucoe feed (50% glucose, atutoclaved, at a rate of 0.7 gram/minute). Af 45 hours a nutrient Addition was made (50 "l Belch's tace teement solution, 14 gram

K

2 11P0 4 14 gram yeast etract, 14 nil vitamin solution, pH =e at 6,60. filtersterilzed). After 70 hours, vimnjrBI was udded up to a final concentration of mg/L. The %oDO was kept at aerobic levels for the first 92 hours. Glucose was present in (small) excess throughout thie run 0-2-12 g/L during the aerobic part (first 92 hears); 0 36 g/L during die Q-hrniue4 pait (from 92-164 hours)).

In a sample taken at 87 hours, the preence of 3-hydmoxypropionaldehyde was suzpected and confired by detecion of t,3-prepanchiol after treating die supemoate smnple. with the reducing agent sodiwnbvrohydride.

p2f dhuT and dMHaB -3 ca se inflcls ?lasrnis for the over-espresion of S~huT and dat-casaflts in u A Sull/hnffi fUgien from plasmid $421, contaningdhafiU Mad dhar, is ligated widi the 5Sit SaVicf pVSOZ vecto to ceate pM2Z. PM422 has dhaB and dhaT under thecapr promnocerin ahigh copy numb-r vcctor.

A Sall/incMi fragen fromn plasmid pMU 1. is ligated with fhe 5.8 It S al/HindIU frngnr-nt from pSS315-B to create $423. pM7 2 3 has dhal wA dhaT under the xpr promoter in low copy nfumber Vector.

Plasrnij:i fon thcsnaztsir ofl djfTg and d&LIa~ 19) csee nBcl pDTl17 is digestd with S acIT ends arc filled with T4 DNA pvlymcrase 1 and the DNA ih digested with Sail to rdeazfc h fagrncz containing dhaT and dlur The framemnt is thien ligated topVSD2 digested with HIndm (ends blunted witfl 74 DNA ponfmcra--ce! and Sall which cre arcd flasmidqz br the ovr-exors ion of a ShaT and&4 dhn NJ-1 ssut oc~us usingr a lac-based inducibleq system.

pDTl18 was digested with Sadl, end cle filled withbT4 DNA polymerase, and DNA is digested with Saill to relea-se the fragment containing elliT and dtwB, both genes containing a Oaolliur consensus tibosonie binding sire. The ftagrnct is then figarrd to pUSH! (Schion a~nd Sdrhuman. supra) dfgetted with HindilI (endsq blunted -with 74 DNA pulym-e rie) and SaI to acatc rid 2 4 Converion of D-Oducoe to 1-3-propandIol by.Rccomtbin Bacifli Growth conditions fra Barilu Growth for dermnsation of 1,3-propwwanel production by Bacifus lichenifonnii and Bacillus sudil jprceeds aerbically at W0C or 35 "C (ws indicated) in shake-flask culture (erlenmncytr fask) and in 15-5 L (toal volume) Bioitttc fernyientrs (working volume 7-10 Cdltorcs in LEG (which contains pert: 16 U tzyptan. 10 g gkcose, 10 g yeast extract, and 5g S CI) shake-flas are stoed by inoculation from air-day old TSA-plawres (Frypticase Soy Agar, B13L #11043).L These shake-flasks ane then used to inoculae either fernnmen or shake-flasks int which the clexnosttaion proper of D-glucose LO 1,3-propanediol converion is detninantfed- Batch culnus in sh*ak-sk Rich media are either -ME Orypticast- soy broth BBlL #117-68). LBGg medi&um B3 MTB suplemented with per L 10.0 g glucose, 2 maL Modified B3alchfs Tr ace-flerrtent Solution in which NTA is replaced. by citric acid, 2.0 g Na 2

CO

3 4.0 g K 2

HIPO

4 I mgvitamin B 2 final pH or medium C (mediumn B, at pH 64). The composition of Modified BacsTat-enn Sotion can be founmd in Methods for General and Molednhu Rarerio Cjediadt et al., eds., p. 158, American Socie-ty for Microbio-logy, Washington, DC (1994)). Minimal media amt either: MM322 (which contains per iter 12.0 gj glucose, 11.3 g K 2 ffP0 4 1.0 g (NI-14 2 30 4 O.Zg Difcv ycas vx-nc, 0.1 g NaGl, 2mg vitamin.BI and 10 inU Modifiedi Balch's Trace-Element Solution modified as above, final -pH 6-7 mediumn D (medium MIM322 supplcmented. with 2 g/L NaZCO 3 final pli or medium E (inediumfl, finaipf OA), Media B and CLand the zninmada media arm filter-teilized, the, othe media ame autoclaved.

The shake-flasks are incubated at 30 0 C with vigorouis shaking for one day, *fter which they are sampled for HPL-Csnplysis of tha mpexmatavt. Gluose is added, the eilftmu is incubated for 1 hr under aerobic conditions, after which the culture is tranferred to 25 rnL vourne glass tubes (which are nearly filld to the lop). Thetse tubes amc subsequendly incubated under anaerobi-c conditions at After inaibation for 1-5 days 9 1 ,3-propanediol in the sapernamant is detcted by I-WLC as described in GENER-AL METODS.

Batchand fedbatcb £uhturcs in fc rienter A 600-mU total volume cuirure ftrm a shae-flask ([BG medium) is used to inoculate a fermenter with 6.4 Lof mecdium, baed in "n arnoclaved for 30 minures (minima media) cr 45 mini (rich mnedia) Typical mnimal media in the fermenter is medium typical 'rich'nmdia is media Di with an additional g yeast extact/L. Filter-sterizd additions (vitamin B 12 or1 cornpension for auxolrop by) mr perfunned after the feminentcr has been aucoclaved, using a syringue and a septum-port in ihe fermente lid Back prcssure (B31, 0.1-OS bar), acradi (L of air per min, 0,4-1 win), stiiring (rm 200-600), teinp-rranie 0-37 0 Diss olved O)xygen and pH by NH 4 'OH and H 2 S0 4 ao- 1 3 P0 4 addition) amc monitored and controlled at the des ired values, as indicatedL After inaoculof ioni, the cell are gown in batch mode for dhe f=rs 14 hi, afr which a glucose feed is started. ]Far anaerobic growtb/producian, the %DO i* allowcd to go to 0% by eithe reduction of xrpm and 1lP, additionally by rpi acing the air going in by N2, as ind-iciated.

Pennenters and shake-flasks ame sampled for 0D 55 0[readings (grt) -andan enzymatic glucose assay an the nqcninte; aupermat i-5 also pitpard fcodiPLeC arnal vs is via our standazd procedurc, as- outtined in GENERAL METHODS. 1,3- Propanediol is present in the sapenamiw EKAMLEa Transformation -of Pimornas, withihaB($ 2.3) AnDLlhaT arj rEM52 1F9 of I 3-p rndiol Producto Constmiaion of plasrnids for co-exprrsian of dha (I 23k n .IT Pseudorpona £0 71he 4-9 kb expression casette for dhali(I AS) and dhaT ftrmpAfl2l was inseried into the vectors pMiMB66EII (Ejiste rt al,, Gene, 43,119 (1986)) and pMMU 4

I

2 07 (Morales 4l., Grne. 971 39 (1991)) using the restriction enzymes EcoRi and Sail to create pDTlO a=d pDT9, respe-ctively- Transfonqaton oftP aeruginoso PAO02845 tth thie VDTh9 exiaPlrnid F. acrrigirwasa PAO 2Z45 cells were prqiarcd for transformation by overnigh, pvwth at 37v( with shaking aw "0 rpm in L-bfud±. A 1:25 inoculation of the cdltmt was made into 25 niL of fresh pzcwanned end pccaratcd L-broth.

Th efrshe curc was incubamd -3 h toearly log pbatp 37 0 M and 200 rprm The CCelST collected by crxtifagsatian, were washed twice in 10 m L of ice cold 0. 15 M MgCI 2 containing 5% dimezhylsulfaxide -mnd resupended in 2 rnL of the dimeliysutlfoxid soiltiorL 't cell suspension (0.2 niL) was combined with 100-200mng pDT9 DNA and placed on icc for 160 win The reaction mixture was hear shacked at 37vC for 2 min and transfeuc4 to ice for 5 m-in- L-brcth (0.5 ml...

was added and the cell ware biVUatcd for 20 mit at 3 7 0 C. Single colonies we obtained from nutrient agar plates supplemented with 37-5 ag/niL chlartnphcnkol.

ConhuWa tws'ufvr f PT I'D int E. aerginoa PAO I The plasrnid, pDTIO0 was mated into PAO I by the method of Figuiski and HeLinski (Proc. Nor!. Acad- Set S. 76. 1648 (1979)). pDflU0 was transformd into E. cali AC80 (Chaki-uny ci. aL, Proc. Natl.Acad. Sri. U. SA., 3109 (1978)) to crieate acnar strain- The heLper sn-tin was E. colt HB 101 containing pRX2Ol3 (Pigursid and Helinsi n=pra) The recipient strain was Psewinoas aeruginos a PAOI (Royle et al_ J. Bacterial., 145. 145 (19B Cultre (5 niL) -of each of the strains; were grown overnight i LB t 37vC, The cells wce washed in 0,9% NaLCL and resuspended in 200 piL. The veils were mixed together and spread on a LA plate (Lurk Agar, Difco). The plate was inceubated at 3,7 0 C far 6 hL The clls were removed from the plat and rranfentd to PIA (Difen) Plates ccntaining 250 ;iagrL carbenciLlin and grown overnight.

Single colonies ere isolated on the sum n mdi.

Pseudomonas aruginoxa PAO I4,DTIO was. grwn ini 25 niL 2XYT (16 g/lT yeast arract 16 g/L uyptone, 5 g/L NCa) pluz 250 pgfrnL curbenicllin, 0. 1 ruM IPTO orvernigh at 3711C. The cell were hat-vested by cencrifugation and esuspended in 1 mLoufl{00mM Tris buffrpH7A4. The cell wec brokcn byFrench Press at 13,000 psi. The, crude enac was. then assayed for glycerol dehydratasc end 1 1 3-propanediol ddiydrog-enmse activity using standard assay&. Protein deterxfmiaton was by B ia-Rad (Bradfor) Frtoin Assay- Specific activity for glycerol dehydratase was 5 U/mrg. Specific activity for 143-propazrcdiol dehydrogenase was 20 U/mg. Simiflarly prepartd 1 crude extract from P. aeruginosa PAC) 2845 ransfonmed with pDT9 contained 'D.05 Wing glycerol dehydratase activity.

Producti2y $2f I noidio] by Pseudornonazy arugtina ccritairtiijc p PsYeudoinnas aeruginosa PAO 2845 contaiing the pDT9 plasud (ATCC 55760) was grown overnight at 37CC and 200 rpmn shaking in ZXYT mediumn squlernenred wit 2-5 pghiL chlormuiphenicoi. PoUowinr overnigju growth, in aliua of the cell snspcnion was uransfen'ed to grow&h medium (3 pants 2XYT medium .l paa IIEP-S0.I nmdium, suppl-enmned ywith 0,25% glucose, 0.2% (w/vO KNO 3 25 pgihiL chlorsmphenicol, 50 mg9. ycas exo-uact, and 8 0 mg/f. nnirit broth) resulting in a cell suspension with an OD66nm of 0,5-0.9 AU. KEPFSO mendium contains the following component

NH

4 C0, 9.52miM; M49C1 2 -6H 2 0, 0.523 mM K 2 50 4 0,776mTM; ILEPES (N-V7 hydazxyahyjpiperazieN -[2ctiesujfopjc acidj), 4 0mM; tricine (N-tris (hydroxynnhyl) methyl glycine), 4 mM; NSCJ 4 -7H 2 0, 0).010 mnMYL K 2 11F0 4 0, 132 rnM; and, trace mziners to give rfna. cnccntration of 6c Ef~lowing cornponems in &Iz sodium cicrate-6H4 2 O 0.001', 130 4 ,7fl 4 0, 0.0005; CoC1 2 6H 2 0. 001; NMnC1 2 A4H7O, 0.00001; ZCiQ 2 0.000005; Na 2

MO

4 -21H 2

O,

0-000025, IMCl2,6I1 2 O, 0.JIX1; Cu.$Or,2H 2 O, 0.00005. After approximately I fi of growth at 300C2 with shaking at 250 rpm, 0.5 mnM HTG (isopropyI-P-Dthaiogeactoside) was axde-d to the growth medium and cl growth was continued.

Aftcr- approximately S h -of additional growit, cell were haivested by cenuifugazion at ro= omrnprarure. Cdll were washed 3x with production medim: IJESO.1 medium supplemented with 0.25% glucose, 0.2% (w/v) KN0 3 25 pigftL chlorarnphenicoDl 50 rng/L yeas exuct, and 80 mgi'L nutient broth. In duplicae 1 the washed cells were suspended at dhe oiiginal harveste volume in production meim containing 0.2%W(4) glY=01,. Cell uspensOziSw~er incubated under a nitrogeni atmosphere at 30YC wit shaking at 250 rpan-- After appoxiately 1 hi, 5 ps/nt coenyme B 12 (5,6-dimethybbenziznidazolylcobuntdc 5-dec xyadenomc) was addd to the MI- suspension and 'he incubation ontnued at 30CC with stwkinsat 25Orpm. Smzule of the cell .uspensn' were cailecied periodicafly for prodnot analy-sis. Upon collectiwi. cells were remove-d from the samples by witrifugaioir and the aqueous supvrn4rnt stored frozen, -209C, unti analyzed.

Analysis by HIPLC with cadibratons basd omnautheti arandards showed that thcsc- ceU susperu ion pioduced 1,3 -propanediul. The results are shwn in Tablec 15. The identity oft die product was conlinc4 by CMS unul ysi L9uL describetd in the GENERAL MUE D-i03 Table Production of 1.3 -propmwaniol bry Pseudomonas Sample Time I(hr) t3-Propancdiol (tiM?) A 0o 0 A 24 5,1 B3 0 0 13 24 5,6 EXAMLflI hvPron of 1.3tpupanediol from D-gl&o= us bnw Pscudomo&Uq2 aergkeo G1eeal rrosnh conditions Pseudonwna aeniginosa strain PAO2845 from PGSC (Pscudononas Genetic Stock Center, East Carolina School of Medicine, Gireenville, NCQ is grown ini basal medium, EPES.1, which die followming components: NLI 4 C1 9.52 mMN; M~gO7 1

H

2 O, 0.523 ruM: K 2 S0 4 0.276 mM; HEPPES hydrowycthytjpizin -N-[2-ehaeulfoflic aci-d]). 40 miA; T-icine (N-tds (hydroxyrnehyl mcthyl glycine). 4inM: FeSO4H 2 O. 0.010 niM: K 2 11P0 4 0. 132 nmlvi and tre nmrals to give funaI cxwmewnions of the following component in &1IL: sodium citrar&6H, 2 0, 0.00t; PeSO-7H1 4 O.0.0005; CoQ1 2 ,61k70, 0.0001; MNfCIr4H 7 O0, (I.OO; ZnCl 2 0.000005; Na 2 MoO 4 '2H 2 0, 1.00002 5; NiQ 2 I6H 2 0, 0.0001I; CuSO21 2

OH

2 HEPES0. 1 is used in all experiznns; suplemnenmaions are noted where they occur.

L01=nsrion of2ZLY21 vc ri Erivenmuant of P. oerumg nosn PJAO 2845 bylgen inflan P- aerugirwsa PAQ 2845 is grown overnight in Nutrient Broth (Difen, Detroit, N]o) at 37 0 C and 200 rp= ahak~nw. Cell arm recovered by verrifugLdon andW DNA extrctd frornmatll usinzg a standard .sline jysis procedur (Searnbrook 1989). The open reading froanc for &Ep (glycerol cataboiin zcgulnory protein gen, Getabk ACCESION #M60805) is mmplified from P.

aenrginora PAD 2845 by PCR using prirriem JJ-gpR-5' put JJ-gIpR-3' (SEQ ID NOS:3O and 31, respectively), incorpratin EcoRi sires at the 5Tends. This DNA fragment is then ligated into plasrnipAROi150 (Parke, Gene 93, 135, (1990)) at its unique Ecofti rerrietion sit,, resulting in plasmidd p1310. E. coil tanzfonned with DNA from the p 13 1 0 ligation mix are spnta on Nutrient Agar (Difco 1 Detroit, W) camaining 5 0 p g/mL ainpidiln and O.0W9 gnL Xgal hromclilro.-und~y~p~gMzp.,j~)Whlite colonics, idcing a Whig probability of gdR insertion, ar picked and transfered to UE meditum suppicmcnted with 50 ggfib ampiciUin. From cells harvested after overnight growth at 3 7 0 C and 200 rpm shking, p7110 DNA is recovcrvd.

Th nsychi cassette region fromn pUC4K (Pharmaia. Cat. No.

27-4958-0 1) is amplified by PCR( using primer (S]EQ ED NQ032 AND 33) appropriately designed to amplify thie region and modify tie tcunbii of die fragment tio be compatible with restriction enzyme Styl (Promega 1 Madison, WII) resulting in the 4 ka frag-ment pUC4K.-scyL The pTJC4K-styi DNA fragment is subelaned into the Srwi site wiidin rue gipR gene of planid p1110, generating plawmid p1I1. E. coil transformed with DNA from the pLT11 ligation ntiztine are spread on LB agar :supplemented with 25 g/rnL kanamycin and 50 jighnL aruipicillin. DNA from 5-20 isolated colorues is individually colicd fullowing overnight Frowth at 37 0 C in LE xmdium supplemented with 25 ptg/nt kmnamycin and 50 pgftnb anipiclin. Thy presence- of thie desired plasmid DNA is confirmed by ge[.eleccrophorcsis.

F- aeruginosa PAO 2S45 is tnnsfoxmed with pill1 DNA fallowing standard protocols. Briefly, P, aerugrinosa cells are prepared for transfounuatioc by overnight growth at 370C with shaking at 200 rpm in [-brorh- A 1:25 inoculation of this overnight culture is made into 2.5 rnL of freh prewmnned and preacrnted b-broth. The fresh cuzltur is incubated far 2-3 hi (to carly log Phase) at 37 0 C and 20D rpmn. Cells ar centzifig-e4 and supemarant decainted. Collected cells are r=suspnded in 10 tnt of ice coldvserie 0.15 MA MgC 2 comalohig disneffylsuifozide and held on ice for 5-10 mii, Cells are centrifuged, separated from the suxperrnatant and resuspnded in 10 mL. oF ice cold sterile 0,15 M M90I 2 co)ntaining 5% dumnthyliufotdt and held on ice for 5-40 min, After afiLnat cetrifugaon and separation from the sirpvruafant, die cellsr euseddi 2 iL -of ice cold steziJc- 0.1.5 M M 8 C1 2 containing 5% ditnethylsulfoxide. A 0.2 nab aliquoc of the cold cell vonceruate is combined with 100-200 rig pil 1 DNA in a precbffld 1.5 mlb polypopylene cntifuge tube mud Ohe nmtunmm held on iY= for 60 mini. The mube is a=e rapidly tranfered to a 37 0 C water bath for 2 mini and knmediazey returned to ice for 5 rin. Approximately 0.5 mnL of L-broth is added and the cells amv irnnbate4 for 0.3-1 hour with genit 8htkg at 37 0 C. Following the recovery ineobatio IQ) 3 L and 50 )zL uL*quts of the cell suspen'rsion ar spread on amrie agar ptlmes suppeninted 'with 5D Vg/mL kaarycim- C6oonies developing on the, selecdtiv meditm ar screcnd for growth Qfl agsr plames with RPESO.1 mediumn suppilementd with 1% SUCCiIic ac id or I1% glyceul. Q Cane s un able to grow on glycerol, but capabl e o f growth on sucdinate, uc rc serd for later use by freezing in 15 glycerol.

Tra risfmntin of gn~a-fjzj'ggirwnsa PAQ 2845 wi~pth P. cer ugizwsa ii prep'ared fur ufansformation by the in~c dod dcscribrd above. A 0.2 niL aliuac of the cold cell concntrat is comnerd with 100-209 zig pDT9 DNA in a prechied 1.5 niL poly-propylene centrifuge tube and the mixture held ion ice for 60 mnii. The tube is then rapidly ruMnsfrvd to a 37 0

OC

water bath for 7- ruin anid imniedktely returned to icc for 5 mdin. Apjproxiately nrOb of b-broth and cells are invubtorc 0.3-1 hi with gentle shaking at 37C.

Following the rmcavery incubation, 10 111 and 50 pL aliquots of the cel suspension are spread on nutrient agar plates supplemented with 37.5 jig/nL, cbloroamphenicol.

S jeeajgip SVA aerugrnosa PAO 2845 n-ans formts for the resec The tranfonnate from above are plated on nutrient apr plates supple-mented with 37.5 pgATmb chloramphwicol grown overnight at 37tC Froml the colonies appearing on these selective plates, apprximtely Mwmy are piced and tranferred to 10 nib Nutrient Bpoth (Difco, Dctroi, AM) containing 37,5 pg/mE chioramphenical and grown overnigh at 37CC and 200 rpm shaking.

To confirm the- prescnce of the pDT9 pLasri~d in the szleced transfarnants plumid DNA is exn-wed, purified and cut with ECOR I (Pmereg, Madison, WI)- The molecular weight of dhe linearized DNA is analyzed by gel ekcuaphoresis.

In addii, PCR anipiftcadon using primer pairs with sequences =omon to dhaT. dhaB), dhcRB2, ond ShaDS (S 13Q ID NO:34 AN4D 35, 36 AND 37. 8 AND 9.4 AN]) 5, repecively) followed by fregmec miokcul weight chairacteriwaion using gel clectropboresis is used to confirm the presence of tec desired gems.

Metabolic Scrrenir-of' rlrR:J' aeruyzirsa PAO 2845sransform~d with p;DJ2 One t wenry dAores Mr selected from the positiv trmsfarmnts above for futher characerization, Cells am grown rrohicaily on Nutrient Broth supplemented with 3 7.5 pgf L ctdcrurnphenicol overnight at 3M9 with shaing at 250 rpm. Cell ant transferred at a 1:8 dilution into the lm= medium with MM =FT (isupmopy-R-D--tiogelanoide) end grown fbr 4-6 h. Cells ame 4=e harveted by centrifgation and washed once with FIEPESGA mdium -Supplemerued With 1o 9/L Slyceral. 0-03 gIL beef extract, 0405 giL peptone, 0.05 g/L yeast extract (all Difco, Detroit, MT) and 0.2% KNO 3 The cells ame then retSspended -at 1/5 the origirwj volumec, with no air spac, in a Email vial and incubaled at 3092 with shaking at 100 1pmk for 18-72 hi. Cc~ls are removed by cenrnfugation and die supemaa anayzed for the prcsence of l,3-prupanediol by HPLC. In additian, the clitnieal identity of I ,3-propanediol i-9 confirmed by gas chronaaRto-aphv-inass specneonchy.

Production of lS-p=Ropnedi 9 j from glucosey by &lpAT-auganosa PAO,2845 transformed with pDT9P (ATCC- 5576(B From the acreenn& procedure above, one to five coems which produce the gtet amount of 1 1 -propanndiol from glycmrl are grown aerobically on nuolerit broth supplemented with 37.5 pgiL clilouiphcnicoI overnight at 3011C with shaking at 250 rpm. Cells are trmfened at a I1PS dilution into the same mnedi um INith 1.5 mM IPYTG, pllowed Ito grow fox 4.-6 I, harv ested by cenrriI&9gdon and washed once- with HEPESO.1I medium suapplemented with A0 gJL glucose, 0.03 &g/L beef extact. 0.05 g/L peptone, 0.05 g/L yeas extrct and 0D.2% KNO 3 A. The cells ame then resuspended at 1/ the aiiginal volume, with no air space, in a small va, Cells arecincubacd atr3OvC wihshaking a: 0 rpm foir aproximately 36 h. Cells ame zrroved by centifugatian wWndrh superanu analyzed few the presence of 1,3-propanediol by HPLC. In addition, the chernical idenfity of 1,3 propanediol is canhanemd by gas chrmtogirphy-mass spe=Ctroety.

EXAMPU~

Co~sinction of2= exsim Or S cstes o =resson of d/ur3 1. dM112. dM33 xd dfaT in AsiergfflusnrW= The IA kb SpelI-lEcoRV fragment from the plaszid pC;lrpyrC (DBerka "Ite development of ge-ne expression systms for ifihrnentoti fungim.

Riotechnot- Adv., 7:127 -154 (1989)). containing sufficient portions for pr~opcr regulation of the Aspergils niger gia A piwuoer and terinator, was Iigated into the Spel and EcoRV sites in the PolYliker of pLlTMUS39 (New Engisid BPiolabs. Be vedy, MA).

Individual clone exprssion oae ,Nfr A. ncr Thei open reading flums (ORE's) for individual Kiebsiella pnewnonlae dhaB subunits and dhaTr were cloned and ligatd into the general e-xpzcs ton vector (pAFX) separately, using the sain doning suategy: Primer pairs for ICR amplification of each individual dWx DRY and the dhadi ORE wer designed to mach the 5' mid 3hends seqnnc for each ORF based on known sequencc of the entire gene operon (dlzaU 1. dha32, 4ha3, dhaflX and dhaT: S EQ ID) NO02 S and 22, 39 and 40, 41 and 42, 45 and 46, 43 atnd 44, mr~peai-vely). In addition to the matching sequence, the prim for the 51 end of ea&ch 01WF wn-c dcsignc-d to ind udec a EcRiq~ rcstrictioc site folowend by aR E3glfl-1restrictioasite atthe5snost tnd ofte sequenlce as well as tliefive base sequencr CAGGA upstream of the!, frst AT(] of each ORE PflMas designed to nmch the 3'endsi of each OR-r- placed an Xbal resffiction site dowiLnai of thy =rnslation sto codan, at the 3'moat endof cidone.

Indivi*Lui -clone fragments for the dM13 and dzaT OR-F's were amplified by PC1 ho-rn the plasmid pffK6-2S, ontahting thec entire K. pnacmonise dba opern 1 usbig the primers described above. The individual ORF clone tgments were isolated based on their respective molecular weights (dMa]11 1540 bp; dhaB2 =607 bp; dhaB3 ;-448 bp,.dhalGW<= 1846 bp dTc -ll1STbp), Using the urilque EcoR I an-d XbaI restrictionf sites desifltd hi the PCR primcrs, r-ach individuial dha8 and dhaT ORFE frgment was ligated into fth EcoRi arid Xbal restrction sites in the polylinker of ptTNMS9 (New EnglandJ Biolabs). The, dh11 2 and dhaD3 3 cloes inL pLITMUSZ9 nt confirmed to be correct by sequencing. A unique 1363 bp Kooi 1-EcoftY restrction fragment firom the codin region of dhaB 1 clone in pLIMUS29 was innoved and implaced with the corresponding resuricutiomrgment from plIK26-28. A unique 783 bip TIM 11 I-MIni I restriction frosnient from the coding region of d/wT clone in pLfl7MUS29 was replaced with the corrsponding resutrim fragment fromn pfIK26-2B8. A unique 1626 bip ECORV resixiction fragment from the coding region of dMEBX clone in pUTMIS29 was replaced with fth coresponding restriction fragment from pM7 (containing the K. pnewmnriae dM113 operon). The S' and 3' en se-quences of the dhaB 1 dhaJJX and dhaT clones, approximatey 250 tip which includes some sequence from the substituted ftriet, was confirmned to be corret by sequencing.

The un~ique BgI UJ-Xbal resition fragmenis containing te ORE-'s of dhau 1, dhaB2. dkaB3, dhaB3X and diaT clones in pLlrIhlUS29 Were limted ni, the B51 [UXbal restricton sites i the gcnenl expression vecr pAEX sepraely, placing exression of each clone imd-r the control of the A. rScer gkzA promoter and rerminator Each resulting vector was tmemd by the zespecive ORF, Lt,; pAERxfAwS1, pAEX:dhaB2, pAEI~dhafl3, pAEKdhaBX and pAEX:dhATf.

Dhai exrtSEsIon calssete vectors for A- iigr The unique Sn~aB I-au Iestdictian tasenr containing the dhaB I expression cassett (consisfing of the niger promoce. the dha2B ORE.

and tenninater) was isolated frota the vector pARX-.baBlI and ligated into the unique, SnaP 1 restricion site in the pAEX--haU32 vecor. The approx. 2, Z kb Seal -Sna I resnierion fragment fom pDH2 (Ward et al., Exp Myc,. 13, 2U9 (1989)) containing the Aspergilus nzidulanspyr6 auxotrophy selectable matker, was ligate inro the tuique Stu I mstriction site in the vec-tor containing the, d&uB 1 and dltgB2 expression cascttcs- This vector was named pA]EX:B 1- The lnique Spt I -Hind III 'rcsriction fragme nts conmaiing the entire exprssioni cassettes for dhaB 3 and dhad] were isoiated from the respective pA.EX:dhaB3 and pAEKdAoT vectors. The cwo expression caseuc frAginerns Wer sirnultmneously ligated, in tsndern. in the uniqne H Ind Mflrstricn sitc in the vectorpUCIS. This vec-tor wins named pAEY-t3+T.

34;nsformnation. isolationio rnfnnm.cnimto fitgaino mprs sion cassettes and eXpMsip of dhaB =nd 4/aT oene in Asvergillus Aspergi Ms nigir strai FS I (PyriC) was co-t-ansfon-ned with the two exrssion vctors pAtX:B I +B2 and pAEXHB34T using the method of (Camphelv et al., ilmproved tnnstorrnation efficiency of A. niger using homologous niaD gene for nitrate reductse, Curr. Genet-, 16:53-56 (1989)).

Transfonnants were seected for by fthir ability to grow on selective, media without uridine. Genoinic DNA of irenfonnan was digested with Hind Ell and SpelIto liberate firagmnents of predicted moleaziar weights, demonstradug integration of intact exprcssion cassettes. Deteccon of each expressiomn cassette was done by Sou theirn analysis, probing with individu al genes separately. The presence of the ShaH 2 pro teini was detected by western wnaysis using anzi-dhaB antibody.

Ecpirssivn of each ORE was tested by growinig trmisfurnws, that have the pAiEXD51+-B2 "ncpAB&-B3+T vectors integrate'd, in 10% CSLmrnedia (~orn stcep liquor (50% solids), 10% NaH 2

PO

4 Ji 2 0, 1.0 gfL; MgSO 4 (1.50 gJL; mialtose, 100.0 gfL; glucose, 10.0 g/L: and Main Arifoan, 0.003% as a seed cultur then aransferring 1/10 volume of the seed cultur toMBWM carbon incdza (NiF{ 2 F0 4 0.70 g/L; K 2 11P0 4 0,70 KH 1 P0 41 0.70 g/L; MgS0j4A 2 O, 1.40 gfL; (INH-4 2

SO

4 10.5 g[L CaC 2 -2HzO., 0 70 w- NJI 4 RoS, 3.50 g/L; sodiumi citrate, 14 gIL; FcC1 2 A4H 2 O, 1.0 mgJL ZnCl2, 5.87 mg/L; QaCQ 2

IL

2 O, 0.42 mglL; M~n~j4U 2 O, 0.2J sng/L; NaD 4 U)71 OJ-I0 0).0)7 mig/L; folkc acid, 0.174 mng/L pridoxneXHi. 6.12 m#J/L; riboflavin, 1L83 mig/i.

pantothenic acid, 23.60 mxJL; nicotinic aci, 26.66 mgtt; biotin, 0.49 mg/L; thianincHCI, 1.39 mg/L; maltose, 120,0 ggL; cadbenicilhn 0.035 rng/L;, streptomycin, 0.035 mg4 tween 80. 0.07% and Man anifoumr4 0.14% for induction of the g&eA promoter. nRNA was isolated from tranom culturs (Fast Track 2 Ki, bwvitrogen Corp.) and Northern analysis performed with cbeta munccnce (Genius Systern, cic hxiger- Mannhcirn) to dete ct transcribed mecssage from each gene. Co-ordinate transcript ion of thdha d11, d&2232, dM4B3 and dhaT gencs in shakcflask cultures was denstr~atcd by Nonhezn hybridizan, probing with gene frgnnsof each dhaB and d/uzT ORr.

Isvl atcd colonies shown to traaib all aof the tramfuimed genvs were chosn to be further transfonned wit pAEXL-haEX. These iwhlten w=r cownufonnd wtthi pABX~dhaD3X "n pAA1O (3.2 kb- AcclI-Asp7lS resuicton frag-ent conaining the Asp erg Was nidudans amdS selectable marker in pUCi18).

These newly ianforrned culturs were selected for on media contaiing acewnide as the sakc carbon soure Trnsfonan colonies able to utiliz acetamide us a sole carbon source wer demonstrated wo have the dhaflX ORF inregratcd by PCR npliflcadior of the dha2BX ORF from genomic DNA using primers KpdhaB X-5' and XpdhnflX-3' (SEQ ID NO:45 and 46).

floduaon f-yccrtq byU A. Nigr-nraimFS1I Aspergillus niger stain FS I was grown in 10% CS L media as a seed culture end iransfented as a 1: 10 dilution co IMM cation inalia 12% altose.

Cltr supernaet was demonstrated to conain 6 g/L glycerol produced by Aspergillus- Analysis of glycerol wos done by HIPLC- Production of I .3-pranefflol 12 reornbin ant A.nje Aspergillus fermentatios were carried out in 15.5 L ita volume Diolafitte femenr, working volume initially 8 L, increasing to I1I L during die rn. Aerobic conditions were inured by aeration with air at a rate of 10 lff4nin., at an1 imnpeller speed of' 700-8 00 rpm and a back-prcsur of 1. 1 bar (mewrb ic conditions z= defined by the Dissolved oxygen (100% D0 defined at ambient pressure). rmured with inwsalle DO-probes: a nfima! value of 35 DO was considered aerobic). The pH was maintained aFt 5.60 by automnatic addition of

H

3 P0 4 or 2E% NII 4 OTL Temperature was snaintained at 32-C.

Trhe foullowing compounds weic- batched into (lie tank and sterilized a' 121 4 OC for 30 min.: 2 gIL JNZ-H 2 POjH 2 O. 17 gfl- (N-4) 2 SO0 4 1 g/L WSO 4 2 SIL Tween 80. 45 g/L Prurnosoy-IDO (a say concentrate of 701% protein), .6 gfr7orn mteep liquor (50% solids). 10 g/L maltose, and 2 gIL MIAZU DF204 (a custom-made antifoam). After sterilizatkn.t 500 gra of the 50% Malirin 150 feed was added 1 togete withi euxbenicilIn and urqomydin (both up to a final concnation of 10 mg/L).

One Ilitcr of a 4.5 h old skvpcrgiflus niger strain (sarain TGR4OD) Cwnfonned with the two expresion vecars pAEX:B1I+B 2 ad pAHM-t34+T, growing insa shzkrfask containin 10% CSL was used to inoclat t femrmetr. The ciriturc was then allowe-d to grw batchwisc, fully aerobic, for 28 hi before a fred (a Maltrin 150 solution, hlea[ sterilized) was startd At aL r-atof 0.9-1.0 gftin. T1he ailtuic was then nm for aniother 2 0 h, daring widch rite %DO dropped to vinnally zero bectase: of the, 0 2 -dcmand -of the cells (the- culture remained at zero to throughout the rest of the am). After that (4-8 h- aftr inocuflation), glycerol was fed in over a pcriod of S hi, up to a final gl ycerol cnncentracion of 163 gIL The malnin feed was atopped 97 h after inoculation, bacpressure and acratie lowered to respective-ly 0.2 bar and 4 [4mhL (0.5 'rvmt, and eo-ermyme B 1

L

2 added to a final concentraton of 10nigIL. When the culture was 122 hbald, broth -was hsrvesrtCd, cencifhgcd, and 0.2 L of ethanpol added to I L of superntaut.

One L of cell-free fermentation broth was vaeuum-disuedT Yedtg about roL aof a dark slurry. The slurry was ce-ntrifuged, anid about 40 niJL of liqui-d supematarnl we= egolected. This3 liquid was thea wewud with 40 tnt of edhanol in iordcr to pftclpitak out residual solids, which were remnoved by cenriifugation. A small samplie of the dreamed liquid was analyzed by [[PLC and found to contain 1,3-propanerdio1: -the idenity -of the propanodiol was confrmed by GC/MS.

Applicants have deposited a reccrnbirtanr Aspergiih's niger strain TCIR4O-1 3. omprising a DNA fragment encodinig dhaB(l dhaBx and dhaT (ATCC 74369).

3Do EZSMPL E z ProdLictioyn of I .3-pnrioI fi9.1 Ew wal~sr p sing recambinalit- A.rfgr AspergdIic frr-meniions mr carried out in 15.5 L totAl volume Biolafite ffermnetcrs, working volume initially 8 L, increaing to I It. during the ru,.

Aerobic conditions ame isured by aeration wini air at a rate of 10 L/ntn, at an impeller speed of 700-800 rpm and ;t back-prcasw of 1.1 bar (aerobic conditions are defined by the [Dissolved Oxygen (100% DO defined at ambient press=r), naeasuzd with intstalled DO-prqbes; a m-inimalzj vipjuet of 35% DO was considenx acrotijet The pH is maintained at 5,60O by automatic addition of 10% 1-l 3

PO

4 oPr 28%i NI4 1 OH. Tem-peratue is rnaimairicd at 32'Cr The followinig compounds =r batched into the tank and steriized at 12l1 C for 30 min: 2 gIL NaH 2

PO

4

AH

2 0, 17 g4L. (NH 4 )ZS 0 4 1 9/L M00O 4 2 EJL Tween 80, 45 gLvLProznosoy-l00 (a soy coocetrn of 70% protein). -6 g/L =on steep liqutor (M0% solid). 10 giL maltose. and 2 SAL MAWU DF204 (a custommade antifuam).- After sicrllintion, 500 usa, of d~ie 50% Maltin 150 feed is added, together with carbeniellln mud scrptomycin (thI up to a final ccentration of 10 nig/L).

One L of a 40-48 b old &spergilhets niger strain transformed with dM1 1.

dhaB 2, dha!33, dhafl4 end dtaT gcnes, rowring i a shakcflask containing CSL (defined in Example 22), is used to inoulat ie fenriecr. Ibec cuvlt= i-s thezn allowed to grow for 30-35 Ii before the feed (ft 50% Maltrin 150 solution.

heat sterilized) is stared, at sire -of I g/tn The eulmrcm is then rn for another 5 hi under 02 limidted conditions (%DO0 zero, un-der hill aration). After that, the Mal nin Iced is stcop- and whcrn dn Inc. I scid tt'Ii1MM 1C S5UF1- L virtually zero, the rpmu is lowered to 150, the HP to 0.2. and aeration is stopped.

The-* fermentzr is flushe wit an aaerobic gas-mixture HZ' 5% CQ 1 N)at -a rate of 7 L./rrdn for 30 iks G3m inlet and outlet is then closed, BIP is maintained at 0.4 baf and co-enzrneD B 12 is added to a final concentratin of zng/L. Throughout, broth samples are cenariged and theu supematant Wr rmpared for TEPLC and UC anialysis. 1,-propanediol is detected in the svpc.-nat&ani.

1PXAMELU-2 Lacrobacillus reweiri (ATICC 23272)t yjn irintaied ggi NMS (Difco, Detroit, WI) plats, CoLonies from a plate were used to inoculate 70 in] LA-etobacillus MRS broth (Dikeo #088 1- 17) supplemented with 25 ruM NaHiCO 3 in a 250 ruL Erleninyer flask- The- flask was incubated in an aaerobic atmosphere H2 CO 2 85-93% 14z) at 12 0 1C, HPLC analysis cFLUactobacrnlus MRS broth showed a componcat with the retention firne of glycerol. Lactobacillus MRS Froth was treated by alkaline boiling e4 ndwzd for glycerol by H-PLC and enzymatic msay. At most, 0.25 glycerol could be detected inthtft i2] medium; if allof this glycerol was trasfored to 1,3 -propancdh1l, 0.21 &IL propanediol could be said to have be=~f produced from glymppL After 10 d of mncubatirn, a sample from the Laceobacillus reweri cdura,= flask was retreved, analyzed by UPLC and GC-MS, and compared to -an inhiajrmedium sample. Correcting foir the glycerol present in the mcdiumi 1354 1,3-prapanediol was produced by baatobacillus reureri from substrates other than glycol.

(I GENERAL INFOUNAT ION: (ji) APPLICJ'JMt WAWI E, 1. DUONT ML )4ThCURS AND CCWANY STREET: 1007 MRKET STREET Ci CITY: NIIMINGTON STATE: DEAAWAWJ& CE) COUNITRY: U.S.A.

(PI POSTAL 00M3 (21P): 19090 TfLEPSOUE: 302-092-811.2 TELEFAX: 302-773-0164 CA) NAME: GEt4DNCOR INTEANATIONAL, INC.

STFEMT 4 CANBRIDQE PLACE irio SOuni wxINTOfl ROAD MC CITY: EO0CHE.SflF W STATE: NEW YOPZX CXXAITYTr IF? POS'TAL COIJ) (ZIPI 1461B TELE9PHONE: (HI TELEFAX: CMt TITLE -OF INVEflICtfl RZ0OOVERS:O OF A rEftinIAflLZ CARSON SOURCE TO I, 3-FP4?NE- DIOL BY A SINGLE H1CRQORGAISM iiii) NTThER OF SEQUENCES: 46 COEPLITR AflDABLE FORM MEDIUM TYPS: 3.50 INCH DISKETTE COMPUTER: IBM MC OPERATING SYSTEM± MICROSOFT WI:NDOWS 3.1 SOFTWARE: N-CROSOF WORD CURAE1F APPLICATION DATA: WA APPLICATION MN4BR; FILINGC DATS± CLASS ZIITO (Vi) PRIOR APPLICATION DATA: APPLICATION NUMBER:, 4W 440, 293 FILING DAME N4AY 12, 1995 ILvi AflORNHUASEN INFOPHAT~t (NQ NAM; LINDA JXAMTUY FLOYD MB REGISTRPLTZOI NU4ER: 33, 692 EEFERENCE/DOaKET Hu~sER: CR-9715-E (2XIFORHILT ICN FovL SEC 3ID NO:1I, jj EAOVEMcE LflIGR! 12145 baS PAirs (D VYPE nuclei-* acid sTRAZ4PEDC(KS: tingle W' TOPOLOGY:1-na (ii) MOLECULE TYE! DMA (genamic wx) SEQUUVCE p~rchiflO1. SEQ IV NiO:1: GT C CLC ACGGTa CrrAflCC AAATTCLGG JATGTCOMGG LATTTG-CATC GooGCLTCaA nAraGcCCCa -GacGCATGC ucoGGcc= G AGCAGGC- CAGCrxGGTC TGATGCAgGG ATTCAGThCAL TCTTCAACAC AOOTTCSATS nC&CTCTCT GTGvGAGcGG CC2GCGATAL ACG2&TCGGGT TTCATTACGAk GGRG&TA TGGCGAAAT TCAGGF'%ThGC CGGCGAAGOG GAGRAAAcCC GGTCAACAC GGATCGCAAT CCaGAGAG CCTGTQTXTC _aTATCAQLAC GTWITcr('CCLC~rGCCGA CGCCAGGQCT CNTC.LTGTCT TAKTTCAATCT CCAktCCA&AT TVCCAAflqAC CTGAlC GGAGAGMJ\ GGGT&LATG TflC*WaAAk~ TGCAGCLTG

GTG'CGGGA

7T~AiTrT IkcArrrrGC! CCTGG XGAT

CCACCTTGOC

'TCAGCTGcGL

GGTZ-AATCAG

GCYTGGGAZ

TGATGhTT

AACATTGCT

GCG1GCTrGGC

GCTCTCA!GC

GATAATC&JOC

CGOCGTOMC

ATAGCCAG

AGCCACCGOC

ATGGGCTTTA

CTTTnCTT

GCGTCILTCG

GGCTGA~C~rGr CaGrATTTTG CIGgcr AUcAG=cTG TGGCGGTCT

DAGILCGCCTT

QAGGTQLkIA

TGCATOGGTT

WCGGTGC

GAC=Cc aTTCAGTGCT

CWGAGGGGTA

AkCGAAX&AAA

CGMCCCTC

CATG-ICCGCT

GGGTCACATA

CCGnTGGAZA

TCGTCTGL.CG

=MlTCCUXN3 TTTC=rATG GnwCrrr TTCTCTGCCA TJLALWCGCGG GGTGGGOAJ~A AATTTTTGC GQAGGATTGT A3GvGCRSA ACTAGGGTTT TTTGTTCL2AI amAtAGCQA AKGATTTTT =GCTCGTc AGGCCGXflCT AkC&TGCtCLC rFATTTCAG ATVTTCAGGiG TOCTGATGCT= oM-CrcT .&TCGCTGRC G=aSCA3AS CCACSATAIT cXOcLAATCXA CCGTCI'QATG TCQGOGTGG TILMACCC TGATfTTG TGcGGCAANG

TATGG&ALJT

IGTTCCCTr~c

TCACTGGCCG

GGAAGG&A

GCTQTCTGT

GLrTT=AAL CuCsCrATG =AciTTTTGC G&ACCx-GA CCGACT6CSCr

GWIQGGATCG

AAA&&ATTA&

CG0CCC TA

GCGCGGATA

rGCTAAQT

TCGGTO.AATA

TWAAUCTGQC

cCGACGGTT

AGGCGATZOG

cc 120 100 240 300 6v 420 450 540 600 660 120 760 640 900 960 1020 1060 1140 1L200 1260 1320 1.35 C TTACTAtCKG flGCGCCGG TGflGTCLT CCCQACCLIC 00VT GACCG ATGCCAAC

GAALAACCG

OTTGCTAT

AGAGCAT

CGC GGTG

GGTGCGAA

CCCGVGAGC

GGCAGT~

TCCAFCT~rC

TGGGAG;TTTAL

GGCkCflfl GGCCfl

TTGCTCGA

TGTCTGGCG

GTGQGXAACT

as-Gca~rcc

GGCALAJLAYA

GCCOGCGC

4 rCGG7GATC

AOCAGOTG

GakgcCcTGQ

C.AGGCTGG

CACCTGMhTC Ar*CCZ)AYGG AfLCTTGCGC GC-TACCTca

GCXGTGGCTA

GccsGC-GGGG

GGOG'IAA&C

TGGAC=G

?CflCLCCGC

CGOCGGTGT

ACC:AGTC

CCCX(3CTAC

?CAATGTTC&

TCCC=YCT

TGAAxTCACGT TAflAACCMT ACAOGAr.C

TGXTGGACAC

3ATGIGCCTC cCCOUAUGCA

TGOGGAGUG

AGOMICAT

C~2c~rC4DA AE2A07CArAA A09AGATTGA

AGATGGGCT

GGCGAIFTATC

CACCTi=TC CGkGGCQAkAC

TGCJIATCCAC

L&CGGTGCAG

CAAAGAGCG

GCCAAGCGC

GVGGrCGA

=TCGGCGG

ACTTACCTCA

AACGQTTTCA

TG&TCTATVC

CGGTAOCCT

GCTGC*CG

GOGGCATMG

nTTCTZO alCTGG2 :zflOD 1500 1660 1920 1980 GGCnTCTGCC- AGOGGTCO3G ATCGMoCGAGA 2ATCGCC CGGRRGGG;GA AAc~cATCAT AA2ATGCOGT rXGCGGAC

CTATCCTCAC

AA&CIM

rTTraCATGG GG'iC~Tflcc

CTGQCAUCG

6CCSCTGcTC ci-ccacGG eCaaaccae CRTTA7CGGC I2GCVCGATDG TGoATAACCAC

CAGCGOCG&C

CGACAG=3~7 C&TGrGMCGAT cocoa CCkGGTCW3GC

?ACAAG=~G

GTOIL'TGcCC

CCGCACCAGG

ACCATCGGCC

CTG=TTATTC

GCCCaCTP CPLTTTTAtroC GGQGTGaTGGrnSGCCAGC oara=CT

AAA&GG&TAT

AGC2TGC-(= cccaGGGcc AGGCGGtCGCT

TTG.ATGAGTC

aTGCCaCIl TGrLC3CTGOC AGMCxT&LCA TeGGCTCTAT

CGCTGGCCAT

CvcRCGTCAh nCGGAACC rN&aTGCTCA rso~pgcGC

AGC&TCAGTT

GT?"AITT

AGTGGCTGGC

rCrccCCC gkC~TlCAG

CCOCTCUGC

GAflCC

GGAAGAJPCC

CGCCTGATC

AM-ZcGCohc

CGCGATGC?&G

GcCAPTGGAGZ crCzCrrTITGC CGCaCCGGAG cCTCaiflCG

AC&GGCG

GGCCAGCCARG

CELTCAAACAC

TGTCGATGCG

GCTGCTGCLT

2040 210 2160 2220 2-280 2340 .2400 2460 2$20C 250B0 2640 210DO 2760 2620 281600 2940 30100 30 S0 3;L20 Grc6oMLA TCTGCATC gaTACC02COugQCGCTGC CQAAGTCacC TZTGMAGTC TGTCG&GGCG CA&OSCAA CCrsnAuc GCMjAT6'r CcocGCGsc( ACCCAGGC~k CRGaATATG

GLAMAATC

ATC&LGTATC

CTC&LCCC&

ACCG-IcDaTC cnCACTCC! AG&TCCGC GCTcaATQ&CC cTrrUChoaL YCCGthAACC GCC=G =CACTTW 'rrCLCktTMA3GALCGG

CCGACLGOGT

GCCG&GcOG TGGcGCCGGA i'CaACsccc TOOCCAAYrCT nGA&ILATC=G CTGQTGCARTA A0CZGGAA GATGACrmCGC TGC.AC-AGCT AG=CGATTG AGAATATCG COSGGLTA!C TCWTCCA AGCCTGIUZIT TTAGCGOCAT G4GCGGT AGGLQLTGTC ITGAAGCAGT ACGAT.ATTGA cGATTCGGC CATGGAG AC vCcCGAtGG ATGCGcX;cr TGCGcGA GTCAGCTGG aaZaCcaci aCgCCATAk Cs7CCaTCcw CToo=caG 0 ZAC3CAG CG3TCTGCTGA TGGcGaGCrA GkCG'C&CGc Gc71GG~CcA(&; CCnTAzCnG GCTGCAacc

GC=CTGATC

GGTGACkr CiTCCCC7CcG

GL&ZTOGRSG

GGTGGCTAC

CATCAGCLGC

GcGGCCCIGc cGAmmAGA

GCAECTGCTC

CGCcAGCCJ±G AGGGCATCCG3

TVUATGCCW

GrcRGAq=CA

?TIGTOGA

COGGGTCAGGC

ATALTGGTGCA

TGGGQA2fCA AUCKJJaCG aahAGATCAG CGLCSCQ1 TCCWZTrlGG GGA&GAGG

GGG!CGGLA

OCOOGOO CAaTCTC GACTTTA.TQ cGUcr=ccc' GCCLLCGSGCG GC&OCTGI7 flCTCTQC- aGGTGATTA co='r~m'G. TGRAGT AACCGCTTA oGCCARG CTGCocoCCC: GAOctChAG AKMcOTTC CETCOCGLAT TOGCG7!Gr GGANTATTT GACALOGGC ACATOCGCT GOGTAGKG CGrTCGCT GCTATTAkTrC tcGCCS ATATCGGCC: GCACCACCCT rrJLAG== AGUATCLGGC ACAGGCQATT CCTGTAGCEFF yckoar-GGcG TTCIMLGCVGA GAnCTC~C C cGGTCACG TAAGCGrCTC CAGGGCGTQ C=GC _AWC sCrOGCtmJ GQCMTCCG C&GCOXGCIm GTTCwICGC GGTGCCOTAU CGTA&CAGGOC GTGGTVCCT yTcCTALLA OGACT

:CACACC=T

LGAGCTCCG

CGTCLACTGC

TCTGGAAEARG

GCAGGOcTG GTACTAT=c

TMTTCCGTCG

TGnGcTCALC GTrAATCT GCTGCCGGcC

GTGGCGCA

TTGAGCGCG

Tc3AGCCCA TCGC009TCG GCCTcGACGC TTATCC4G

TTAAOCAGLCT

3180 3300 3420 3640 3660 3720 3700 3! 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4550 4620 4 680 4740 4000 AAi GA&TkLTGC TGCGr,=QATA

OCCGCGT

=cnGCcIG 0CCt=TACC YTTCTCOATS CGCTGOCC GATAES&ATAC ATTCAGI ?TC GrWCaAioC GTAc--CRo'rl GTZCCGC-CGGC AGCTGGCM caLcccUcAaG GCKGCMT UY±TCGTzCr OC JTCGGTSACQ TOTCCGGTAG uam'GAGG TA=cTO G G'ACCGC=GPt 1COGC~ccX GOGCC~AGC GCAGOG~rXGT C.ACt=CCrrCC GTCAT!AGGr1 n~kCTcArr GATGGC G~ =G4tflaR CGAC&TGMkC GGCAATX TGCTC&TT.C CATCrCCaCC ATGflGGGGA AJLTACcOTA TCLTCCACA AGTGLCAG7 COGGT&T GTTGA'rC AMGTAGC CGCTCG TACJIGGOCGC- =TTGALAA ccT(CrCc &rrnaTcca AGGCCACGQ GGcG~CxisG TCCGTCAXCG CATGGTGffCC GTccacr CfTTCmG

GACGCGTGAI

?2CGCCAT

CCAGCTGGTG

CCAGCNA(A

C=CCCGA

GGTTAG=cG GcGGGTnOGT

CEATCGAT

CGCcTTc AUGGOFACCOC V7=CCTCC AGFCCCAGC CGCGCQQAYC CTALAAAGG CGTACCZG TLAACTC&U WGCWCTUGO GGAAAMTCGC GnVSAG&A C acraccor CTfT&TGRGC nirTAcO s1=cect GATGCcw AGMSG&T GcZITTTTVQO CAG'nZCAIX ATATCGGCGA CGGCaGCAGG AacaaarOG CoCcarscs TSC&CGAGC tGCATAZGCC- AflrTOXT G'nacasGCc A-TGGGGA flTGGAGATA TAGGCCTCTh

CAGGCATCC

ATCGTTGLTA

TTcGG)GTTG GAflCGCSACG ALTCGMCrA

GCCCAVGGTG

GflGGTGTCT

CO=GCAATAA

GC fAcpAGC

GGCGTTGGGG

AA2AACTZ flAAn'STCc

AAMLALCT

ACCG TACAGP.

GOIQGCAGGCO

CATC=GOGG

CTTATCflA

GTCAGGRACC

ATAGWOCA

ACGAITGATG'r TrCGGG~rTCQ TGCRGGGTTwr

AXGCTTT

CCAAAAAACT

C7CCTCAG rsczcskccu

GTGCACQCCGC

GKTZGTrCCG

CIAGCT

AflAJGCAGCG GmGTZ G&WaccGT cr-kGcrGUcc

GA=CCCG

CAGGGCTCG

GCCaTTG=c GOGGCGA1AC eCrorC;C enCAharGA fTCtvCOGC GcCATCTTTA TcCIrtCCcLG CA=GGCAG ThACQY2TGG ChcC&GATAA GTTALThflCC GAAAAAA ThocroCCGT &&CGOCGAOG CGOCAAAAT AMTAATTcG GCTGGC ncaarT Tl"ATTCa AATATCOCALG TCTTQXTC GCSQTCAA.C TnacCCAC0G CGCCfTT TTAC~rpACA

GCGGTGCC-GG

ATTCCGGCAT

CAATcOTGCG

ACGGCGAGGC

GCCCA

CTTGCtUA

TCAAACATAC

TCCAGGCGCc GCGCCAflTA

TATQGGOTAC

AcxAAAAA

CCGCGVTCGG

ATTTOIV=G

CCAC CT

CQAGQTGGC

GaCCM 2CrCCTGCLFl

CGGCGTGGT.

TCAGCAGTGG

7VJcacrT= CMMTGTAT7

ACTGGTACLO

csasGccscc CGCCCAt TC=GGcT

GGCTGTC

CT&LArmLLT r2ATAUCTCAT

ACT~GGCAA

CCTGCTCATT

TTAGCTGCAC

cvrrCasCEL

AQQCCTZCGTC

ccAX-ccccAc flGGOCAGGT 4920 5040 5100 5160 5220 5200 5340 5400 5460 5520 5580 5640C 5700 5760 5020 5880 59-40 6000 15060 6120 6180a 65210 6300 96 0 6420 6450 6540 6600

GATTGTCTGP.

GCG1ATArTC

TTTGTCILGC

GLLANCGTA

TT=T=~l CAAAlflAAk

TGCCGGTAAT

ACflG7TTGGC, 'TCTTCTCAT ACTAGGCGGC GALTACTAMJ GTTATTTGT CGCCOCGC.LT AflflGGO YTTTATTA.

ruMCCscrsa GTfl.AGTTTC CGAATAA T?lflrwrr GGQCXOGGOGG cfCWr-.C=C canTczaCTc CGCAVCAGA na=artcc ATCfGTTr GATTTAGTCAk AT&G41GTfl& AIYGAT"fl ATCLLTGCSG ATAGTGAAA-C TSTCGCAQA TECA1X4CT'GO CTCATflLAAA GGCCrOGCGT ArrcGorAcc GATCAAkAACG P=TGCAGTA AGTGWXTSA AAGQGGCTG nRAGT&C;Ai CGGTCTG&TC TOGaCCCAT? TfCGCCQAT TTC&fCTTT GAGCOLTGA GCOCC-GTCAA TCAGG&OGOO AC&GCOOZT TGACCCGQaTC ACQCALLCG CCGGQCCAG ACGTTGAOM CACL&LGCAG flGLTATTCAt COTC&GOOG ASGGTCL~ wGATGCG TGCGTGCC CCGGLCCCOC ACAATJfLLL

CTEGATTGGCG

TCTI'C.&TAA

TTTG-ACATG7A

GCAATCGC

GAGGkTCA

CAGATGAAC-G

TCCAAtCCAGT 2GGAGGCGMS

TTGCA!TCAC

TGGGGLT

GCCACGTCAC

ICOAATTc cGCITGACGoc GCVGAkGGCCG GG&TE2CGG CGGTCGGTAT CCSCGCTAC GCGGCCGCCC CGGCGTGTTG SCLTGC*3TGG CTAACCAZGC TT-A4GC CCf&TA&CC GCGGGTTGLAA -AATGCGCAC &G&GCAAGTC .GATGCTCTAC ?TCAGGGACT 'GCJJLACQPGC GAflCGQOUC GGTCCO CCGCCAACOA CCAGACTTIC AG&TGCTCGC GGQ!ILGAC- A CkTaI-rczC GCTCc~ai GTQACTGAT SC?? OAOGGC XCACcLAAxC;C GCcGCGG GCGWtGTTTA aarCCCGGC ACGCACTGCT CGCTUOLAGA TACVGCAGA CS9GTGrT CCGTGGTC&A ASC43GTTtCT ACCTCJDGCL CCGjAT COO CCG&ATCGC Coaoaaas oCGPoaoia OTATOOSCAT GAAAACCZGA TCGCCTCZAT TCCCAC CtG ATATTOOCCG rTTrn r-Ta cGGCTzACAG TT!CQATGCGG nGfLThZA GGCCIGCGYC COTOLCCA ATCCcG T.TlCOGCGA OGAAATAGCr, TACCOOCAL2C

GAT

1 GGCGf

CAATCAA&

CTTCAGjAA GCTGrTCGTC

GCCGAG

COCCTOGOrCO AGCGAZ G GCTG=tC?

!GA!TACAAC

GCTGGGOCTG

TAALCQCC

CTTCCTTTAT

AAT*GCGCG

oorATCAt2LT orvaacc

GT=CTGGGLT

CTGClCC

ATCGCCATGG

GTCGaLCTGG

TJGATCG

ACCGGCCA

CTGCAGAAGA

CAGGAGICCL

GGVI'CGCAGT

CTQS= oCcG

GALAGOG=TT

TACCTCC

GCTATTCGO

OCCCOSGGG

OGrocGTCGG

G&CTGT

ACCCTGRLTGC

ANCACGhCA

ATCCTGCAGC

ATTGCCI2'TC CrX2ITATCG 67OV0 6840 6900 6960 70 2C 700 7140 720g '7260 7320 7 340 7440 7620 7,740 7000 9D040 0160 0220 0340o C=CG~GAGG& GTGCGOcc GOCACCTACG ACGTGGGGA GGATC*ZGACTGDGGTGAur OW.CcGGCAc

AGATGATW=

CALCACPATG CVWCCGjtk- GCGACTC ACCGG3CaCT 8400 8460 ATRTTGTCG CGcOaGGC CGCLGCCGCa TTGAGGATAT TGCTG0x3ccA GwCGGGTCAOC gGGATTACC TCAACCT

CCGCCAGCAAT

SWCCATZTC

C4GGGC

ATTCTCJLALTA

GflCOGnGT ACCGGaATc TCSGGrTGGT cGSTGCtG GCATCTCC GLAGCT cCrLTYGAATA AGCG'fT~ GtGntcGccc vGccTrcnA CQL2cCTCAA AGAGC=rAnj GCATTcqx 'OZCG&C CGGCATaC= CATCGGTATC TGcGTCIIG CAAcCIGrAG CGflQLTTGG CnAAAACGCTX TGGAACGAA TCAGNT(GG AAS&AGDCCAL ACLTrVG GGTxAC AGGAaar CCcGGAGCAT ATCCTGACG C GscTaac G&AGGTGGC GQFCGCLGATT GCCGAGCAQA GG&QCTMhTC GOCTTCCT:G ccaci'Ccrc CAt;GCGGCSi2 QAC&GGT ODDGcCTrJC CCWASAGL ACnsACW GGC.cCAVC &FIGQTGSOGC CGGGATCGC GOCAGACOCa CGOGCTGWSJ CAGGCO TCTTAALCSAA GCCGCGQDG LAS4ACATC&k A?1CTATC&I NCG C JLSbLalTzS=t nanarcc osc'scn C2IQOCCGAVA rCasTcAAC~ AGACA-ccca nnLTcAGCc TCTTTTAOCC AaoCnT= CCGrrG&Jtc CGC9Ac Gkf GTCGGATCG.

AAACACC&GC ATCkCAcYCC SATCG&TATG cca'iazc OCPGOOG AACA&AG CCTTCACOCC CorG~qsC =CTcnn 1TGGGGA tGCC A&C CTGLGCGOCC CAGCCG GaiCCACcor CATCtiA.n CCGATCTGC *CTGTTCTCCC AGGGCGGCT GCT7aLGCTG GAQACCTACC SCGCGCXMG CGCGCAAfl. GTCACCTTG COGGTgCcOg CGGccwAAnz TrATnvcckJU AGCCGC rTTTCATALCA CAkW.AcGCCG mnoaDGcRc cCTCACLTC GACTAGTAAk LL3XATGCG OG? rCAGGAT iPATrGnTAG CCflCXCGCTG CTACcOGCAA A=TIrCACC GATAflACCC TCISGAACJ t cCQCAGGIATGT G&QrrCnCC CG3CCAGA2JC TEG&C-TACCA TGCAGCGa.A TOVGGTGGCG VC rcGCG CGCGGCW=G ACGAGcGCII raGGcfrc TATAAC.GCO TGcGcCGrT TGCZGGOGT VGCCGACG&G CTGGAGCACA caLGGcATGC TCCrGGTnc GGCGGAAGTC TATCAOC.GC GG;CATnAGCT AGGPcA 4t GCC=rAATA GV3CG&2-IQr ATAT=WSaAM TGCGw CTACGCCAG Gcaosxaw n'rrGccaa GCALTGAAAGG GkcGOG~kCp AAT&TCGCVG- GG&CCTC CGAAMACkCC GTGGrxkiTj AGC~3&TGTC CTC~CKTaA TtATTGGCA TGTOLZ&ATC IM&CCATCA OCQGAA=&? 8640 8700 5520 9940 9000 9015 0 9;20 s1i 9240 9300 9360 9420 9480 9S40 9600 S co 9720 9780 99410 9900 9960o 10020 TflZC&A~GA TVGACCA2GA TCGQTCA7AA CCCGACG CCQGGOGG TGGCGTQG 08 10080

CGTGGGGAC-G

GGGGTGGATC

TrlGGCT

CTGGTG&AAO-

GCAGGt.CCC

GATCCG

GGCCJ!TCGTC

cccOCAGGGG AAAnGGCCOGC C=CcTGC=a TGAkcGQGG

CCALTCTG

CQ1Q.TGC 12ATGCA~CC7T

CQTGQACGA-CC

GGCG&T=C

G-aTAALCGG;CG G=GGGcfC GCGAAGca CAGCCaGG*G

TZLOGWCAC

TGTlCACCAAC

CGECCTTTGTG

GGAAG('CCTTG

ccGMCAAT TC=GCCAkGC

ACCTTA&CCG

cccacrAG ATTaJCTGCGG ACTATCGCrC GTACfrl&YTG GAccGGGGGA AAccCXrrGC

GAGGGGGTAN

QATGTOcAGT GGxAG&CGGCCQ

ATGGILGATG

ArCQcdoccG

AACATGGCCA

GACCOGGC.G

UTCCAWECTCG

aAG&TCT

TTCA=C

arGTrrcnA CCGCTGGAAAk YGccT~CCCFC-

GTGCI'GOTV;G

WG9CACTATG GDGGTCGcAo aCTCaCTz

GGCGTG=X

TCGCTGCGCT

GCCTTGGCGT

ACGACOCCO

GTAkAca0 ?GGcrGGT

CZATCGCA

GCG=CCTGA2 CGoXr4iGTGAT

ATGTCGOGA

ACATCCGCGG

A-IACGVTTAT

CCr;TCLGAT AAV7GAOC cCr;CTcGAC CCGGGcmGG CrGCTGGCGGA GTCAcGAGLA AAGTGG'Z3TA AAACaT

CGCGCGCC-A

GCGGCTCATC

GCCa=TCJGC

AC&-~C-TAT

G;GCcGAGTT GC0GCMGGCC aGCACrC= GCGGZGQrcC CGATlTTT7 =tCCCGfT GGCvGMtGAC CTCAAA;G GQGGg= AaGQ GGAAGGGC UCQQWGGGXG crTTClCGGQ CTAAO)CCC= TssCRa~C=G TCCGCGGTG CCC GGOGGC AACCMTCAt oscsGO n GcCACAflC CGILAOCGGGC- ALCCACGCCG sZcOGxCCAt GAGATGFGcG TCCCIeA GTGCLGG GGCGGCGATG GTGAMSGCSG CCQLCflCLGZ ACCGKgMGTL MTTLACCACT COCQGCT=G GGPTCCGCG ATCGrICUCG LAATATgGT AGCCIGEA AGCMATLU A mTACCLX TG FC!XGGTG GAGT7CTTTC: C&TaCAaWM GQCG1AAA=G CuTGCjOc=G CaGGQajAAAG GGrTaTCACCC GGCGGTTCCAL GCIGGACTTT GAQATCCSO'- CGQGCAGGGC lA.kTTTCGGG, AaG(3CMtGT CAGGCGAATT 2TCA GGATrOC tGTAhITGL arQGC cc G&TTCICAT GGGQCCAOCT TTGL1TCA.~g ATaACCTGAk CtCIDGCTt AGTXArGCGA 1014MJ..

GGTGCTJ '10200 ACCGCG 21260 CGaC2GTGGA 112320 AGTGGTGC 1038Q AAcACanCCA 1G440 TG=CA&G&C 10500 TTAMGQCQA 10560 AGGATGLrG 10620 GC*=GCT 100 CGLTATACA7 2.074Q CTGATGGCCQG 10000 ATCGTCTGCA 10050 TQCTOQGG 10920 CGG~rGCCGCT 10980 cGl(i±GGGGCA 11040 rr&RAACCGA 11100 ToG~CAAAGT 111-60 GSGAtRCCCT 1L220 TGcCrrCfl 11260 AGJAACWOTT 1L.340 TTCCCCtTAT 1L1400 AGc=lAknc X;r460 GAAXCAGAdCG 12.5WO

AAI

4 CGGOGC 1ie00 CCAGaACTZCI 12A940 TTTCTTCGGC 11700 SQGATCLGT 11760 CTCCOGOGCG t10820 CACCTAAATC CGGQGSaTGAC CATTGOCC= TG43a=rrG CCTGflG& AOGCCGCAAG -1lee8a GTGCTCcr 2-ATWGrrC CCAS&CGGCC GQ4GGCTC GOCCOGCCC GaMGTAtr-*Z l4 GQGCfTCr GOCACT&T TCTVGaTCnT 4AACASASLTC ASCAIATfl SCGaCf-ACT )2000 O00CCAGC flL&CCTGG CG=acl-a TCCACGACC CGCATVCACA TATCLCTT'I 12060 AflCAGCGT T79CWfGCA GC4CLOCM'.1C AOG.GCAKTCC nTGGc.CkVr GACA!GGCC 12120 CTGCCGACG ACGQCkCGG AATTC 121t45 (21 IflWAION FOR 6190 IV NC;2-.

W1 SEQVEfL2 C~HARCTERIRTI CS!, flEGTi{: 30 bage pairs TYPE: nuclii *eid SThANDEDNESS; aIhgla TZOoLoaY lineam 4W t4$LECOULE TYPE: DNA SEQUENCE DtSCRIPTION: SEC 11) N0:2: GSAATTCAkT AAAGRZCL Lk&CLIATflGu nIFORMTbON FOR SEQ ZD NO.,3: SFQUDJCS. CHAflCTERSTrCS: ILENGTHz. 29 bas lpaIrs Yf"2: nwfleg4c c IC) STRANDEDWS:Sa1 ID) TOPOLOGY:, lineat (11t NOLUCULE TYPE: DNA igenoriC) MilP SEQUJENCE DE"CRiPfION: SEQ IV NO: 3: GQGAATTCTT &fLTCAATSGT GTCQGCZQG 29 1NFORHAkTIOt4 FOR SEQ ID uo*4c SEQUENCE LEnGTH: 3D base pairs (BI TIPE- Duc1SdLC aid (C STflHDSESS r sinle (DO TOPOLOGY: linear (ii) kCLECXIE TYPE: DIN (qOr9m±C) (xi) SEQUENCE DESCRIPTION: SEQ ID NOA 4 MflaATTCAT GCAACAG&C& A-CCCAAATTC INFOR4MATION FOR SEQ IV VO: ()SEQUENCE. CEARATEflSTICS: (A tLENXlH- 2S bna pairs 74 TYPE: mu.jeic viJd STFlNDEDKI4Es s ingle (DI TOPOLOGY: lliear (ii) MOLECULE TYPE: DNA (gefol±C) (xi) SEQUENCE DESCRIPTION, SEQ IV NWn SGaAnTC&C TCOCTTACTA AGTCG THYOPMLTZCN FOR SEQ TD 11± -G: (13 SEQUENCE CHCARACTERSTICS IAh LENGQTH: 27 bas. pairs M~ TYPE: nuclteic acid C STRANDEDNES Singl@ M1 TOPOLOGY: linear (i4j3 MOLECULE TYPE DNlA 4geflomic (xi) SEQUENCE DESCRIPTIONL SEQ IID NO: GCGAATTCAT GAkGCTATCG3T ATGTFZG 27 UWNOfATION FOR SEC0 ID £100: SEQUEWMC CHARACTLRISTICS:.

LENGTH: 28 base pairs (B3) TYPE.- nucfle±r. acid STPANDENZss± sinqle TOPOLOGY: linexr (ii) MOLE=UL TYPF, DNA4 Igenzmhco (zi) SEQUtENCE IJESCRIFTION: SsQ ID NO±7?: GOGATTCAG AAiTGcaGWC W&Ruur 26 j24YOPMATlOtl FOR SEQ ID NO!8± SEQUENCE CiIAflCTERISTICS: (NI LENCTB: 28 bane pairs B) TYPE: nwinleic acid {CI STRANDEDNESS: sfitgl-e QJ3 TOPOLOGy:; linear (ii) motm~uL TYPE: DIII (genonc (x±3 SEQUENCE ]DESCRIPTION: SEQ 1ID NOI-61 QGCzA.nncn a&ocGAenn ACC&TGCQr- 210 fl4YOEZ4TION FOR SDO ID NO:9:: (i3SWXnIC-Z CHAPACTERISTICS: WA LENGTH: 27 base p&±rs B} TYPE nueceic acid rC) SnncUiEaMSS! single i[N TOPOLOGY: linear MOLECULE TYPE: flfl 19enmmc) I xi) SEQUENCE DESCRIPTICtI; SEQ 1: D NOLS GCQAATTCTT AGCftCI ACGCACC 121 INFOROTIOJ FOR SEQ 11) 1C~j1U.

Mi StQUItflC CHAR.ACTRISTICS; WA LENCTH:- 94 base pairs, TYPE± nuclekic aid STRuMmnnESS! single TOPOLOG7: linear (ii) MOLECULE TYPE: DNAl (qe-ncta±c) mlx SIEQUENCE DtSCRIPTION; S:EQ ID N01l0: k!9Cf2sSGL TCTAGLLTAT TGLGCTCGUIA TTCctGGGCA TGCGGTACCG GATCCLGAAA LflGCCCCA CCTGCAjGTG CG(3GCT I TTTT INFORM#TION FOR SEQ 'It) NO: Xfl (lI SEQUENCE CHARkCTRISRICS LENGTH: 33' base pairs MB TYPE. nucleiu ac-id $SIMlEDNE85: aingle TOLcOOT: linar ML)- MOLECULE TYFlfl MMA 1genmicc) (xi) SEQUECF.ErSCtnnMON; SEQ ZDfl:1 escc-aoCrr AAG(GAG=fA ATAAATGa AltO ZM)"NORIATION FOR SEQ TD NOL12:.

(i SEQUENCE CHAPACTERZSTICS 4A) LEHGTMK: 26 base pai-rs 4B) fE: nucleic acid STnNDE.DNr.5,&, stogie MOLECULE TYPE: DLM (gencmic) (x4i) SEQTJE IDESCRflTEOff SEQ ID 1O1 GCTCTAGA2T ATTCAMrGGT GTCSSG INFOR~MATION FOR SEQ2 ID t4O-13.- C SEQUENCE CNAACTflI1flCS: LENGTH: 42 base paiLrs MB flit! nu LOivaci 76 26 STRAIJDDHES: Single ID: T'OPOLOGY. linear (ii) M3LDflIT TYPE2 DNA (gmncaic) W l) SEQUENCE DESCR3PTZONt :SEQ XD 240:13: SO=CGTC7A GAAtTATGkG CTATCGTJ.TG ?ZTQLTTATC TG 42 (21l ZNORNA2ION rOR 6M TZ) R4O1lU SEQLTEN CflRLkCTERZ9flCS: LENUYH: 35 ham. Fairs TYPI;- nucleic acid W) STRIWDEVDqSS: sringle CJ~ TOtOLOtYr: lin-ear (WI HOLECULC TYPE! DNA (ysoMin) lyZ± SCUnct DESCPXPTIO&z SEQ TD $Q;14; TCTG.ATACGG GATCCTCAGK ATGCCTGCG GAAAI? 36 fl4FORMATIN FOR SEQ ID Q~ WI SEOUEZSE OfAJflCTEflfTIOS;.

(Al LENGTH: 102 bulge pair.

TYPE: nucleic ac-id (C STR=lWflNES$:. SiLnqjo TOFOLO*fl linear U.0l nOLECULE TYPE: DNA cgODnoMiC) SSQUC~CE DESCRPTION: SEQ ID H40:15: CSiATCTGTGC TGVTTG3CCAC GGTATEOAOC ACGGA GATnATGQOC TXCLCADCTC GACTGTCGGA CGGGGGCACT GGLACAZA Gflcaoccsas CCsTvACccc CTTGACAATG 1203 C=CATCCTfG AGCA-IITAAT TCALACCACTA AACJ&AATCAA CCGC-GTTTC CGGIGGTAAC ISO0 C 151 IROPMNTioN roa SEQ ID NO.16: W SEQUEOCE cHAnAcTFalSflcS: IA LENGTH: 149 b~as pairS E) TYPE; oacil- acid s7raaauprvss: sinqr~a TOPOLOGY: 3-inar {i111 MOLECULZ TYPE: WNA (genomip) (xi) SEQUENCE DESCRIY IIW: SEQ ID tlO:16: CCACTCTGC ?GTfOCCAC- G'GTATGCA3C AiZCAGCGWa GATThTGGG TcCOCCGOTC Q&CTGTCGGA CGGGGCtCT GGAACATGCC AMCTMGA CR&AATTC AACCACAAA 120 77 CAnrTcnLcc COGTTCCCG rmA=GAhc fFORIATX1rI FOR SEQ ID iiO.-1: (iL) SPQUEA=cE WA LENQ1!: 33 bams pairs ?YPES nuclei1c acid STRAbEENES.S: single MD TOPOWOGrh l1-ear (ii) H)LECULE TYPE:- D11A (genceia) fxi) SEQUENCE DELCR~flION., SEQ ID MC: 17: GCALTCACT AG~CTLCTC TCMCTTC CAC X2JFOVMATX:Ot roa SEQ ID klO:le:- M1 SEQUENCE C9kACT=ERI8'fCS: LENGTH: 33 base pairs Trprr nucleic0 acid STRLNDEDNESS:- single TOPOLOG; Zua (l MOLECULE !YPE± DNA (gencaic) jxi; SEOLU.WCC DZSmj=TIOCf: 6Z 11) 00:l6: C3G-ACCTT QGTTACCTCC OGQAAAC.GCG=T INFORPM&TION FOR SEQ I1D MO.29: SDOtiNKE CHARACTERISTICS: LSWGTkI: 16 base p4;irs WS flVPE± nucimic acid STRANDSDULSS5: Single (BD) TOPOLOGY: linear WOi MOLECULE TYPE: DNAZI (qenan-) (xi} SEQUNCE DESCRIPTION* SEC ID NO: 19: TOG&CCACAA GGAGGA INFOPiMATION FOR SEQ ID 14O;20.

I i) SEQUENCE CMAAlLCTlTCJ;.

LENGWH: 16 taut pars (BI TYPE: nucleic acid CC) STMJ4DYEDNzSS:. single TOfL'OLOGY: linear (W1 MOLECULE TYflt- DNA (ganumici (xi) SEQUENCE DESCFJPfljOt; 5D0 ID N0.20; CAGTCCTCC TDGTGG '-1L43 42 ZmTOEXLATZON FOIL SEQ ID K0.21: SEQUENC CEAPACVEEZSTICS: LflIGTE: 4S base pairs TYPE' nucleic aid STBAHDEDXESS: single CD) TOuOLOy l'inear (ii) MOLECULE TYPE- lDX& IgtflCmio) SEUENCE UFSCRI[flOl SEQ IID N01 21; ACTGGCCGTZC GTTTTACTC(, AGTCGTGACT QOMkFACCC TGGCG (2h INFORMATION FOR SEQ ZD NO:22: ±3 SEQUENCC CHARKCTEPISTXVIS: LWGTM: 14 base p,&trs TYlPE. nucleic acid- C)STP.A2MENESS, uing)-e i)TOPOLOGS:. lin-ear C1-± HOLECUE~ TYPEt DNA (gnnaic) IXi) SEQUEN9CE DnESCnnzON: SEQ ID 110 22: Jt&TTCAiflGG &GGTr NTFORT-ftTIOE FOR SWQ Ili N0,23! S5eQUENCE CJEAR&CTEFtITICS: LEIXGTE: 14ba pairs C I TYPE: nucleiy aoid STRANDEDNESS: F,;ngle TOPOLOGY: linear (-ii MOLECUL TYPE:- DNA (genamic (xi)4 SEQUENCE DLSCIUETXON: SEQ ID N0123; *1- 4! 14 CakGACCTCC TTTG (21 )INFORMATION FOR SEQ ID NO--24-' SEQUENCE CHARKCTRISTICS' LEUGTH: 1S base pairS TYPE: nucleic acid STRIMDDESS: single (JD TOPOLOGY: Slnear (ii) MOLECULE TYPE: DNA (VrenMiC) sscumtt DESCRIPTI ON: SDQ ID N0),24: AQCTTGCCA CCATQ-AAAA ZNFOMRLATDN MOR SEQD ID NO: 00~ SEOUrNCc CHRACaMnTICS: LENOTft 19 base pairs WE TYPE: nucleic acid 6TRAMUEDNES&5 sinle (Dp. TOPOLOGY: linear W1i )OLEW=Fm TYPE: rm Lgencmi Wx) SEQUENCE DESCEIPTCUE: SOO ID QA2CTTTTCL T4flTCGACA (21 IHYORMAflON FOR SEQ ID NO: 265: Wi SEQUZNCt CHARAa-,ERISWICS:- LEflGH: -3 base pairs ME TYPE; nucleic amidS STEANDEDt4ES: Single tPPLOGY: 1innr (ii) MOLECULE 'flPE,- DNA (9enomic) NOi ENICE DESCRPfIw~z =E ID NO:2E: TCGAOCCAl GSA IMFOPnATION~ FOR SE ID H0:27z WU SEQUENCE CHARnCTEISY ItS: LENGTH: 13 b~ase pairs TYPE: ntuclqeic acid 4C) STRA1NDEDNESSa Single TOPOLOGy: linear MOLECULE TYPE- DNA (genom±cJ (nj) $ZQUECE DESCRIPTION: SEQ 11D NO.n27, CAGTCCTCC TG INFOfl4Txonq FOR SEQ ID 140-28R U)i SEQUENCE C~HARACTEBZTST:CS: LENGTH: IS base pairs TYPE: nucleic acid STMUIDEDNESS:- single ONI TOPOLWtY. Lnemr (ii) MCLECUL TYPE: JDHK (gcncmic) (xi) SEQUENCE DESCRIOQg: SEQ ID MO:28t TCGACGAATT CAGGAZ91A '-9 (~INFOKOSIO FORP SEQLI XV No.29: Wi SEUENCE cBAnAnRflTlaS: LENGTE: 1B ba~e pairs TYPE., nucleic- acid SRnMWxESS: single Mb TOP'OWGXZ: ILnear (i1t) WMJECULE flit: DM~ (gergmuich -SEQUENCE DBESLWJPTION: SE ID HO: 29; CTAG'CCTCC TGAATTCG 1 IFORM4ATION FOR SEQ =D NO0:3-0: (iJ S CQUEDCE CHARACTERS SfCS:, [,ENGTH: 23 base pairs TYPE! nucleic acid MC STRAk4DEDRSSS: single TOPOLOGY: linear (ii) MOLECULE flE: DNA (qenfl-nCi (74) SEQUELNCE DESCRIPT.ION! SEQ ID iO.-3O: ATGTAC.AAG TCCGTCGC CGA 23 INRNAPTION FONk SEC ID N0.31; C1) SEOQfNCE CHAPACTflXSTSCR: LU4QTI: 22 base paiLrs t: nucleic ac44d STRANZ4DDIS: s0angle TOPOLOGY: linear (ii) MOLEZCULE, TYPE. DNKA (genccici CS~iI SEQUJENCE DSSCRIflION: SE0 IV NO: 31, TCAGCGGCGC AGGflAGGCG CQ 22 W2 INFI'DPTODN FOR SEQ ID NO:32: Wi $ECLTNCZ CHARACTERISTICS: CA) L5NGTa: 31 base ME TYPE: -nucleic acid SZRANunxDssSS: single (LI) TOPOLOG9Y: Ii~hb~t MOLECULE TYPE: DHA (geinrsicj (2ti) SDEKO DESCRIPWXON: SE* ID NO:32: ATGACCIAG G GCCSGATC.CG TCGACCTOCA Gm 31 IHFON4&TIO&T FOR SEQ ID No, 33, M- SECRnia CRAARACTEMTZCS: LDIGTH: 32 bane pairE ?Yntt nucleic acid CC) flMNDENZS atug1o W)TOPOLOGY: linear CU-I) MOLECULE TYPE: IDNA (ganamic) W-i) SmQvmEE DSaculmflON SEQ ID no: SS: C'DACCCTTG CCCCGGAYCC GTCGACC'IGC AG 32 INFORMATION FOR SE'Q ID NO:34: USEQUENCE CHAsflC7ZERIST1CS: (A LENGTH: 23 base pairs (EJ TYPE:, nucleic- acid STRMWKDPWE5; mingle TOPOLoGYv. fl1nemr MOVLECULE TYPE: DNA (genmoudC (Xi) SLQ~tNtE DZ$OR2EPTIQ*K: $EF* =r NO034: CACGGCCTG CGCAGGTTG GGG; 23 (21 iXFOPmiIO FOR SEQ nD LENGTH: 21 base paixs TYMt nucleic acid $T;AJ4DlKY.S: tinrle TOL5-OLOCY: linear (11) MO0LE=U TYPE BRA (qenOwmiC) (Xi) SEQUENCE DZSCRIPTIlN: SEQ ID SGCP*GCC SC ACSPATTGCGG C 21 INFORMATION FOR SEQ ID no:36 SEQUENCE CKLafCTERISTICS: LDIMrs: 21 base pair.

WS TPEL: nucleic acid ST MtDNmS: single (DI TOPOLOGY: linear (±M1 MZXrLSCz TYPE: DKR (genoiaic) (xiS) SZQUENCE DIESCRIPflOH: SEQ JED NO: 19.

GCGGAA ACC ra(XTGCATCG C 21 ZNFOPHArION F SEQ ID NO: 37: (i SEQUENCE CHARACTtRZSTZCB; W LE INGTH: 21. base paL-ta TYPEZ: nucLeic actc SThANDEDEss: Isingi.

TOPOLOGY:, Itnear MOLECULE TYPE: nNA (gencmic) (xi SVNCCM DESCRZ1TZItM S80 ID MC :37: OC*rZCAGG4G ACrGCAALAC G 21 C21 INFORM2ATFION FOR SEQ 1c 80:38i: 0L] SEQUEWNCE CHRACTERISTICSE WA LEGT=H: 35 banme pairs MYE: nucleic acid STBAI4DEDtIESS: single TOPOLOOX: linear ii MOLECUJLE TYPE; DVk Cgenomio Wzi SEQUENCE DESCRIPTION: SEQ ID 11:35$: GGAA&TTCAOA T'C'CAGCAfLT GALLAGATOA 7AAM 1 INFORMtTION FOR SEQ ID NO:3S: JI) SEQUENCE CHARAC~TRISTICS: LENGTHC: 34 bane pairs M3 TYPE. ntuoLeir, acid (Cj STRAXNEPNE S: single TOPOLOGY: linear UnPMOLECULE TYPE: DNA (gemc)c iw-P SEQUENCE DESCRIPTION; SEQ IDW 3 GCz~r7CACA TCCAGCAAr GCJLLCAGACA AlC= 34 INYORMATIOU r011 SK~ ID SEQUENCE CBARAC=tUSTICS; LENG TH: 28 bae pairs rrPE2 nucleic aciLd STPRNDEUNSS: Single ToPOLOGY: ine- a r (ii) MOLECULE TIME; DNA (ge1IcudlC itml SEQUENCE DESCRIPTIONh SEQ TV N;4(i: GCTCTAQATC ACTCCCCTA CTAAOT'CG flMronIClo FOR SEC 10) NO: :41- SEQUENcE CHARZRER~ncs; WA LENG'Pj; 37 taco Pairs TYPE± nucleio acid STRANnDN=S. single T0 OOOGY: litazm (ii0 MOLECULE TYE: =kA -(ymno) (zi) SflguNcc DESCRIPTION:~ SEC ID NO:41: GGAAMrAs&A TCTCAGCAM7 aGcsmh.AA nccAisc ThCRMTIOJ FOR SEQ It) P1:42!- (a SEQUJENCE CHARACTERISTICS: 4A) LtXGTJR: 27 baise pa~irs TYPE: nucleic acid STaANDZNsrnwss single TOPOIXfl: 1ltnear 11LECUE TYPE± DNA Ig -enom-ic) (xi) SEQVWCE DESCMIPTILQ: S5Q ID D: 4.2: GC=fAGT AGCITCCryT ACflJZc fl4FORMAXION F'0R SErQ ID LJo±CJT (±SEQUEMC& C~hAPACTERTICS: (Al LENGTH: 99 bae pairs CE) =lE: n~ucleic acid STRAKDnES single rOPOLOGY: in-ear (ii) ICLECULE TYlPE- DNA (geomit) Xi) SEQUDICE DESCRIPTrION: sEQ zD No:4- GGAATTCAGX TCTCAGCAA&T GArTATCGT ATOTTYMi ItUVORMAIZCN FUJR SEQ ID uoW 44:- Ii SEQZJEWC CAARkCTE~ISflCS: LENGTH: 24 base pairs MB =Z:3 nuclic acid $TADlEBSS: oaule TOPOLoGY: linear ni)hftzcum TYPE.- DNA (goacaic) (xi) SEQUERt"n DESCRPfxii: SEQ 11)tfQ;44: GCZCTAG&TC Ac3&jT ,G GCGG INFORMATION FOR SEC 1B Wi SEQUENCE cHAPAcrtiSTICS: LETH;, 3S base pairs W TYPE: ntuclpei- acid (Cl -SflANDEDNDSS: aingle TOPOLOGY: linear (1i HDLECYL TYPE: DNA (genM~iC) (xi) SDQUENCE JDESCRIPTION: SEQ IV NO! GGJLAflCAGA TCTAGCAATG CCGTTAATAG CXZGGs fl37PMlflION FOR SEQ ID NO:46: Wi SEQUENlCE CUABACTERITZCS: LENGTH: 25 banS pairs TYPE: nucleic a034 STRANDEDUI.SS Single TOPOIO(7Y: iInenar Ui MOLECULE TYPE: DNA (anm~ic) (,xi SEQUENCE DESCRIPTXION4 ZpQ ID mO-.d6: GCTCTAS ATT AATWCGCCTG ACOGC Where the terms "comprise". "comprises", '"compri sed" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, stps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, compornent or group thereof, The present application is a divisional application of Ausrtlian Patent Application No.

71565/00 (the "parent application") the specification of which is herein incorporated by reference. The parent applicalion is itself a divisional application of Ausiralian Patent Application No. 5679/96.

I 1.Y3*- 1 77pp

Claims (3)

1. 3. A cosmid comprising a DNA fragment of about 35 kb isolated from Klebsiellapneumoniae wherein the DNA fragment encodes an active glycerol dehydratase enzyme having the restriction enzyme digest in Figure 1, columns 1 and 2.
2. 4. A transformed microorganism comprising a host microorganism and the cosmid of claim 3. The transformed microorganism of claim 4, wherein the host microorganism is E. coli, the microorganism designated by ATCC Accession No.
3. 69789. 6. A transformed microorganism comprising a host microorganism and a first DNA fragment isolated from Klebsiella pneumoniae, the first DNA fragment encoding an active glycerol dehydratase enzyme having the restriction enzyme digest in Figure 1, columns 1 and 2, and at least one second DNA fragment isolated from Klebsiellapneumoniae, the second DNA fragment encoding an active functional protein other than a glycerol dehydratase enzyme. 7. A recombinant Pseudomonas sp. comprising a DNA fragment encoding dhaBI, dhaB2, and dhaB3 and dhaT and designated by ATCC Accession No.55760. 8. A recombinant Pichia pastoris comprising a DNA fragment encoding dhaB dhaB2, and dhaB3 and dhaT and designated by ATCC Accession No. 74363. 9. A recombinant Saccharomyces cereuisiae strain pMCK1 /10/17 (MH) comprising a DNA fragment encoding dhaBl, dhaB2, and dhaB3, and dhaT and designated by the ATCC Accession No. 74370. 86 COMS ID No: ARCS-160826 Received by IP Australia: Time 14:50 Date 2007-09-13 13/09 2007 THU 14:52 FAX +61 3 9859 1588 CALLINAN LAWRIE 2010/015 0 0 A recombinant Bacillus lichenifbrmis strain BG188/pM26 (Clone comprising a DNA fragment encoding dhaB1, dhaB2, and dhaB3, and designated by ATCC Accession No. 98051. 11. A recombinant Bacillus subtilis strain BG2864/pM27 (Clone comprising a DNA fragment encoding dhaB, d, dha dhaB3, and dhaT and 0 designated by ATCC Accession No. 98050. 12. A recombinant Streptomyces lividans strain SL14.2, comprising a DNA V. 10 fragment encoding dhaB 1, dhaB2, and dhaB3 and dbaT and designated by ATCC 0C Accession No. 98052. r 13. A recombinant Aspergillus niger strain TGR40-13, comprising a DNA Sfragment encoding dhaB 1, dhaB2, dhaB3, and dhaT and designated by ATCC Accession No. 74369. 14. A bioconversion process of claim 1 to produce 1,3-propanediol which process is substantially as herein described with reference to any one of the Examples and/or accompanying Figures. A cosmid of claim 3, substantially as herein described with reference to any one of the Examples 1 to 6 and/or the accompanying Figures. 16. A transformed microorganism of any one of claims 4 to 6, substantially as herein described with reference to any one of Examples 1 to 6 and/or the accompanying Figures. 17. A recombinant Pseudomonas sp. strain of claim 7, substantially as herein described with reference to Examples 14 or 20 and/or the accompanying Figures. 18. A recombinant Pichiapastoris strain of claim 8, substantially as herein described with reference to Example 7 or Example 8 and/or the accompanying Figures, 19. A recombinant Saccharomyces cerevisae. strain of claim 9, substantially as herein described with reference to Examples 9, 10 or 11 and/or the accompanying Figures. A recombinant Bacillus licheniformis strain of claim 10, substantially as herein described with reference to Examples 17, 18 or 19 and/or the accompanying Figures. 21. A recombinant Bacillus subtilis strain of claim 11, substantially as herein described with reference to Examples 17, 18 or 19 and/or the accompanying Figures. 87 COMS ID No: ARCS-160826 Received by IP Australia: Time 14:50 Date 2007-09-13 13/09 2007 THU 14:52 FAX +61 3 9859 1588 CALLINAN LAWRIE 0011/015 C) Co, 22. A recombinant Streptomyces lividans strain of claim 12, substantially as herein described with reference to Example 15 or Example 18/6 and/or the accompanying Figures. 23. A recombinant Aspergillus niger strain of claim 13, substantially as herein described with reference to Example 22 or Example 23 and/or the accompanying Figures. DATED this 13 1 day of September, 2007 E.T. DU PONT DE NEMOURS AND COMPANY and GENENCOR INTERNATIONAL, INC. By their Patent Attorneys: CALLINAN LAWRIE 13n 907o a3772.speOip- s COMS ID No: ARCS-160826 Received by IP Australia: Time 14:50 Date 2007-09-13