AU619193B2 - Preparation of novel protein sweeteners - monellin type - Google Patents

Description

T o WORLD INTELLECTUAL PROPERTY ORGANIZATI PCT WL International Bfreau AU-AI-19397/88 6 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

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1< (51) International Patent Classification 4 C12N 15/00, C07H 21/04 C12N 1/20, C12P 21/00 A23L 1/236 Al (11) International Publication Number: (43) Internaut nal Publication Date: 29] WO 88/ 10303 December 1988 (29.12.88) (21) International Application Number: PCT/KR88/00010 (22) International Filing Date: (31) Priority Application Number: (32) Priority Date: (33) Priority Country! 2 June 1988 (02.06.88) 064,341 19 June 1987 (19.06.87) (71) Applicants: LUCKY, LTD. [KR/KR]; 20, Yoido-dong, Yongdungpo-gu, Seoul 150-010 LUCKY BIO- TECH CORPORATION [US/US]; 4560 Horton Street, Emeryville, CA 94608 (US).

(72) Inventor: CHO, Joong, Myung 4238 Treat Blvd., Concord, CA 94521 (US).

(74) Agent: KIM, Yoon, Bae; Y.B. Kim, Lees Associates International Patent Law Offices, 7th Floor, Garden Tower Bldg., 98-78, Unni-dong, Jongro-gu, Seoul 110-350 (KR).

(81) Designated States: AT (European patent), AU, BE (European patent), BR, CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (European patent).

Published With international search report.

A.O. J.P. 16 MAR 1989

AUSTRALIAN

19 JAN 1989 PATENT OFFICE (54) Title: PREPARATION OF NOVEL PROTEIN SWEETENERS (57) Abstract Novel proteinacious sweeteners are provided comprising an amino acid sequence based on the sequences of monellin subunits. The single protein may be prepared by recombinant techniques, to provide for a stable strong sweetening agent, which may be utilized in a wide variety of contexts.

1 PREPARATION OF NOVEL PROTEIN SWEETENERS TECHNICAL FIELD Novel proteinacious sweeteners are provided produced by recombinant techniques.

BACKGROUND ART Monellin is an intensely sweet material present in the sap of "Serendipity Berries", the fruit of the West African plant, Dioscoreophyllum comminisii.

The material has been purified to homogeneity and shown to be a basic protein with a molecular weight of about 1.1 x 104 and is completely free of carbohydrate.

,,:inellin is the first well characterized material among several sweet or taste modifying substances found in tropical plants. It has been characterized and shown to have two subunits of about the same size held together by non-covalent bonds. The two subunits-are not identical and the flavor modifying ability of monellin is dependent upon the presence of both subunits and a single mercaptan.

PCT/KR88/00010 u I WO -88/10303 2 group, which if blocked abolishes the sweetness.

Because of the uncertainties and cost of extracting natural products from plant sources, an alternative route to the production of protein sweetners is of substantial interest. Recombinant techniques offer an opportunity to synthesize proteins of varying types.

However, in employing recombinant techniques, one is required to develop a strategy for producing the gene, demonstrate successful expression of the protein in a cellular host, and isolate a product which is shown to have physiological activity. In many instances, it is necessary or desirable to modify the naturally occurring sequence, which substantially increases the uncertainties of success of the production of a useful product.

The relevant literatures are as follows Morris et al., J. Biol. Chem. (1973) 248 534-539 describe the characterization of monellin. See also Cagan, Science (1973) 181 32-35 Wlodawer and Hodgson, Proc. Natl. Acad. Sci. USA (1975) 72: 398-399 Bohak and Li, Biochimica et Biophysica Acta (1976) 427 153-170

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i.e 3 Hudson and Bieman, Biochem. Biophys. Res. Comm. (1976) 71: 212-220; Jirgenson, Biochem. Biophys. Acta (1976) 446 255-261; and Van der Wel and Loeve, FEBS Lett. (1973) 29: 181-183 for further characterization. U.S. Patent No. 3,998,798 describes the preparation of natural monellin.

SUMMARY OF THE INVENTION According to this invention there is provided a DNA sequence encoding a polypeptide comprising an amino acid sequence having at least 80% of the amino acid sequence of the subunits of monellin covalently bonded together through a peptide linkage.

Novel DNA open reading frames, constructs employing the open reading frames and expression systems are provided for expressing novel proteins having sweetening capability, where the proteins employ a substantial proportion of the amino acid sequence of monellin. The proteins are a single molecule as distinct from the two subunits of monellin, so as to define a single sequence.

DISCLOSURE OF INVENTION Novel proteinaceous sweeteners, methods for their production, and intermediates used in the methods, particularly nucleic acid intermediates are provided.

The sweeteners are modelled after the naturally occurring sweetener monellin, where the two independent subunits of

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WO 88/10303 PCT/KR88/00010 monellin are .joined together in a continuous sequence.

The two subunits may be joined end to end, by modifying the amino acids adjacent the juncture between the two subunits, or by introducing a short bridge extending the sequence.

The amino acid sequence will be in substantial part the amino acid sequence of the subunits of monellin, usually having at least about 80% homology with the monellin sequence, more usually at least about 90% homology with the monellin sequence. The sequence may be varied by insertions, deletions, or substitutions, where insertions and deletions will usually not exceed about 9 amino acids, more usually not exceed about 6 amino acids, and substitutions may be conservative or non-conservative, where the following table indicates as conservative substitutions those amino acid on the same line. For the most part, polar amino acids will not be substituted for non-polar amino acids and aliphatic amino acids will not be substituted for aromatic amino acids.

u;:3 r:i WO 88/10303 PCT/KR8!8/00010 5 Amino Acids Aliphatic non-polar G, A, P V, I, L polar neutral S, T, C, M, N, Q charged basic K, R, H acidic D, E Aromatic F, W, Y For the most part, conservative substitutions will be preferred and the cysteine and methionine at positions 41 and 42 of subunit II will be retained. (In the numbering of amino acids relating to monellin as distinct from the subject constructions, the numbering of the natural monellin subunits will be employed.) (9 i WO 88/10303 PCT/KR88/00010 6 The protein may have either subunit II or subunit I as the N-terminus, particularly subunit II. Depending on the construction, the product may or may not have an Nterminal methionine. The two subunits may be joined by a short bridge, usually of not more than 10, usually not more than 8 amion acids, or may be joined directly, or preferably the amino acids at the juncture will be modified. The amino acids at the juncture forming the bridge will provide for a polar juncture, that is, at least 50 number usually at least about 75 number of the amino acids will be polar and conveniently, at least about 25 number generally about 50 number will be amino acids naturally present at the subunit terminal. The amino acids may come from a loop of subunit I.

In referring to the juncture, the juncture will include as a bridge not more than about 10, usually not more than about 6 amino acids of the naturally occurring sequence of the subunits. For the joining of the C-terminus of subunit II with the N-terminus of subunit I, the juncture will be at Ile(46) of subunit II and Gly(6) of subunit I with the intervening amino acids, if any, as the bridge.

I- 1 -7- S 1* I Where subunit II is the N-terminus, one or more of the wild-type amino acids at the juncture may be removed or substituted, usually not more than about 10 amino acids will be removed or substituted, more usually not more than about 6 amino acids. Generally not more than 75% of the removed or substituted amino acids will be associated with one of the subunits.

Bridges of interest will include .a 2 a 3 4~ 5 6 7_ 8 aax- aax -x x aac aax aax aax 10 where only one amino acid need be present, and the individual amino acids as defined are as follows aa, is A, D, E, K, R or Y aa 2 is Y, A, D, E, N, Q, K, R, T or S; aa 3 is N, Q, S, T, D, E, A, R or Y; aa.

4 is F, W, Y, S, T, D, E, K or R aa is D, E, K, R, L or T aa 6 is D, E, V, I, L, K or R aa 7 is G, A, V, I, L, K or R aa 8 is K or R where x is 0 or 1, at least one x being 1.

i(mix i t.lIogu of' intureat inuludu iuquunues where A 0 0 I -8a is Y or E aa is D, E, Y or K aa 3 is N, T, A or Y aa 4 is R, S, K or E is E, D or T aa6 is D or R aa 7 is G, I or L aas is K or R For sequences having the first two amino acids Y arnd E, there may be from 0 to 4 x's that-are 0, while for chains having different amino acids as the first two amino acids there may be formr 0 to 5 x's that are 0. That is, the above chains will usually be from 3 to 8, more usually 4 to 8.amino acids.

Of particular interest is removal of the phenylalanine where the junczture will be Y-E-N-E-R-E-I-K. Other bridges include Y-E-N-R-E-D--f-K Y-K-T-R-E-D-I-K Y-E-R-E-I-K Y- E-N-I-K Y-E-I-K Y-Y-A-S-D-K-L-K Y-A-S-D-K-L Y-A-S-D-K Y-S-D-K E-D-Y-K-T-R-O--R and E-D-Y-T-R. Usually there will be at least one Y, E, K or R present in the chain, QLI~ more usually a least one of E, D, X or R. Preferred amino WO 88/10303 PCTfKR88/00010 9 acids for the bridge are Y, I, S, T, D, E, K, R, N or Q, where greater than 50% of the amino acids of the bridge will be selected from this group.

The total number of changes, insertions, deletions, and substitutions will generally not exceed a total of 12, more usually 10 amino acids, where substitutions will be counted first, followed by deletions or insertions to arrive at the total.

The subject compositions can be prepared by recombinant technology. In order to provide for expression, a gene must be provided. Sequences for subunits I and II may be obtained from the natural source as genomic DNA or cDNA.

Alternatively, a strategy may be developed for preparing single stranded sequences which may be ligated together to provide the desired double-strand. The sequences are designed to minimize heteroduplexing, so as to substantially insure that the resulting ligated double-strand DNA has the proper open reading frame. The strategy employed in the Experimental section is particularly preferred.

Once the double-stranded sequence has been designed, the various single-stranded fragments may be synthesized and WO 88/10303 PCT/KR88/00010 ligated together in accordance with conventional techniques.

The coding region may then be used to prepare an expression cassette. The expression cassette will comprise a transcriptional and translational initiation regulatory region at the 5' terminus in the direction of transcription of the open reading frame and a translational and transcriptional termination region at the 3' terminus of the open reading frame in the direction of transcription. j Today, there are many vectors which include transcriptional and translational regulatory regions of a wide variety of genes, where the initiation and termination regions are separated by a polylinker, so that an open reading frame may be inserted between the initiation and termination regions to be under their transcriptional and translational regulation.

Depending upon the particular expression host, vectors are commercially available or have been described in the literature and may be prepared from available segments having the necessary functions. For the most part, the vectors will include a replication system, which may be low or high copy number, usually having copy numbers of fewer than about 1000, although in certain situations, runaway vectors may be 2. A DNA sequence according to Claim 1, wherein said polypeptide has a sequence of subunit II as the Nterminus and subunit I as the C-terminus.

2- in WO 88/10303 PCT/KR88/00010 1 employed. Alternatively, instead of having extrachromosomal maintenance, one may provide for homology between the vector and the host genome, to enhance the opportunity for integration.

Where integration is involoved, one may provide for an amplifying gene in tandem with the expression cassette.

Amplifying genes include dihydrofolate reductase, the metallothioneins, thymidine kinase, or the like. These genes will be accompanied with an appropriate transcriptional and translational regulatory region to provide for expression in the expression host. With prokaryotes, a polycistronic message may be employed, where the amplifying gene and the sweetener gene may be under the regulation of the same transcriptional and translational regulatory regions.

Usually, the vector will include a marker which allows for selection of those host cells containing the expression cassette for expressing the subject protein. Markers may Sinclude biocide resistance, particularly from antibiotics, heavy metals, or the like; complementarity to an auxotrophic host to provide prototrophy; resistance to viral infection; etc. One or more markers may be present, particularly where one marker is used for insertion of the construct, so that aa 6 aa 7 aa 8 wherein x is K, D, E or R is G, I or L is K or R is 0 or 1, at least one x being 1.

Ifc ?s^ /3 S4 4 PCT/KR88/00010 WO 88/10303 j:- 12 loss of the particular capability will indicate the presence of the expression cassette.

Transcription intiation regions which may be employed include those associated with such genes as trp, lac, gal, his or viral promoters such as /\PR and P 4 promoters, yeast promoters such as those associated with the genes adh-1, adh-2, mat, gal, pgk, pyk, pho5, mA, gapdh, amy or dbfr, etc.

Joint promoter regions may be employed, such as the tac, adh- 2/gapdh, gal/gapdh, cye/gal transcriptional initiation regions.

See, for example, U. S. Patent Nos. 4,418,149 4,304,863 4,350,764 4,363,877 and 4,366, 246.

Specialty sequences may also be used, such as enhancers, to enhance the level of transcription. A wide variety of enhancers have been reported in the literature associated with a wide variety of genes in a range of hosts.

Another specialty sequence is a signal leader; which provides for secretion and processing of the protein. Again, a large number of signal leaders have been described in the literature and have been shown to be effective with a broad spectrum of proteins. Thus, if one signal leader is not efficient, other available signal leaders may be tried. As i Si r wo 88/10303 PCT/R88/00010 S 13 exemplary of signal sequences are U.S. Patent Nos. 4,336,336; 4,338,397 and 4,546,082.

The signal sequence, if any, will be joined to the open reading frame coding for the sweetener at its 5' terminus and will provide the methionine codon, where the open reading frame will be in proper reading phase with the methionine.

Thus, the precursor protein will include the signal sequence, the processing signal, and the protein sweetener in going from the N- to the C-terminus, where the signal sequence and processing signal will be enzymatically removed as the precursor protein is secreted. A number of processing signals are known, based on the host and the enzymatic system employed for secretion and processing whereby the signal sequence is removed.

A wide variety of hosts may be employed, both prokaryotic and eukaryotic. Common hosts which are exemplary include E. coli, B. subtilis, 8. licheniformis, S. cerevisie, K.

actis, N. crassa, Streptomyces, Aspergillis niger, and the like. Other members of each of the genera may also be employed. For the most part, microbial expression hosts will be employed, particularly procaryotic.

eployemoy, se rticuary procssic.

I WO 88/10303 PCT/KR88/00010 14 Depending upon the nature of the host, various techniques may be employed for transforming the expression host with the expression cassette, either by itself, or as part of a vector or other construct. The introduction of the expression cassette may be as a result of conjugation, transformation, transfection, transduction, fusion, etc.

Intact host cells, protoplasts, partially regenerated protoplasts, or the like may be employed for the introduction of the exogenous DNA.

Once the host has been transformed, it may then be grown in a selective medium, so as to select for those hosts having the marker or associated expression cassette.

Where antibiotic resistance is involved, the nutrient may contain a level of the antibiotic cytotoxic in the absence of the antibiotic resistance gene. In the case of auxotrophy complementation, the nutrient medium lacks the necessary metabolite. Where the product is produced and retained in the cytoplasm, after sufficient time for the cells to grow, the cells may be lysed and the desired protein obtained by conventional purification procedures.

These procedures included liquid-liquid extraction, HPLC, non-covalent bonds. The two subunits are not identical and the flavor modifying ability of monellin is dependent upon the presence of both subunits and a single mercaptan

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WO 88/10303 PCT/KR88/00010 S 15 chromatography, electrophoresis, etc. The product may then be subjected to further purification, such as gel exclusion, chromatography, etc.

The resulting product may be used in a variety of ways as a sweetener. It may be used in canned products, in conjunction with various carbonated drinks, as a powder or liquid for addition to various beverages, such as coffee, tea, or the like, in cooking, chewing gum, toothpaste, mouthwash, dental hygiene products, pharmaceuticals, meat products, e.g. ham, sausage, etc., instant soups, yogurt, desserts, cereals, animal food, etc.

The subject proteanase sweeteners may be formulated as a liquid or powder. As a liquid, other additives may be combined, such as stabilizers, buffers, bactericides, protease inhibitors, or the like. An aqueous medium will mormally be used where the sweetener will be from about 0.1 to 90 weight of the composition. For powders, various excipients may be added which are conventional food extenders.

Rather than providing the sweetener as an independent product, the expression cassette can be prepared for use Cagan, Science (1973) 181 32-35 Wlodawer and Hodgson, Proc. Natl. Acad. Sci. USA (1975) 72: 398-399 Bohak and Li, Biochimica et Biophysica Acta (1976) 427 153-170 M N WO 88/10303 PCT/KR88/00010 16 in plants. Particularly, expression cassettes can be prepared where a constitutive or regulated transcriptional initiation region functional in a plant may be employed, so that products, such as fruit, vegetables, melons, or the like may hav enhanced sweetening. A wide variety of constructs are described in the literature, demonstrating expression of a wide variety of genes in plants, using either constitutive or regulated transcriptional initiation regions. Transcriptional initiation regions include the various opine initiation regions, such as octopine, mannopine, nopaline, etc. Alternatively, plant viral transcription initiation regions may be employed, such as the cauliflower mosaic virus promoter. Other transcription initiation regions, particularly inducible regions, more particularly regions associated with cell differentiation, include the small subunit or large subunit transcriptional initiation regions of ribulose-1,3-biphosphate carboxylase, fruit specific promoters, heat shock promoters, etc.

The following examples are offered by way of illustration and not by way of limitation.

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7. T~he sweeteners are modelled after the naturally occurring sweetener monellin, where the two independent subunits of WO 88/10303 PCT/KR88/000 17 Example 1. oligonucleotide Synthesis and Purification The following oligomers were synthesized using Applied Biosystems 380B DNA Svnthesizer.

5' 3' U1 TATGGGAGAATGGAATrATCGATAFIGGACCArI'ACTCAAAAC (46mer) U2 FGGGTAAG'TCGCTGTrGACGAAGAAAACAAGATT1GGTCAATAT U3 GGAATAGAAG'T 1GCAGAGAAG(45mer) U4 AO ATAcGAAAACGAAAGAfAAGGGGTACGAATACCAA (4 Smer) U5 ITTTGIFGGIICrGACAAGC'TCAGAGCTGACArTCT U6 GAAGACTACAAGACCCGCGGTAGAAAG GGAA=ITAACGGT U7 CCAGTrCCACCACCATAATAG (2lmer) Li CGi~AA'TCCA'1CTCCCA (2lmer) L2 CGTCMACAGCGAMFIACCCAAGI1AGTGAATGTCCAATAT L3 cOTGrGAAAGTAATCTACCATATTG'ACCAAT=rG TrCF (4 Smer) L4 C CG'1ITGTAAAT7AGTCFTCTTATACATGGTCTAATAA TGTCAGAAGCGTAMACATAcAATGGTATICGTACCCCrAArI (4 Smer) L6 TAcGGGGT IGTAGTCT1'OAQATCGCTAAGT L7 TCGACTArrATGGTGGTGGAACTGACCG'ITGAATCTOAACAAU=C (48mer) The oligomers were isolated by urea-poly.acrylamnide gel electrophoresis and purified by passing through a tuted for aromatic amino acids.

WO 88/10303 PCT/KR88/00010 18 Seppak C18 column (Whatnian).

The following is the amino acid sequence encoded by gene indicating the monellin subunits, the bridge, and the ligation strategy.

(Ligation strategy) Fused Moneilin Met Gly Giu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gin Asn Leu Gly Lys Phe Ala Val Asp Giu Giu Asn Lys Ile Gly Gin Tyr Gly Arg Leu Thr Phe Asn Lys Val Ile Arg Subunit II Pro Cys Met Lys Lys Thr Ile Tyr Giu Asn Giu Arg Giu Ile Lys Gly Tyr Giu Tyr Gin Subunit I Leu Tyr Val Tyr Ala 'Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly NPro Vai Pro Pro Pro of amino acids relating to monellin as distinct from the subject constructions, the numbering of the natural monellin subunits will be employed.)

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WO 88/10303 PCT/KR88/00010 19 NdeI SalI U1 U2 U3 U4 U5 U6 U7 LI L2 L3 L4 L5 L6 L7 2. Annealing, Ligation of oligomers, and Isolation of Fused Monellin Gene Each oligomer was phosphorylated at 37 oC for 45 min.

in a reaction mixture of 30 P 1 containing 50 mM Tris-HC1, pH 8.0, 10 mM MgCl 2 10 mM DTT, 1 mM ATP, and 5 units of T4 polynucleotide kinase. Each reaction mixture was pooled, extracted by phenol/chloroform, precipitated with ethanol, and dried under Speed-Vac. The dried pellet was dissolved in 50 Y 1 distilled water and 7 JJ 1 ligation buffer (0.2 M Tris-HCl, pH 7.5, 0.1 M MgCl 2 S0.1 M DT) added. The solution was placed in a 95 °C water-bath and cooled slowly to room-temperature overnight.

To the mixture was added 7 1 of 10 mM ATP, 40 units of T4 DNA ligase (New England Biolab Inc.) and 2 P 1 of water.

The reaction mixture was kept at room temperature for of subunit II with the N-terminus of subunit I, the juncture will be at Ile(46) of subunit II and Gly(6) of subunit I with the intervening amino acids, if any, as the bridge.

min., extracted by phenol/chloroform, precipitated, dried and redissolved in 85 P 1 water. The ligated oligomer mixture was treated with restriction endonuclease NdeI and SalI (New England Biolabs, Inc.). The 290 base pair fragment was isolated by electrophoresis with a 7% polyacrylamide gel, the band electroeluted and purified using the Elutip-D column (S S Co.).

3. Molecular Cloning M13mp19RF was used for cloning the fused synthetic monellin gene. First, M13mp19RF was cut with XbaI/Sall (New England Biolabs, Inc.). The large fragment was isolated and purified as described previously. A synthetic XbaI/NdeI adaptor was synthesized.

XbaI Ndel CTAGAAACTGCAATGTTGAATAAACGCTGATTITCGATCA 3' 3' TTTGACGTrACAACTTATTTGCGACTAAAAGCTAGTAT 5 (38mer) The adaptor was purified as described above. The NdeI/ SalI digested, annealed fused synthetic monellin DNA fragment was combined with XbaI/SalI-treated M13mp19RF where xis 0or 1, at least one x being 1.

(!omjitok3Hon of' interet. hIulude uquences where L

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WO 88/10303. PCT/KR88/000 21 and XbaI/NdeI adaptor in 10 YP 1 of 20 mM Tris-HCl, pH mM MgCl2, 10 mM D LT, 200 units T4 DNA ligase E.

Biolabs, Inc.) and incubated at 4 OC overnight to provide Ml3mpl9 MCN-1RF. The transformation was done by adding 5 Y 1 of the ligation mixture to 200 JP 1 of E. coli JM101 competent cells (Messing, J. Methods in Enzymology (1983) 101 :20-78). The dideoxy DNA sequencing and Ml3mpl9 NON-iRE preparation were done as described in Messing, J. (1983) Methods in En-zymology and Sanger, T. et al. Proc. Natl. Acad. Sci. USA (1985) 74 5463-5467.

4. Construction of Expression Vector Synthetic fused monellin DNA (293 bp) was isolated from Ml3mp19 MON-1RF and purified. The vector pDR72O containing trp 0, P (Pharmacia, Inc.;Cat. #r 27-4930-01) was digested with SmaI/PvuII and blunt-end ligated to produce ptrp322. The ptrp322 was digested with H-pI/ Sall and a 2.5 kbp large fragment isolated. A synthetic H-Pl/Ndel adaptor, will be at least one Y, E D, K or R present in the chain, ~IL? more usually at least one of E, D, K or R. Preferred amino WO 88/10303 PCT/KR88/00010 22 AACTAGTACGCAAGTTcACGTAAAAAGGGTAATACA 3' (36mer) 3' TGATCATGCGTTCAAGTGCATTTCCCATTATGTAT 5' (38mer) Hpal Ndel was synthesized using Applied Biosystems DNA Synthesizer Model 380B. The ligation reaction of the 293 bp synthetic fused monellin, HPaI/SalI-treated ptrp322 vector and the HpaI/NdeI synthetic adaptor was carried out in the presence of 10 Y 1 of 20 mM Tris-HCl, pH mM MgC1 2 10 mM DTT, and 200 units of T4 DNA ligase Biolabs, Inc.) at 14 *C overnight to give ptrp322H MON-1. The transformation of E.coli W3110 (ATCC 27325) with this plasmid and screening of recombinant clones was done as described above.

5. Identification of Expression of Synthetic Fused Monellin Gene For a gene expression study, an overnight culture of 50 Y 1 of ptrp322H MON-1 in W3110 with Luria Broth was inoculated into 5 ml of M9 media containing 0.4% casamino acid, 10Y g/ml vitamin B 1 40J g/ml ampicillin and cultured at 37 °C in a temperature controlled- Experimental section is particularly preferred.

Once the double-stranded sequence has been designed, the various single-stranded fragments may be synthesized and -23 shaking incubator until OD 650 n m reached about 0.5. Then 0.1 mg of indoleacrylic acid was added to the reaction mixture to a concentration of 50 p g/ml and the mixture incubated further for about 8 hrs. The cultured cells were pelleted at 2500 rpm for 5 min. in a Beckman J6 centrifuge. Laemmli protein sample buffer was added to the cell pellet, followed by heating at 95 0 C for 5 min.

and the DNA loaded onto a 15% Laemmli SDS-polyacrylamide gel (Laemmli, Nature (1970) 227 680-685). The electrophoresis was run at 300V for 2.5 hours. The gel was stained with Coomassie blue brilliant dye demonstrating a product having the correct molecular *weight. The expressed product was isolated and shown to have a sweet taste. (Morris et al., J. Biol. Chem. 248 p584 (1978) and US Patent No. 3,998,798).

5 Following the above procedures, modified DNA sequences were prepared, where the amino acids at the juncture were varied. The following sequences indicate the sequence joining the isoleucine of subunit II (amino acid 46), (Bohak and Lee, supra, numbering) to the glycine (amino acid 6) of subunit I.

2. Y-E-N-R-E-D-I-K 8. Y-A-S-D-K-L *4 include a replication system, which may be low or high copy number, usually having copy numbers of fewer than about 1000, although in certain situations, runaway vectors may be 3. Y-K-T-R-E-D-I-K 9. Y-A-S-D-K 4. Y-E-R-E-I-K 10. Y-S-D-K Y-E-N-I-K 11. E-D-Y-K-T-R-G-R 6. Y-E-I-K 12. E-D-Y-K-T-R 7. Y-Y-A-S-D-K-L-K The codons employed were the same as the codons indicated for the MON-1 construct, with the exception of the sequence indicated as 3, where codons preferred by S.

cerevisiae glycolytic enzymes were employed. That sequence is AAG, ACT, AGA.

It is evident from the above results, that novel proteinacious sweetners based on the monellin sequence may be produced as a stable single-chain protein for use in a wide variety of ways. The product can be produced efficiently and economically by employing microbial hosts, so that a stable uniform supply of the sweetener can be obtained, as distinct from isolation from natural sources.

In addition, various changes may be made in the structure of the amino acid, without affecting its sweetening characteristic, while providing for other advantages, such as chemical and physical stabilty, storage life, ease host etc.

one to provide prototrophy; resistance to viral infection; One or more markers may be present, particularly where marker is used for insertion of the construct, so that i t i\ i:' i8; ia~ i i -i -::cti- WO 88/10303 PCT/KR88/00010 25 of formulation and purification, enhancement of sweetness, etc.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skillEi in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

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Claims (11)

1. A DNA sequence encoding a pol3peptide comprising an amino acid sequence having at least 80% of the amino acid sequence of the subunits of monellin covalently bonded together through a peptide linkage.

2. A DNA sequence according to Claim 1, wherein said polypeptide has a sequence of subunit II as the N- terminus and subunit I as the C-terminus.

3. A DNA sequence according to Claim 2, wherein in said polypeptide sequence the interventing sequence from tyr(41) to leu(3) of the monellin wild-type sequence is modified by substitution, deletion or insertion consisting of a total of not more than ten amino acids.

4. A DNA sequence according to Claim 3, wherein said intervening sequence is of the formula -LL-fT li- i cells to grow, the cells may be lysed and the desired protein obtained by conventional purification procedures. These procedures included liquid-liquid extraction, HPLC, -27 wherein: aa 1 is Y or E aa 2 is D, E, Y or K aa 3 is N, T, A or Y aa 4 is R, S, K or E aa 5 is E, D, R or T aa 6 is K, D, E or R aa 7 is G, I or L aa 8 is K or R wherein x is 0 or 1, at least one x being 1. a

5. A DNA sequence according to Claim 4, wherein said intervening sequence is Y-E-N-E-R-E-I-K; Y-E-N-R-E-D-I-K; Y-K-T-R-E-D-I-K; Y-E-R-E-I-K; Y-E-N-I-K; Y-E-I-K; Y-Y-A-S-D-K-L-K; Y-A-S-D-K-L; Y-A-S-D-K; Y-S-D-K; E-D-Y-K-T-R-G-R; or E-D-Y-T-R.

6. An expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, a DNA sequence according to any of Claims 1 to 5, wherein said DNA sequence has methionine at its 5' terminus, and a translational and transcriptional termination region, wherein said regul- g U30 o,/ food extenders. Rather than provriding the sweetener as an independent product, the expression cassette can be prepared for use 1 .A WO 88/10303 PCT/KR88/00010 28 atory regions are functional in a microbial host.

7. A mincrobial host comprising an expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, a DNA sequence according to any of Claims 1 to wherein said DNA sequence has a methionine at its terminus, and a translational and transcriptional termination region, wherein said regulatory regions are functional in a microbial host.

8. A method for producing a proteinaceous sweetener having at least about 80% homology with the two subunits of monellin, said sweetener consisting of a single chain, said method comprising growing in a suitable nutrient a microbial host comprising an expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, a DNA sequence according to any of Claims 1 to 5, wherein said DNA sequence has a methionine at its 5' terminus, L i specific promoters, heat shock promoters, etc. The following examples are offered by way of illu- stration and not by way of limitation. k i4, 29 29 and a translational and transcriptional termination region, wherein said regulatory regions are functional in a microbial host, whereby said DNA sequence is expressed to produce said proteinaceous sweetener; and isolating said proteinaceous sweetener.

9. A DNA sequence encoding a polypeptide comprising an amino acid sequence having at least 80% of the amino acid sequence of the subunits of monellin which sequence is substantially as hereinbefore described with reference to the Example. An expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, a DNA sequence according to claim 9 and a translational and S 15 transcriptional termination region, which expression cassette is substantially as hereinbefore described with i reference to the Example. S11. A method for producing a proteinaceous sweetener having at least 80% homology with the two subunits of monellin, said sweetener consisting of a single chain, which method is substantially as hereinbefore described with reference to the Example. day of September 1991 LUCKY LTD LUCKY BIOTECH CORPORATION By their Patent Attorneys GRIFFITH HACK CO -4 T1z 1 C* INTERNATIONAL SEARCH REPORT Ittnalonaf Application No PCT/KR 88/000101 1. CLASSIFICATION OF SUNJECT MATTER (if several ctaasificxtlon syrmt~olsaooly. indicate all) Accord is to hWerNatlonal Paetnt Classification (IPC) or to both liational Classfication anid iPC IPC C 12 N 15/00, C 07 H 21/04, C 12 N 1/20, C 12 P 21/00, A 23 L 1/236.

11. FIKLOS SEARCHED minimum Documentation searched Classification System IClassification Symbols Int.Cl. 4 C 12 N, C 07 H, C 12 P, A 23 L. Docuimntaion Searched other than minimum ocumentation to the Extent that such ocuments are Included In the Fielda Searched I

111. DOCUMENTS CONSIDERED TO 5E RELEVANT' Categoryz *Citation of Document, with Indication, where appropriate, of the relevant passages Rtelevant to claim No. i3 D,Y F'EBS LETTERS, Volume 29, no. 2, issued (1) January 1973 (Amsterdam), H. VAN DER WEL et al. "Characterization of the Sweet- cummtinsi (Sotapnf)o Dis,se pages181 -Tasingi Protinfro Diosoeophylslum1 184; especially page 181, introduction; page 183, tables 1,3. D,Y SCIENCE, Volume 181, no. 4094, issued() July 6, 1973 (Was 'hington R.H. CAGAN "Chemostimulatory Protein: A New Type of Taste Stimulus", see pages 32-35. Y EP, A2, 0 054 330 (UNILEVER NV), 23 June (1) 1982 (23.06.82), see abstract; fig. 1. Y WO, Al, 87/03 007 (BEATRICE COMPANIES, ING.),i (1) 21 May 1987 (21.05.87), see claims 1-12. D,A BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNIr- (1,2) CATIONS, Volume 71, no. 1, issued 1976 (New York, London), G. HUDSON et al. "Mass Spectrometric Sequencing of Proteins: The Structure! Iof Subunit I of Monellin", see pages 212-220. Special categories of cited documents: Is later document published atter the international fi'ling data document def'ining the general state of the art which is not oar Priority data and not in conflict wit" the application but cited to understand the principle or theory underlying the considered to be ot particular relevance Invention earlier docijmeint but published on or slter the international docur"enrt of particular releance; the cimed inivention filing date cannot be considered novel of cannot be considered to document which may throw doubts on priority claim(*) or involve an inventive step whicrh is cited to establish the publication data of another documeint of particulair releivance;* the claimfeid invention citation or Other special reason (s specified) cannot be considered to Involve an inventive stop when theo document refarring to an oral disclosure, use, exhibition or document IS Combined with ofe or more other such docu. other means mefits, such combination being obvious to a person skilled "Is" document publihed prior to the internatIonal filing date bu In the on. later than the priority date clairmed document member of the same pateont farmily IV. CEIRTIPICATION Date of the Actual Completion of the International Search Data of Malling of this Interitatonel Sc44rch Repor 03 August 1988 (03.08.88) 10 August 1988 (10.08.88) internatIoala Searching Authority Sig~nstre Of Authe~ii~dl Oftqc AUSTRIAN PATENT OFFI(CE4 Form PCT/ISAM (20 second sneet (isimidiy IMS) To the mixture was added 7 Y 1 of 10 MM ATP, 40 units of T4 DNA ligase (New England Biolab Inc.) and 2 JJ 1 of water. The reaction mixture was kept at room temperature for F I International Applicationl No. PCT/KR 88/00010 Ill. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) categoryj Citation of D>cumnent, with! indicatiOn, wh4V9 sporo~ate. of "m releant Passages .Relevant to Claim No D,Aj US, A, 3 998 798 (CAGAN et 21 December, (1,2) 1976 (21.12.76), see abstract; claim. Form PCT.ISA.210 (extra shooet) (Jettuay 11985) l' The adaptor was purified as described above. The Ndel/ SalI digested, annealed fused synthetic monellin DNA fragment was combined with Xbal/SalI-treated M13mp9RF C. i ;y i .i Anhang zum internatio- nalen Recherchenbericht Uber die internationale Patentanmeldung Nr. In diesem Anhang sind die Mitglieder der Patentfamilien der im obengenannten in7,rna- tionalen Recherchenbe- richt angefUhrten Patentdokumente ange- geben. Diese Angaben dienen nur azur Unterrich- tung und erfolgen ohne Gewihr. Annex to the International Search Report on Interna- tional Patent Application No. PCT/KR 88/00010 This Annex lists the patent family members relating to the patent documents cited in the above-mentioned Inter- national search report. The Austrian Patent Office is in no way liable for these par- ticulars which are merely given for the purpose of in- formation. Annexe au rapport de recherche internationale relatif i la demande de brevet international n'. La prbsente annexe indique Les membres de la famille de brevets relatifs aux docu- ments de brevets ciths dans le rapport de recherche inter- nationah vis& ci-dessus. Les renseignements fournis sont donnbs i titre indicatif et n'engagent pas la responsa- bilit& de l'Office autrichien des brevets. Im Recherchenbericht Datum der Mitglied(er) der Datum der angefUhrtes Patent- Veridfentlichung Patentfamilie Verbffentlichung dokument Publication Patent family Publication Patent document cited date member(s) date in search report Date de Membre(s) de la Date de Document de brevet cite publication famille de publication dans le rapport brevets de recherche 054 330 23/06/82 AT-E 22 327 15/10/86 054 330 22/09/82 054 330 17/09/86 BR-A -8 108 074 21/09/82 CA-Al-1 192 151 20/08/85 IE-B 52 417 28/10/87 JP-A2-58-004799 11/01/83 JP-B4-61-005392 18/02/86 JP-A2-61-043996 03/03/86 WO-A1-87/03 007 21/05/87 AU-A1-65 433/86 02/06/87 JP-T2-63-501264 19/05/88 255 823 17/02/88 US-A -3 998 798 21/12/76 None