CN101787352B

CN101787352B - Poly butyric generates the Sphingomonas mutant bacterial strains of defect and the method for clarification of sphingans and combinations thereof thing

Info

Publication number: CN101787352B
Application number: CN200910166669.7A
Authority: CN
Inventors: S·鲍威尔; E·布克; N·哈丁; Y·N·帕特尔; J·C·施耐德; D·玫斯尼尔; N·莫里森; R·比赞森
Original assignee: CP Kelco US Inc
Current assignee: CP Kelco US Inc
Priority date: 2000-03-02
Filing date: 2001-03-02
Publication date: 2016-12-14
Anticipated expiration: 2021-03-02

Abstract

The present invention relates to Sphingomonas mutant strain, wherein at least one gene of the protein that coding participation poly butyric (" PHB ") synthesizes has sudden change so that mutant strain generates the sphingans lacking PHB.The present invention is also actively working to the method for the sphingan solutions for preparing clarification, including aqueous sphingan solution (particularly lacking the sphingan solutions of PHB) is heated to clarifying temp about 30 DEG C about 70 DEG C, and with clarifier and ferment treatment solution.It addition, the present invention is also actively working to comprise food or the industrial products lacking PHB's and/or clarification sphingans.High acyl group Ji orchid sugar that one embodiment of the invention is devoted to clarification, that lack PHB and production method thereof.

Description

Poly butyric generates the Sphingomonas mutant bacterial strains of defect and clear The method of clear sphingans and combinations thereof thing

Background of invention

Invention field

The present invention relates to because of null mutation internal storage polymer, polyhydroxybutyrate (polyhydroxybutyrate, " PHB ") generate defect but the Sphingomonas of the capsular polysaccharides (sphingan) of generation normal quality (Sphingomonas) mutant bacterial strains.The invention still further relates to clarify the Sphingomonas mutant being generated defect by PHB The method of the sphingans that bacterial strain generates.The invention still further relates to comprise food or the industry lacking PHB's and/or clarification sphingans Product.

The discussion of correlation technique

Sphingans is by the capsular polysaccharides of the bacterial secretory of Sphingomonas.Sphingans is structurally relevant but not phase With.The Common members of Sphingomonas and the sphingans of generation thereof include: generate many natural ponds of lucky blue sugar (gellan, S-60) Sphingol single-cell (Sphingomonaselodea) ATCC 31461；Generate the Sphingol single-cell of welan (S-130) (Sphingomonas sp.)ATCC 31555；Generate the Sphingol single-cell ATCC 31961 of rhamsan (S-194)；Generate The Sphingol single-cell ATCC 53159 of diutan (S-657)；Generate the Sphingol single-cell of the polysaccharide (S-88) not yet named ATCC 31554；Generate the Sphingol single-cell ATCC 31853 of the polysaccharide (S-198) not yet named；Generation is not yet named many The Sphingol single-cell ATCC 21423 of sugar (S-7)；Generate the Sphingol single-cell ATCC of the polysaccharide (NW-11) not yet named 53272；The Sphingol single-cell FERM-BP2015 generating alcalan (Biopolymer B-16) (claimed Alcaligenes latus in the past (Alcaligenes latus)B-16)；Like this.Description about Sphingol single-cell and the polysaccharide of generation thereof can be joined Read U.S. Patent number 4,377,636,4,326,053,4,326,052 and 4,385,123 (about ATCC 31461 and S-60 Polysaccharide)；U.S. Patent number 4,342,866 (about ATCC 31555 and S-130)；U.S. Patent number 4,401,760 (about ATCC 31961 and S-194)；U.S. Patent number 5,175,278 (about ATCC 53159 and S-657)；U.S. Patent number 4,331,440 and 4,535,153 (about ATCC 31554 and S-88)；U.S. Patent number 4,529,797 is (about ATCC 31853 and S-198)；U.S. Patent number 3,960,832 (about ATCC 21423 and S-7)；U.S. Patent number 4,874, 044 (about ATCC 53272 and NW-11)；U.S. Patent number 5,175,279 (about FERM BP-2015 and B-16) (will They are collected herein by reference).

Sphingan polysaccharides is relevant on the primary structure of its main chain, comprises sugar D-Glucose, D-Glucose aldehydic acid and L-Mus Lee's sugar (or L-mannose).Such as, the lucky blue sugar i.e. primary structure of S-60 comprises D-Glucose, D-Portugal with the molecular proportion of 2: 1: 1 Grape alduronic acid and L-rhamnose, they connect together in the following order formed tetrose repetitive: glucose, glucuronic acid, Glucose, rhamnose.In native form, lucky blue sugar on identical glucose residue by acetyl group and glyceryl substituents Modification.On average, in lucky blue sugar, each tetrose repetitive has a glycerate substituent, each two tetrose weight Multiple unit has an acetic acid substituted base.Another kind of sphingans, the diutan i.e. primary structure of S-657 is different from Ji orchid sugar, its In an extra L-rhamnose disaccharide side chain be attached on a glucose residue, be consequently formed hexa polyose repetitive.S- 657 comprise acetyl group in the position 2 and/or 6 of another glucose residue.

Sphingan polysaccharides (also referred to as carbohydrate gum) is mainly used in making aqueous solution thickening or gel, and generally falls into two classes: thickening Agent and gellant.Typical thickening agent include starch, guar gum, carboxymethyl cellulose, alginic acid, methylcellulose, xanthan gum, Karaya and Tragacanth.Common gellant includes lucky blue sugar, gelatin, starch, alginic acid, pectin, carrageenan, fine jade Fat and methylcellulose.

Gellant is in numerous applications for food industry, including confectionery jellies, fruit jam, dessert gel, sugar-coat, dairy Product, beverage, like this.It addition, gellant can serve as the composition of microbiological culture media.Gellant is at the condition of use and gel Quality aspect is different.These distinctive features of gel cause some gellant (if starch is at confection in specific products In fruit jelly, in sugar-coat and alginic acid is in sweet green pepper bar (pimentostrips) at dessert gel, agar for gelatin) exclusive make With.

Although some gellant is used for specific products, but there is shortcoming in conventional food formulations.Such as, dessert glue it is usually used in Freeze the gelatin of formula from animal, need cooling to solidify, and apply be restricted because it is unstable after the heating.It is usually used in Dessert gel, confection and the carrageenan of fruit jam/jelly prescription, carrageenan and locust bean gum blends, pectin generally limit In fragile and inelastic formula, poor storage stability, and on using, it is subject to geography in some country (such as Japan) Limit.The starch poor transparency and the fragrance release that are usually used in confectionery formulations are poor.Therefore, it is intended that exploitation can be used for not being gelled by routine The gellant of the food formula of agent relevant issues puzzlement.

A kind of useful especially gellant is lucky blue sugar (S-60), i.e. by antibacterial many natural ponds Sphingol single-cell ATCC 31461 The capsular polysaccharide generated.Commercially, this carbohydrate gum be by under aerobic conditions with many natural ponds Sphingol single-cell inoculation fermentation Culture medium and formed.Fermentation medium comprises carbon source, phosphate, organic and inorganic nitrogen-sourced and suitable trace element.In nothing Ferment under the conditions of bacterium, strict control of aeration, agitation, temperature and pH.After having fermented, before reclaiming carbohydrate gum, by thickness Meat soup pasteurization to kill survivaling cell.But, also promote internal storage for generating the optimal conditions of fermentation of lucky blue sugar The generation of polymer, polyhydroxybutyrate (" PHB "), it hinders the final clarification of lucky blue sugar and reclaims.During the fermentation, PHB closes Become and obtainable carbon source is competed in the synthesis of Ji orchid sugar, and PHB synthesis is at war with the synthesis of Ji orchid sugar.

According to the method being carried out reclaiming by fermentation broth, different features shown by lucky blue sugar.Directly reclaimed by fermentation broth And the lucky blue sugar generated is in natural or high acyl form, it is modified by many natural ponds Sphingol single-cell, at a glucose There is on residue acetyl group and glyceryl substituents.Generate soft, soft with the lucky blue sugar that this natural or high acyl form separates Gel tough, resilient.The lucky blue sugar of low acyl form can be thus provided by hot alkali treatment by Ji blue sugar deacylated tRNA base.With The lucky blue sugar that this deacylated form separates generates hard, firm, fragile gel, which has limited its business application.Natural With the gel that deacylated tRNA Ji Jilan sugar mixture generates intermediate texture.

Some application needs limpid lucky blue sugar.But, it is merely able at present clarify deacylated tRNA Ji Jilan sugar.At deacylation process In, at the high-temperature alkali carry out lucky blue sugar, reason Ji orchid sugar removes acyl substituent and cracks many natural ponds Sphingomonas cell. Then solid and cell debris are filtered to remove, generate limpid, the lucky blue sugar of non-acylation.Also can not pass through at present The lucky blue sugar of natural or high acyl form is clarified in filter because required high solidification temperature (carbohydrate gum forms gel after the cooling period Temperature) and organic capsular nature so that can not be smooth separated lucky blue sugar by many natural ponds Sphingomonas cell.In order to Need the application of native gellan sugar, can be by chemistry or Enzymology method cracking many natural ponds Sphingomonas cell；But produce eventually Thing will exist remaining PHB, and make the solutions turbid of generation rather than limpid.

Except lucky blue sugar is as the purposes of gellant, other Sphingan polysaccharides has found useful business to apply the most.S-657 Polysaccharide gives polar solvent (such as water) with significant pseudoplastic behavior so that S-657 can be as rheology modifier such that it is able to Suspended particulates, reduction friction, stable emulsion and foam, disposal filter cake and control are filtered.Therefore, S-657 have found industry Purposes, i.e. as rheological additives for multiple bonding system, as U.S. Patent number 6, disclosed (inciting somebody to action in 110,271 It is collected herein by reference).

Except weakening transparency, the PHB found in sphingans has an effect on the rheological properties of its carbohydrate gum.Specifically, PHB in S-657 carbohydrate gum affects polysaccharide and regulates rheol ability, such as oil field, Qi Zhongliu in porous media flows system Change is learned at oilwell drilling, is completed and keep in repair in fluid to play a significant role.It addition, the PHB remnants in S-657 may be at bank Forming process causes infringement, thereby increases and it is possible to reduce the productivity of oil well.The existence of PHB also limits S-657 carbohydrate gum in family with individual Application in people's care product, wherein outward appearance for obtain consumer accreditation for it is critical that.

Accordingly, it is intended to the PHB in elimination sphingans generates.Alleviate the problem hindering PHB to generate in Sphingomonas strain A kind of method be in bacterial strain chemical induction random mutation with suppression PHB generate, such as U.S. Patent number 5, institute in 300,429 State, which disclose LPG-2, a kind of PHB of suppression generation but still be able to generate many natural ponds Sphingomonas mutant of lucky blue sugar Bacterial strain.Many natural ponds Sphingol single-cell is formerly referred to as many natural ponds pseudomonas, and refers to same organisms.LPG-2 bacterial strain is preserved in The i.e. American Type culture collecting center of American Type Culture Collection, numbering ATCC 53967.Although LPG-2 bacterial strain generates lucky blue sugar, but its quality is inconsistent, it may be possible to there occurs additional mutations during due to chemomorphosis.

Genetic engineering be for generate PHB generate defect Sphingol single-cell null mutant strains more selectively Method of mutagenesis.Genetic engineering can to PHB synthesis by way of in a kind of gene carry out selective mutation or disappearance, then can be complete Full suppression PHB generates and does not affect the quality that carbohydrate gum generates.

Therefore, it is highly desirable to exploitation synthesis PHB capability defect, simultaneously sphingans generate maximize thus can alleviate want Seek the Sphingomonas mutant strains of the effort being removed PHB by sphingans.

Summary of the invention

The present invention relates to Sphingomonas mutant strain, wherein coding participates in what poly butyric (" PHB ") synthesized At least one gene of protein is by selective mutation or disappearance so that mutant strain generates sphingans but do not generates PHB.

Another embodiment of the invention is devoted to by multiple Sphingomonas species (i.e. ATCC 31461 He 53159) DNA separates, the isolated DNA sequence of coded protein PHB synthase.

Another embodiment of the invention is devoted to the method that preparation lacks PHB, clarification sphingans, including following Step: Sphingomonas mutant strain is fermented, and the sphingans of PHB is lacked by fermentation broth clarification.

The embodiment that also has of the present invention is devoted to the method preparing the sphingan solutions of clarification, including following step Rapid: sphingan fermentation broth is heated to clarifying temp about 30 DEG C-about 70 DEG C, process sphingan fermentation meat with clarifier Soup, then with soup in ferment treatment fermentation.

The embodiment that also has of the present invention is devoted to the method preparing the sphingan solutions of clarification, including following step Rapid: sphingan fermentation broth to be heated to clarifying temp about 30 DEG C-about 70 DEG C, processes fermentation broth with chelating agen, with molten Bacterium ferment treatment fermentation broth, with causticity or oxidizer treatment fermentation broth, and uses Protease Treatment fermentation broth.

Another embodiment of the invention be devoted to prepare clarification, lack PHB, there is high-gel strength Many natural ponds Sphingomonas mutant strains of the lucky blue sugar of high acyl group (natural).

The present invention also have embodiment be devoted to comprise lack PHB's and/or clarification sphingans food or Industrial products.

The summary of figure

Fig. 1 describe from Rhizobium meliloti i.e. rhizobium melioti (U17227) (SEQID NO:1), Alcaligenes eutrophus i.e. Alcaligenes eutrophus (J05003) (SEQID NO:2), Acinetobacter sp. are the most motionless Bacillus strain RA3849 (L37761) (SEQ ID NO:3), Rhodobacter spaeroides i.e. hydrogenlike silicon ion (L17049) (SEQ ID NO:4) and Methylobacterium extorquens i.e. turn round demethylation bacillus (L07893) (SEQ ID NO:5) PHB synthase protein sequences, use LaserGene (Madison, WI) software DNA star MegAlign Compare.Select region 1 and 2 as having the conserved region of moderate degeneracy, and for offer about 400 base pairs (" bp ") Polymerase chain reaction (" PCR ") product.

Fig. 2 shows the sequence (SEQ ID NO:6) of the 408bp Insert Fragment in plasmid pEB1.

The schematic diagram of Fig. 3 is exemplified with for cloning and build internal disappearance in the Sphingol single-cell phaC gene of many natural ponds Step.

Fig. 4 describes the sequence (SEQ ID NO:7) in phaC district.The restriction enzyme sites of underscore mark PstI (CTGCAG).Arrow instruction primer binding site.A part of phaC gene is extended to TGA termination codon by first PstI site Son (runic).It is described separately in mutant the base of disappearance.XbaI site (TCTAGA, double underline) substitutes in mutant Disappearance district, as described in text.

Fig. 5 is that mutated phaC gene enters many natural ponds Sphingol single-cell chromosome by homologous recombination and excises integration load Body, is only left the schematic diagram of complete or sudden change phaC gene in chromosome.

Fig. 6 is the schematic diagram of plasmid pL02.

The schematic diagram of Fig. 7 demonstrates the vector integration comprising phaC disappearance and enters many natural ponds Sphingol single-cell chromosome.

Fig. 8 shows the cell counting by being measured by the broth samples coated plate from 10 liters of fermentations with graph mode.

Fig. 9 shows through EcoRI digestion the Sphingol single-cell base of the probe hybridization with ATCC 53159phaC gene Southern hybridization because of group DNA preparation.It (is λ HindIII and λ HindIII+ respectively that swimming lane 1 and 2 comprises size criteria EcoRI).Swimming lane 3-6 comprises the base respectively from Sphingomonas ATCC 53159,31461,31551 and 31961 Because of group DNA digest.

Figure 10 is phaC gene and the DNA sequence of flanking region of ATCC 53159 (SEQ ID NO:13).Underscore marks BaMI (ggatc), EcoRI (gaattc) and the restriction enzyme sites of NotI (gcggccgc), the overlapping primer position of double underline mark Point.Arrow instruction primer sites.Runic highlights phaC gene.

Figure 11 describe phaC district genetic map and for PCR amplification primer.

Figure 12 describes Strategies For The Cloning, wherein is used for building by PCR and only comprises phaC flanking region and remove whole phaC base The product of cause.

Figure 13 is the concentration of potassium hydroxide schematic diagram on the impact of light transmittance.

Figure 14 is the concentration of potassium hydroxide schematic diagram on the impact of gel strength.

Figure 15 is the schematic diagram of the impact of Calgon Concentration On Transmittance.

Figure 16 is the schematic diagram of the impact of Calgon concentration on gel strength.

Detailed Description Of The Invention

The present invention relates to through genetically engineered, because inactivateing what internal storage polymer, polyhydroxybutyrate (" PHB ") synthesized Null mutation and synthesize the Sphingomonas strain of the capability defect of PHB.The Sphingol single-cell of the shortage PHB of the present invention Mutant strain can synthesize the most useful sphingans, and according to turbidimetry qualitative determination well-known in the art, it does not contains PHB (embodiment that sees below 4 and U.S. Patent number 5,300,429, their content is collected herein by reference).PHB be High-carbon and accumulation in Sphingomonas cell under low nitrogen condition (identical with the condition of the sphingans generating optimum level) Storage polymer.

In many organisms, have studied PHB synthesis, and identify at least three kinds of genes of PHB synthesis (Anderson, A.J. and Dawes, E.A., Microbiol.Rev., 54:450-472,1990).PHB with three steps derived from second Acyl coenzyme A (CoA).The first step is catalyzed by 3-ketothiolase (phaA), causes the formation of acetoacetyl CoA.At second step In, acetoacetyl CoA is transformed into beta-hydroxy butyryl CoA by enzyme acetoacetyl CoA reductase (phaB).Finally, beta-hydroxy butyryl CoA is formed PHB by PHB synthase (phaC) polymerization in the third step.Coding participates in the protein of poly butyric synthesis extremely Few a kind of gene may be caused the Sphingomonas lacking PHB by the sudden change of selective mutation or disappearance.

Such as, Sphingomonas mutant strains as herein described is the result of at least two sudden change: (1) coding PHB closes The disappearance all or in part of the phaC gene of enzyme, this causes the reduction of sphingan production unexpectedly；(2) sphingan production is recovered Spontaneous mutation.Present invention also offers optional preliminary mutation, including increasing Sphingomonas mutant picked-up plasmid DNA The spontaneous mutation of ability, i.e. S-60wtc in the Sphingol single-cell of many natural ponds suddenlys change.

Chelating agen, causticity or oxidant and for cell lysis and digesting protein is used it addition, the invention discloses Enzyme clarifies lucky blue sugar and the method for other sphingans of the shortage PHB generated by Sphingomonas mutant strains.The present invention Also disclose to comprise and lack PHB's and/or clarification the food of sphingans or industrial products.

In order to illustrate the details of the present invention, describe by genetically engineered many natural ponds Sphingol single-cell and sphingol list Step involved by born of the same parents bacterium ATCC53159, but, as will be explained hereinafter, the invention is not restricted to transform many natural ponds Sphingol single-cell With Sphingol single-cell ATCC 53159, it is also not necessarily limited to any specific gene that coding participates in the protein of PHB synthesis.

Use the degenerate primer of two conserved regions design according to protein coded by phaC, obtain many natural ponds sheath by PCR The interior segments of ammonia alcohol aeromonas strain ATCC 31461phaC gene.The nucleotide sequence (being shown in Fig. 2) of this fragment is used In design inverse PCR primer such that it is able to separate greater part and the 3 ' flanking sequences of phaC gene.It is said that in general, inverse PCR Technology clones the flanking region (see Fig. 3) of purpose nucleotide with the rightabout of natural orientation.Inverted PCR fragments is arranged in them The cloning procedure of natural orientation cause the disappearance of 232 base pairs (" bp ").This fragment and the equipotential base of chromosomal phaC gene Generate because exchange eliminates the PHB in the Sphingol single-cell of many natural ponds.Internal 232bp disappearance can reduce lucky blue candy output unexpectedly.

By the large-scale culture of saltant type many natural ponds Sphingol single-cell separate that lucky blue candy output is restored spontaneous derivative Thing.The derivant of the shortage PHB of the present invention is without foreign DNA but has the 232bp from native chromosomal and lacks and non-table The spontaneous mutation levied.PDG-1 and PDG-3 bacterial strain is preserved in American Type Culture Collection i.e. U.S. typical case Culture collecting center, numbering be respectively ATCC_ _ _ _ _ and ATCC_ _ _ _ _.

For generating concrete Protocols in Molecular Biology (the i.e. inverse PCR of the Sphingomonas mutant produced for PHB And deletion mutation) be not critical to.Use conventional molecular biological technology to generate Sphingomonas mutant to belong to Within the ken of those of ordinary skill in the art.Can be used for suddenling change phaC sample gene in different Sphingomonas Other useful Protocols in Molecular Biology include but not limited to transposon mutagenesis, point mutation and insertion element sudden change.

PhaC gene be PHB synthesis by way of in a gene；Thus possibly through selective mutation or disappearance ginseng With PHB synthesis by way of other gene generate there is the Sphingomonas mutant of expectation phenotype (i.e. PHB generates defect). PhaA (3-ketothiolase) and phaB (acetyl can be included but not limited to so that the purpose base generating expectation phenotype is tired with selective mutation Acetyl-CoA reductase).

Once generate Sphingomonas mutant, make them grow in the aqueous solution of referred to as fermentation broth or ferment, And in fermentation broth, secrete sphingans with capsular polysaccharide form.It is fermented at the Sphingomonas mutant making shortage PHB After, it is possible to use technology well-known in the art precipitates sphingans by meat soup pasteurization and with alcohol (such as isopropanol), by This prepares sphingans.

Preferably, after fermentation, can by sphingans clarification and with suspended solid and cell debris (fermentation broth ring The part in border) separate to generate sphingans that lack PHB, that clarify.It addition, the defecation method of the present invention can be applicable to above Any sphingans bacterial strain beyond the sphingans of described shortage PHB.As described herein, defecation method includes fermented meat Tonga Heat, and process fermentation broth with one or more chelating agen, one or more causticities or oxidant or its mixture, use subsequently Any lysozyme and/or any protease process.

For lucky blue sugar, PHB is generated many natural ponds Sphingomonas mutant of defect and the defecation method of the present invention Associating such that it is able to generate the clarification Ji orchid sugar of high acyl form.Height shown by the lucky blue sugar generated by this mutant and method Transparency and high-gel strength, this for make dessert gel, confection, beverage, like this be useful.

In one embodiment of the invention (hereinafter referred to as " first scheme "), can be by the side comprised the following steps Method carrys out clarification of sphingans aqueous solution: with one or more optional surfactants, one or more chelating agen, one or more Causticity or oxidant or its mixture process sphingan solutions, then with at any lysozyme and/or any protease Reason.

In another embodiment of the present invention (hereinafter referred to as " alternative plan "), can be by comprising the following steps Method carrys out clarification of sphingans aqueous solution: processes sphingan solutions with one or more chelating agen, uses any lysozyme subsequently, subsequently It is one or more causticities or oxidant, is followed by any protease or proteinase mixture processes.

In first scheme, the method that can in a step-wise fashion carry out the present invention, first live with chelating agen, optional surface Property agent, causticity or oxidant or its mixture process sphingan solutions, then enter with any lysozyme and/or any protease Row processes.In alternative plan, Step wise procedure can be carried out, first process sphingan solutions with chelating agen, followed by appoint What lysozyme, followed by causticity or oxidant, the most any protease (with this order) processes.

Advantageously, the method for the sphingan solutions for generating clarification described herein provide can (the need to) Suitably use after dilution, molten without any sphingans the most chemically or mechanically processing (except pasteurization and precipitation) Liquid.Applying for some, the sphingan broth separation sphingans can clarified by these according to routine techniques, including by meat soup bar Family name's sterilizing, is adjusted to meat soup expect pH, and precipitates sphingans with alcohol (i.e. isopropanol).

This sphingans rehydration in water and dissolving provide the limpidest sphingan solutions.According to this Bright, the limpidest sphingan solutions (1%w/w) have exceed about 60%, preferably greater than 70%, most preferably more than 80% Light transmittance.Routine techniques and equipment (such as the spectrophotometer of commercialization) can be used to survey at any wavelength of visible spectrum Amount light transmittance.Generally measure the light transmittance at wavelength about 600nm-about 650nm.The sphingans that can measure several types is molten The light transmittance of liquid: meat soup that untreated meat soup, part process (as only with chelating agen, causticity or oxidant, chelating/causticity or The meat soup that chelating/oxidant mixture processes, or only with lysozyme and/or the meat soup of Protease Treatment), process after meat Soup or the sphingan solutions rebuild.The limpidest solution (having the light transmittance exceeding about 60%) described herein is bag Aqueous solution containing about 1% (mass ratio) sphingans, described sphingans is to be divided by the meat soup after processing according to the method for the present invention From.

The sphingan solutions that the method for the present invention can be used to clarify includes that the whole fermentation broth comprising sphingans is (described Sphingans is the microorganism by fermenting and producing sphingans in Nutrient medium and obtains), by hydrotropism's medium adds The solution entering the sphingans separated and obtain and partially purified sphingan solutions.Comprise unexpected fermentation solid, can be used for The aqueous sphingan solution of the inventive method can comprise the about 0.01%-about 10% sphingans (matter relative to solution gross weight Amount ratio).Any aqueous solution comprising any known sphingans can be used in the practice of the present invention.

The first step of two kinds of defecation methods of the present invention includes, by routine techniques, sphingan solutions is heated to clarifying temp, Temperature in such as jacket canister controls, open steam is injected, like this.Preferably open steam is injected, so that contracting heat time heating time To the shortest.The scope of clarifying temp is about 30 DEG C-about 70 DEG C, preferably approximately 50 DEG C-about 60 DEG C.Sphingan solutions is added Heat to preferred temperature required time significantly can change along with the size of pending sphingan solutions and volume.Such as, by little Volume (such as 50ml) sphingan solutions is increased to about 60 DEG C by room temperature may have only to a few minutes, and by 40,000 liter of solution (there may be as in batch processing) raises similar temperature may need several hours.

One of two schemes according to the present invention, next step of the inventive method include with selected from least one chelating agen, The clarifier of at least one causticity or oxidant or its mixture processes aqueous sphingan solution.Or, can as mentioned above will Sphingan broth adds clarifier while being heated to clarifying temp.

In first scheme, next step is to add chelating agen in sphingan solutions when there is causticity or oxidant.Logical Often, the scope of the time of contact of chelating agen and causticity/oxidant be every kind about 0.5 hour-about 2 hours, preferred sequestrant About 1 hour, causticity or oxidant about 0.5 hour-about 1.0 hours.Generally, in sphingan solutions, causticity or oxygen are added The concentration range of agent is about 0g/L-about 2g/L, preferably approximately 0.5g/L-about 1.5g/L.Generally, to sphingan solutions The concentration range of middle addition chelating agen is that about 1/0000000th (" ppm ")-about 3000ppm, preferably approximately 1000ppm-is big About 2000ppm.

After the clarifier of first scheme processes, sphingan broth is carried out ferment treatment step, the most separately or simultaneously Lysozyme and/or protease is added in sphingan solutions.Generally, enzyme contact sphingan broth time range be every kind at least About 0.5 hour-8 hours, preferably every kind at least 1 hour, most preferably every kind at least 2 hours.Generally, the concentration range of lysozyme It is about 11,000MCG unit/L-44,000MCG unit/L, preferably approximately 20,000MCG unit/L-25,000MCG unit/ L；Generally, the concentration range of protease is about 65,000Delft unit/L-260,000Delft unit/L, preferably approximately 100,000Delft unit/L-150,000Delft unit/L.When for the application, " MCG unit " refers to pH6.6 and when 37 DEG C Micrococcus lysodeikticus (Micrococcus lysodeikticus) relative to the heating rate of reference standard (as Genencor Described in International Inc.)；Similar, " Delft unit " reference and sample are molten provided by seller Genencor The concrete algoscopy of the extinction ratio in liquid.

For the enzyme of ferment treatment step, solid cellular debris is degraded into soluble compound, thus improves sphingan solutions Light transmittance and contribute to defecation method.The protease being applicable to this method can be from antibacterial, fungus or plant origin Acidity, neutrality or alkaline protease.The Exemplary acid protease enzymes that can be used for the inventive method includes but not limited to by aspergillosis The protease that microorganism belonging to genus (such as aspergillus niger) generates.The neutral protease that can be used for the inventive method includes but not limited to by bud The protease that spore bacillus spp. microorganism such as bacillus amyloliquefaciens generates.The alkaline protease that can be used for the inventive method includes But it is not limited to by the generation of bacillus micro-organism such as bacillus subtilis, Bacillus licheniformis and Bacillus pumilus Protease, Streptomyces spec such as streptomyces fradiae, streptomyces griseus and raw silk rings streptomycete the protease generated, by withered The protease that grass Bacillus protease such as subtilisin Novo, subtilisin Carlsberg obtain, including egg White enzyme such as subtilopeptidase A and bacillus subtilis peptidase B.The lysozyme being applicable to the method includes purchased from Genencor The Multifect of International Inc. (Rochester, New York)Lysozyme or can be by plant, animal or micro-life Thing derives any lysozyme that source obtains.The source of any protease or lysozyme that can be used for the present invention is not most important 's.These enzymes and obtain their method and be well known in the art.

As described in first scheme above, can simultaneously or separately add ferment treatment and (carry out with lysozyme and/or protease Process) included by enzyme.Process simultaneously and refer in sphingan solutions, add protease and lysozyme with random order and spend Random time, condition is to there is both enzymes in processing procedure in sphingan solutions.When added at the same time, at lysozyme and albumen Enzyme is the most active and carries out the method for enzymatically treating of the present invention under conditions of providing expectation enzyme function.Can temperature about 30 DEG C- About 70 DEG C, pH about 5-about 9 (preferably approximately 6-about 8) carry out method of enzymatically treating while this embodiment.Although The actual temp of this embodiment and pH scope can change along with enzyme used, but embodiment this while In, the method carrying out the present invention under conditions of relatively mild temperature and weakly acidic pH so that lysozyme and protease (acid, Neutral or alkaline) all will show that the activity of acceptable level is with clarified sphingan solution.

Preferably, it is carried out as follows ferment treatment, in sphingan solutions, is i.e. added separately to any lysozyme and/or albumen Enzyme.Most preferably, (it is acid big to neutral pH range i.e. pH scope about 3-for lysozyme in its Optimal pH condition respectively About 7.5, be neutral to alkaline pH range i.e. pH scope about 6.5-about 9 for protease) under add in sphigan solution Each enzyme.Different lysozyme show that with protease the temperature of optimal clarification activity may be different with pH scope.If it is it addition, necessary Making a choice between the lysozyme or protease of ferment treatment, the most preferably ferment treatment includes one or more albumen Enzyme.

In alternative plan, be the ferment treatment of any lysozyme after chelation step, be followed by one or more causticities or The process of oxidant, is followed by the ferment treatment of any protease.As it appears from the above, ferment treatment can be at lysozyme and protease Reason.This variable order makes any lysozyme can play a role to condition of acidic pH in its preferably neutrality, and makes Any protease can play a role to alkaline pH in its preferably neutrality.Can be by the phase described in first scheme above With lysozyme and protease, and chelating agen, surfactant and causticity or oxidant are used for putting into practice alternative plan.

Need not stir sphingan solutions, certainly, at one's leisure can be gentle or the agitation sphingan solutions of interval, to avoid Solid sedimentation and the contact of promotion enzyme.

Be applicable to the chelating agen of the inventive method be can by with polyvalent metal ion formed multidentate complex and Metal ion forms precipitation or adsorbing metal ions and chelates the polyvalent metal ion in sphingan solutions (such as Mg²⁺、Ca²⁺、 Deng) compound or compositions.Preferably, chelating agen is soluble in water or the compound of water-alcohol or compositions, and is to have Machine and/or the alkali metal of mineral acid or alkali salt or the organic/inorganic hydrochlorate of alkalescence (containing amine) organic compound, and Organic and/or mineral acid or alkali compounds self.Other chelating agen that can be used for the inventive method is cation exchange resin With carbon acid plus carbonate.The most useful especially salt compound and compositions include ethylenediaminetetraacetic acid, phosphoric acid, Metaphosphoric acid, carbonic acid, citric acid, tartaric acid, gluconic acid, glutamic acid, pyrophosphoric acid, polyphosphoric acid, Metaphosphoric acid, glucosaccharic acid, second two Alcohol two (beta-amino ether)-N, N, N ', N '-tetraacethyl (EGTA), ethylenediamine, 2,3-diaminobutane, 1,2-diaminourea hexamethylene Alkane, triamido triethylamine, salt like this.Suitable, useful salt can include the mono-, di-of above-mentioned acid, three and/or four Slaine and the mono-, di-of above-mentioned alkali or trisalt.Preferably, the chelating agen for the inventive method includes ethylenediamine tetrem The salt of acid, citric acid, phosphoric acid, pyrophosphoric acid, polyphosphoric acid, carbonic acid, pyrophosphoric acid and ethylenediamine.The example of useful chelating agen include but It is not limited to disodiumedetate, ethylenediamine tetraacetic acid,dipotassium salt, tetrasodium ethylenediamine tetraacetate, ethylenediaminetetraacetic acid four potassium, lemon Lemon acid trisodium, potassium citrate, sodium hexameta phosphate, hexa metaphosphoric acid potassium, sodium polyphosphate, potassium polyphposphate, sodium pyrophosphate, pyrophosphoric acid Potassium, mono phosphoric acid ester sodium, mono potasssium, disodium hydrogen phosphate, dikalium phosphate, tertiary sodium phosphate, tripotassium phosphate, sodium bicarbonate, sodium carbonate, carbonic acid Hydrogen potassium, potassium carbonate, cation exchange resin, dihydrochloride, diacetate, lithium salt, ethylenediamine dihydro Iodic acid, like this.It is further preferred that sodium hexameta phosphate is used as chelating agen.

As described in embodiments hereinbefore, surfactant optional associating with causticity, oxidation and chelating agen makes With, thus improve the light transmittance of final lucky blue sugar product further.Being applicable to the surfactant of the inventive method is can be There is hydrophilic and that form aqueous emulsion during hydrophobic substance (solid or liquid) compound or compositions.Preferably, live in surface Property agent water soluble or the compound of water-alcohol or compositions.The example of useful surfactant includes but not limited to SDS, polyoxy Ethylene sorbitan monooleate (Tween 80, ICI Americas Inc., Bridgewater, NJ), lecithin, single the sweetest Grease, tartaric esters of monoglycerides, phosphated monoglyceride (such as single sodium salt), lactylated monoglyceride, acetylated monoglyceride, amber Amber Acylated monoglyceride, ethoxylated monoglycerides, sorbitan ester, polysorbate (polysorbate), polyglycereol Ester, sucrose ester, sodium stearoyl lactate, propylene glycol ester, like this.

Any time during chelating agen, causticity or oxidizer treatment adds optional surface in sphingan broth Activating agent, the scope of time of contact be every kind about 0.5 hour-about 8 hours, preferably approximately 2 hours.Generally, to sphingans The concentration range adding surfactant in solution is about 0.0g/L-about 3.0g/L, and preferably approximately 0.1g/L-is about 1.0g/L.Generally, adding the concentration range of surfactant in sphingan solutions is about 1/0000000th (" ppm ")-big About 3000ppm, preferably approximately 300ppm-about 1000ppm.

Be applicable to the escharotic of the inventive method include but not limited to potassium hydroxide, sodium hydroxide, tertiary sodium phosphate, all so Class.Potassium hydroxide is preferred escharotic.Or, it is possible to use oxidant replaces escharotic.Can be used for the present invention side of clarification The oxidant of method includes sodium hypochlorite or other hypochlorite, chlorine dioxide (chloride dioxide), hydrogen peroxide, crosses second Acid, ozone and other oxidant well-known in the art.In the present invention, it is preferred to oxidant be sodium hypochlorite.

It should be noted that, it is molten by processing sphingans with chelating agen, surfactant, causticity or oxidant or its mixture Liquid and the clarification degree that realizes may affect the enzyme concentration required for follow-up ferment treatment or time.Such as, increase for this The amount of kind of the chelating agen of method, surfactant, causticity or oxidant or its mixture can reduce that to realize clarification of sphingans molten Enzyme amount required for liquid and/or time.In order to obtain sphingan solutions, preferably adjust and balance chelating agen, surfactant, severe Property or oxidant or the concentration of its mixture and the time of process and/or lysozyme and/or the concentration of protease and the time of process, To optimize the production of shortage PHB described herein, clarification sphingans.

Described herein lack PHB's and/or clarification sphingans can be used for numerous food or commercial Application.Such as, lack PHB's and/or clarification the lucky blue sugar of sphingans the most natural (high acyl group) can be used for improving food product such as dessert gel, sugar Really, fruit jam and fruit jelly, beverage, thin film, sugar-coat, taste like this, quality, stability and outward appearance.As extra model Example, lack PHB's and/or clarification sphingans such as S-657 can more effectively as rheology modifier for industry answer With such as oil drilling or bonding system.The present invention other lack PHB's and/or clarification sphingans also will be at food product The larger range of application with in industry.

The following example provides the illustration of the present invention, and should not be misinterpreted as limiting by any way the model of the present invention Enclose.

Embodiment

Embodiment 1: generate many natural ponds Sphingol single-cell phaC fragment

In order to identify the high conservative region of PHB synthase gene, by National Center forBiotechnology The gene bank retrieval of Information i.e. NCBI (" NCBI ") is from the PHB synthase of different organisms Sequence.Select and study from rhizobium melioti (gb:U17227) (SEQ ID NO:1), hydrogenlike silicon ion (gb:L17049) (SEQ ID NO:4), torsion demethylation bacillus (gb:07893) (SEQ ID NO:5), true oxygen Alcaligenes (gb:J05003) (SEQ ID NO:2) and the sequence of acinetobacter calcoaceticus bacterial strain RA3849 (gb:L37761) (SEQ ID NO:3).PHB synthase base is selected in comparison The protein sequence of cause, as shown in Figure 1.In conserved region, according to its position and relatively low degeneracy, select region 1 (rhizobium melioti 411-417 bit codon) and region 2 (rhizobium melioti 535-541 bit codon) provide about The PCR primer of 408bp.

According to conserved protein sequences and the apparent codon-bias of many natural ponds Sphingol single-cell ATCC 31461, design letter And PCR primer carrys out the sequence between amplification region 1 and region 2.Codon-bias is by from many natural ponds Sphingol single-cell ATCC Exo polysaccharides (exopolysaccharide) the biosynthetic enzyme coding region of 31461 (is checked order by Luis doctor Ielpi, does not sends out Table) five kinds of genes and from closely-related Sphingol single-cell ATCC 31554 (it generates S-88 carbohydrate gum) (Yamazaki Et al., J.Bacteriol., 178:2676-2687,1996) the codon of complete exopolysaccharide biosynthetic enzyme genes bunch use Rate is inferred.

The N end primer being referred to as PHADG5 (SEQ ID NO:9) comprises 5 '-AGTT clip districts, TCTAGAXbaI site and target To TTC GAY CTS CTS TAY TGG AAY 3 ' the degeneracy hybridization region in region 1.It is referred to as the C of PHADG7 (SEQ ID NO:10) End primer comprises the CCA III SGG CCA CCA GCT GCC of 5 '-GTAT clips, ACTAGTSpeI site and target area 2 Degenerate region.In SEQ ID NO:10, " I " refers to trophicardyl, a kind of nucleoside compatible with other base any (i.e. A, C, T or G) Acid.

By primer PHADG 5 (SEQ ID NO:9) and PHADG7 (SEQ ID NO:10) for from non-stick liquid sample bacterium The chromosomal DNA of strain Gps31 is the PCR reaction of template.Gps31 is the S-60 mutant not generating lucky blue sugar.Taq polymerase with Taq StartAntibody (ClontechLaboratories Inc., Palo Alto, CA) is that PCR provides thermal starting, 100 μ l reaction system comprises 2.5mM every kind dNTP, 4mM MgCl₂, and 50pmol every kind primer.Temperature program(me) is: 96 DEG C of 5min； 96 DEG C of 1min of 30 circulations, 58 DEG C of 1min, 72 DEG C of 1min；72 DEG C of 5min again, then by be cooled to 4 DEG C terminate reaction. PCR reaction causes the single band being positioned at expection 416bp size (408bp+ clip).After XbaI and SpeI digestion, by fragment gram Grand in the pUC19 carrier that XbaI digestion, calf intestine alkaline phosphatase (" CIAP ") process, to generate plasmid pEB1.From Article two, the DNA sequence (SEQID NO:6) of the 408bp Insert Fragment of chain is shown in Fig. 2.This fragment comprise EcoRI, KpnI and The restriction site of PvuII.Cloned sequence after translation proves to have cloned PHB with the comparison of other PHB synthase protein Synthase.

Embodiment 2: build phaC by inverse PCR and lack

It is used for Southern hybridization determining and the suitable limit of Sphingol single-cell S-60phaC gene larger piece section will be provided Enzyme processed, its biggest must being prone to is reclaimed by inverse PCR.According to QIAGEN(Valencia, California) DNA purification tries The method that agent box describes, is separated chromosomal DNA by Gps31.Use by 408bp Insert Fragment (the SEQ ID being cloned in pEB1 NO:6) the Southern analytical proof that the probe generated is carried out, in the PstI digest of many natural ponds Sphingol single-cell DNA, 408bp phaC fragment (SEQ ID NO:6) is positioned in the fragment of about 2kb.

By the sequence (SEQ ID NO:6) of 408bp Sphingol single-cell S-60phaC fragment for selecting outside reading code PCR primer, as shown in Figure 2.Primer PHAC12 (SEQ ID NO:11) reads the N-terminal to protein coded by phaC, has Clip 5 '-GTTC, XbaI site TCTAGA and hybridization region GGC GCG ATC AGC TTG TTG TC 3 '.Primer PHAC11 (SEQID NO:12) reads to the C-terminal of protein coded by phaC, has clip 5 '-GTTC, X baI site TCTAGA and miscellaneous Hand over district GAG TCG CTC GAA TCC TTT GTC 3 '.With PstI digestion many natural ponds Sphingol single-cell chromosomal DNA, and 200 μ l volumes connect 0.5 μ g DNA to be cyclized.The KpnI digest of linear DNA molecule is generated as template using being used for React for inverse PCR, to generate the 1.7kb fragment of 408bp phaC fragment (SEQ ID NO:6) flanking region, as Fig. 3 institute Show.

1.7kb fragment is included in their the PstI end two flanks with the contrary Opposite direction connection relative to natural orientation District.The cutting instruction flanking region of PstI site has similar size, for 850bp and 980bp.In order to fragment being changeed back natural taking To and simultaneously generate lack initial 408bp clone most fragment, with XbaI digest 1.7kb fragment, under diluting condition Self connects to be cyclized, and then digests with PstI.The fragment of generation is cloned into and processes through PstI digestion, CIAP In pUC19, referred to as pEB4.

The order-checking of embodiment 3:phaC clone

1.7kb Insert Fragment in pEB4 is checked order, and combines the sequence of 408bp fragment (SEQ ID NO:6).Merge 1920bp DNA sequence (SEQ ID NO:7) is described in Fig. 4.A part for this sequence, i.e. by PstI site to TGA termination codon Son (1-1200 bit base), coding and the protein (SEQ ID NO:8) of other phaC gene c-terminus 2/3 homology.Sequence Comparison confirms to have cloned correct gene.

This phaC be cloned in 408bp fragment have 232bp disappearance and corresponding to XbaI site 6bp i.e. TCTAGA insert Enter.This disappearance and insertion cause the frameshift mutation changing carboxyl terminal, and at the 1102nd bit base, place are imported new end Only codon.

Embodiment 4: build integration vector and be transferred to Sphingol single-cell by homologous recombination

For phaC deletion mutation being transferred in the Sphingol single-cell of many natural ponds, using can be in the place being applicable to plasmid construction " suicide " carrier replicated in main (such as escherichia coli) but can not replicate in Sphingol single-cell.In Sphingol single-cell Select by the antibiotic resistance of plasmid surface, identify the bacterium that those plasmids are incorporated in chromosome by homologous recombination Fall.As shown in Figure 5, the selection lost antibiotic resistance identifies those duplicate blocks and is recombinated and may cause sudden change The bacterium colony of the reservation of (i.e. disappearance) or wild type gene.Can pass through phenotypic expression (PHB synthesis) measure have disappearance gram Difference that is grand and that have between the clone of wild type DNA.In order to measure the phenotypic expression of PHB, use qualitative turbidimetry: by some Meat soup (about 1ml) is added to the Clorox of 9 times of volumesIn (Clorox company, Oakland, CA), and it is incubated at least 4 in 37 DEG C Hour or overnight.There is PHB in the appearance instruction of white precipitate.

For the ease of detection the second exchange recombination event, positive selection system amendment is used for many natural ponds Sphingol single-cell. Bacillus subtilis genes sacB (encoding levan sucrase) can be transferred to (Kamoun, S. in gram negative bacteria Et al., Mol.Microbiol., 6:809-816,1985).In sucrose, cultivate these antibacterials can promote the synthesis of levan, And levan is poisonous to antibacterial.Therefore, if there is sacB gene on carrier, then sucrose can be cultivated and be used for identifying those Lose the separation bacterium of carrier.

By obtaining pLO2 plasmid at the Steven Slater of Cereon, Monsanto.As shown in Figure 6, pLO2 plasmid exists Have kalamycin resistance, ColE1 origin of replication and RP4 transfer starting point carrier on comprise sacB gene (Lenz, O. et al., J.Bacteriol., 176:4385-4393,1994).PLO2 plasmid can be used for carrying out metastatic gene by natural engaging process.Should Plasmid can replicate in escherichia coli, but can not replicate in Sphingol single-cell；And comprise for driving plasmid to shift Site, but be free from forwarding function.I.e. pLO2 plasmid can drive, but can not self transmission.The second plasmid carries For the gene for engaging forwarding function, and play a role with trans.Although this embodiment uses pLO2 plasmid, but it make With being not critical to.Those of ordinary skill in the art will recognize that and how to design and transform suitably other plasmid, and use It is transferred in Sphingol single-cell by routine techniques (such as electroporation, conversion, like this).Similar, that is mould to use card Element is not critical to as selected marker.Those of ordinary skill in the art will recognize that and how to select other suitable selection mark Note.

The 1.7kb PstI fragment comprising phaC disappearance is connected in the pLO2 of PstI digestion, referred to as pLO2-phaC Or pEB11, and by using the conversion of electroporation to transfer to escherichia coli YMC9 (F-Δ lacU169 thi endA hsdR) In.Purification coli strain, and with many natural ponds Sphingomonas S-60wtc and the large intestine bar that carries plasmid pRK2013 Bacteria strain JZ279 mix, latter provide engage transfer function (Ditta et al., Proc.Natl.Acad.Sci.USA, 77: 7347-7351,1980).S-60wtc is the derivant of strain S. elodea ATCC 31461, and it is as picked-up Spontaneous separation bacterium that the ability of plasmid DNA increases and select.Stable phase (overnight) culture is used to carry out engaging transfer, i.e. 1mlYMC9/pLO2-phaC_Δ, 1ml JZ279/pRK2013 and 2-3ml many natural ponds Sphingol single-cell.Mixing culture is also being filtered Concentrate on device, be then placed on TYE culture dish (8g/L tryptone, 5g/L yeast extract and 5g/L sodium chloride), and in 37 DEG C insulation 7 hours.Then cell is suspended from deionized water to be placed on Selective agar medium.After being incubated about 7 hours, adding 25 μ g/ml streptomycin (selecting escherichia coli in order to counter) and the YM culture medium (3g/ of 7.5 μ g/ml kanamycin (in order to select plasmid) L yeast extract, 3g/L Fructus Hordei Germinatus extract, 5g/L peptone and 10g/L glucose) above select the kanamycin of S-60wtc to resist Property transconjugant.Weighing according to kalamycin resistance, the occurrence frequency of integration is 1.5 × 10^-6。

Embodiment 5: select the second exchange deletion mycopremna

Two kalamycin resistance intergrants of purification, and non-selective YEME culture medium (0.25% yeast extract, 0.025% Fructus Hordei Germinatus extract) in pass on three times, be then coated on 7.5% sucrose to select permutoid.Obtain seven and block that Mycin sensitivity permutoid, but be all that PHB is positive.It is used for confirming the phaC that carrier phaC inserts chromosome by PCR test District and determine the Insert Fragment position relative to wild-type phaC genes.Devise and lack flanking region and carrier end homology Primer.As shown in Figure 7, restructuring can two kinds of orientations be carried out, and causes respectively: (1) has disappearance on the left side of carrier, the right is The phaC gene of phaC genetic fragment, this will generate PHB negative clone；Or the left side of (2) carrier is complete phaC gene, the right side While be phaC, this will generate PHB positive colony.

It is all the second, possible preference, the PHB positive that six pLO2phaC single exchange intergrants are tested proof Orientation.Possible strong preference is recombinated in the side of disappearance, or PHB positive strain grows better than PHB feminine gender recombinant. Plasmid with less preferably mode i.e. the first, orientation negative for the PHB bacterium colony that carries out integrating the most more likely enters in preferred sites Row the second recombination event, causes the dual exchange retaining mutant phenotype.

Screen transconjugant by PCR, and test PHB expression, be in the first or the conversion of PHB feminine gender orientation to identify Body.In 24 bacterium colonies of test, it is PHB positive integration body that PCR result proves 21, and 3 is PHB negative integrants.PHB surveys Examination confirms these results.Select this 3 PHB negative strains (3,15 and 22), purification, under non-selective conditions, cultivate three Generation, and be coated on sucrose.In five kanamycin sensitivity bacterium colonies of each parent, one is that PHB is negative.By This, mutant that isolated three lacks PHB, that kanamycin is sensitive, referred to as NPG-1, NPG-2 and NPG-3.

Embodiment 6: characterize the lucky blue sugar biosynthesis of mutant

Test NPG-1, NPG-2 and NPG-3 by carrying out 10L fermentation in 14L fermentation tank, and compare with LPG-2 Relatively, LPG-2 is the shortage PHB type mutant (U.S. Patent number 5,300,429) by chemomorphosis isolated.Fermentation stage With used medium and U.S. Patent number 5, similar described in 300,429, simply second stage culture medium is used for all seed rank Section.Three seed stages were used before inoculating final culture medium.Transfer volume is 2.5-5%.Use different organic nitrogens (Quest Nzamine EKC, Chicago, Illinois, 0.41g/L) replaces promosoy (0.5g/L).In seed stage Corn syrup level be 3% rather than 3.75%.Final 10L fermentation is similar to seed culture medium, but comprises less phosphate (0.5g/LK₂HPO₄), and control pH as desired by adding KOH.Organic nitrogen (1.1g/L) and inorganic nitrogen NaNO₃ (1.5g/L) the highest.Add defoamer H-60-K to 0.6ml/L.Corn syrup level is 3.85%.Prepare in deionized water The culture medium of terminal stage, and add calcium and magnesium.

As shown in Table 1, according to total precipitate (" TPM ") and viscosity, the lucky blue sugar that NPG mutant generates considerably less than LPG-2.Broth viscosity measures with 60rpm with No. 4 axles in Brookfield viscometer.Total precipitate is by by meat soup In pressure cooker, heat 10 minutes, then measure with the isopropanol precipitating of 2 times of volumes being dried.These results have and can weigh Renaturation.The analysis instruction of broth samples in sweat is generated substantial amounts of organic acid.Therefore, the lucky blue sugar of NPG mutant Low yield becomes two carbon and three carbon intermediums and carbon dioxide relevant with the substantial amounts of carbohydrate inversion when lacking PHB synthesis.

Embodiment 7: separate the mutant having recovered the lucky blue sugar productivity

The metabolic intermediate (such as organic acid) accumulated because of the blocking-up of PHB synthesis may have unfavorable shadow to lucky blue sugar synthesis Ring.Estimate in the incubation in the culture medium promoting lucky blue sugar synthesis, it is possible to anaphragmic occurs so that lucky blue sugar synthesis Reach normal level.Be coated with from 10L fermentation (embodiment 6) some fermentation broths with measure cell counting (Fig. 8).Observe Arriving, at the end of fermentation is soon (i.e. 44 and 69 hours), the bacterium colony of about 0.5%-2% is bigger and more tacky than NPG.Purification these Bacterium colony, and in shake flask fermentation, test the generation of PHB and Ji Lan sugar.The new bacterium that separates is a lack of PHB, and lucky blue candy output is higher than Initial NPG mutant.The total precipitable thing of the optimal compensation mutant is comparable with LPG-2, and the 80% of >=wild type (is estimated TPM reduces about 10-15%, it may be possible to caused by the forfeiture of PHB mass).These bacterial strains are referred to as PDG mutant: PDG-1 Derived from NPG-1, and PDG-3 is derived from NPG-3.PHB and the Ji Lan sugar evaluating every kind of bacterial strain in shake flask fermentation generates.

Table 2, experiment 1: lack the shake flask fermentation of PHB bacterial strain

Bacterial strain	TPM(g/100ml)	%S60	%LPG2	PHB
					S60-wtc	1.52			+
LPG-2	1.42	93		-
					NPG-1	0.72	47	50	-
PDG-1	1.44	94	102	-
					NPG-3	0.51	34	36	-
PDG-3	1.37	90	96	-

It addition, use Brookfield viscometer with No. 4 axles with 60rpm mensuration second batch S60-wtc, LPG-2, PDG-1, Broth viscosities with PDG-3.Table 3 below shows broth viscosities.

Similar to shown in U.S. Patent number 5,300,429 for the culture medium of shaking flask.The first attached bag culture medium Han YM.The Two and terminal stage be each 500ml shaking flask equipped with the previously described culture medium of 100ml, the phosphate being only intended to buffering is higher (K₂HPO₄It is 2.8g/L；KH₂PO₄1.2g/L) and organic nitrogen be 1.0g/L.

Being not limited to theory, new sudden change can be to limit glucose degradation to become the spontaneous mutant of organic acid.Spontaneous to coming The analysis instruction of sample in the circulation of ferment tank, the organic acid of PDG-1 and PDG-3 generates big with control strain S-60wtc and LPG-2 Cause identical.

Bacterial strain PDG-1 consistently generates substantial amounts of high viscosity Ji orchid sugar, and TPM ＞ 14g/L.

Evaluate these cultures colonial morphology on flat board to check the stability of bacterial strain, the most spontaneous PDG- 1 mutant and the original NPG strains with low lucky blue candy output.After YM agar is cultivated about 60 hours in about 37 DEG C, PDG-1 shows the form dramatically different for parent NPG-1 with it.The meat soup fermented from PDG-1 is not observed and has The bacterium colony of NPG-1 type form, the stability of indicator strain.

Embodiment 8: the existence of homology phaC gene in the Sphingomonas beyond the Sphingol single-cell of many natural ponds

Sphingomonas beyond the Sphingol single-cell of many natural ponds identifies the homologous genes of phaC, thus demonstrate,proves Bright Sphingomonas beyond the Sphingol single-cell of many natural ponds generates the feasibility lacking PHB mutant.

Four kinds of Sphingomonas are carried out Southern DNA hybridization: generate the ATCC of diutan (S-657) 53159；Generate the ATCC 31555 of welan (S-130)；Generate the ATCC 31961 of rhamsan (S-194)；Lucky blue with generating The ATCC 31461 of sugar (S-60) is as comparison.By every kind of strains separation genomic DNA, and digest with enzyme EcoRI.To disappear Genome DNA sample (1 μ g) after change separates on 1% agarose gel, and uses Schleicher and Schuell Turboblotter(Keene, New Hampshire) is transferred to nylon membrane by capillarity in neutral conditions.

Use degenerate primer PHADG5 and PHADG7 (see embodiment 1), by Sphingol single-cell S-657phaC gene The PCR amplification in region, portion, with Digitoxin-11-dUTP according to scheme (the Roche Molecular of manufacturer Biochemicals, Switzerland) prepare the probe of Digitoxin labelling.Use from Roche Diagnostics (Mannheim, Germany) DigEasyHybHybridize in neutral conditions according to the scheme of manufacturer.In 44 DEG C (than calculated T_{Most preferably} Low 10 DEG C) filter membrane is hybridized.These conditions anticipated by cause the DNA molecular same more than 90% hybridize (Birren, Et al. B. compile, " Genomic Analysis, A Laboratory Manual " i.e. genome analysis, laboratory manual, 1997). As used herein, term T_{Most preferably}It is defined as T_m-20, wherein T_mIt is to be defined by formula " 50+0.41 (%GC)-600/ probe length " , wherein %GC is 65%, and probe length is 400 nucleotide.Filter membrane is cleaned two in 44 DEG C in 2xSSC, 0.1%SDS Secondary 15 minutes, and use anti-Digitoxin-alcaline phosphatase conjugate and Digitoxin detection kit according to manufacturer Scheme (Roche Molecualr Biochemicals) develop the color.

Fig. 9 shows results of hybridization.In Sphingomonas ATCC 31461, detect that there is expection size (2.6kb) EcoRI digests band.Sphingomonas ATCC 53159 and ATCC 31961 generation has identical The band of size.Sphingomonas ATCC31555 comprises and the 2.4kb fragment of phaC probe hybridization.Thus, Southern DNA hybridization confirms these three bacterial strain and comprises phaC sample gene, and can generate according to methods described herein and lack Weary PHB bacterial strain.

Embodiment 9: build ATCC 53159 mutant strain with phaC disappearance

Use recombinant DNA technology, build the Sphingol single-cell ATCC53159 mutant strain of disappearance phaC gene. It is carried out as follows the structure of mutant strain: by the flanking DNA district of PCR amplification phaC gene, and be cloned in suicide vector, pass through Engaging transfers in ATCC 53159 cell by the suicide vector comprising flanking PCR products, then by kalamycin resistance The selection of (by vector encoded) realizes whole plasmid adjacent phaC upstream in Sphingol single-cell ATCC 53159 chromosome Or the integration at the homologous genes seat in downstream.By Chromosomal excision phaC locus and carrier DNA by encoding on carrier The result of the follow-up second exchange event that sucrose sensitivity selects.

In order to separate the clone comprising phaC gene and flanking region, prepare genome dna library, and use PHADG5 and PHADG7 primer (see above embodiment 1) is screened by PCR.Constructing two genomic libraries, one is at carrier Inserting NotI in pZERO-2 (Invitrogen, Carlsbad, CA) and limit enzyme fragment, second is at pLAFR3 (Staskawicz et al., J.Bacteriol., 169:5789-5794,1987) inserts Sau3A partial digest fragments.By often Individual library separates a positive colony.Sub-clone from the BamHI-NotI fragment of these plasmids, and to suitable sequencing fragment with Measure phaC gene and the DNA sequence of 5 ' and 3 ' flanking regions.Plasmid p21-7 and pJCS104-2 comprises the 5 ' of phaC gene respectively With 3 ' end and flanking regions.

Figure 10 shows the DNA sequence of phaC gene and flanking region.Figure 11 shows genetic map.Opening code-reading frame is logical Cross the initial and termination codon existed and combined the use Borodovsky analysis (Lasergene from GeneMark GeneQuest module) and P_aeruginosa_3.mat Matrix prediction coding region BLAST analyze and determine.Sequence is Obtained by the Insert Fragment sequence connection in clone p21-7 and pJCS104-2.Joint between two kinds of sequences is positioned at NotI position Point.

Figure 12 describes how to be used for generating by PCR and only comprises phaC flanking region, lacked whole phaC gene The product pJCS105 of (1737bp, by last nucleotide of the first of start codon nucleotide to termination codon) 112-1.Two kinds of external primers (primer 1Xba and primer 4Xba) are engaged with the expectation crossed between phaC upstream and downstream district Primer (overlapping 1) be applied in combination.Table 4 shows primer sequence.

According to the scheme of manufacturer, plasmid pJCS104-2 and p21-7 (every kind of 200ng) and primer 1Xba and 4Xba is (every Kind of 50pmol), overlapping 1 primer (2pmol), dNTP and from Clontech Laboratories company (Palo Alto, CA) Advantage HighFidelity 2PCR kit the most senior high-fidelity 2PCR test kit in Taq polymerase mixing.Then Matrix thermal cycler expands: 95 DEG C of 1min；95 DEG C of 30sec of 5 circulations, 44 DEG C of 30sec, 68 DEG C of 2min；20 30sec, 53-68 DEG C of 30sec of 95 DEG C of individual circulation, 68 DEG C of 2min；68℃3min.The DNA fragmentation expanded by gel-purified, and use Same primers expands to generate more product again.Amplification condition is: 95 DEG C of 1min；25 circulation 95 DEG C of 30sec, 64 DEG C 30sec、68℃2min；68℃3min.Then SNAP gel purification kit (Invitrogen) is used to be separated 1.1kb by gel Band, and use topoisomerase, the carrier with 3 ' T jags and Competent TOP10 cell according to Invitrogen Scheme be cloned in carrier pCRII-TOPO (Invitrogen), to generate pJC105-112-1, as shown in Figure 12.

Gel-purified is from the 1.1kbXbaI fragment comprising phaC disappearance construction of pJCS105-112-1, and is cloned into In the pLO2 of XbaI digestion.Reclaim the Insert Fragment of two kinds of orientations, referred to as pJCS106-5 and pJC106-16.

Label switched is used for building the shortage PHB bacterial strain of ATCC 53159.As described in example 4 above, by switching Close and plasmid pJCS 106-5 and pJC106-16 is imported ATCC 53159.As described in example 4 above and 5, carry out the first He Second exchange deletion mycopremna selection, first select the integration shown by kalamycin resistance, be then coated on sucrose with Select the permutoid sensitive to kanamycin.Deletion crossouts (versus wild type), referred to as NPD-is obtained by diagnosis PCR detection 3 (derived from pJCS106-5) and NPD-6 (derived from pJCS106-16).

NPD-3 and the NPD-6 bacterial strain generated has accurate phaC chromosome deficiency and does not remain foreign DNA.But, this The gum production of a little deletion mycopremnas is greatly reduced, but after fermentation culture, isolated recovers pressing down of gum production subsequently Gene processed.

Under conditions of promoting S-657 synthesis, cultivate NPD-3 and NPD-6, and select that there is pressing down of large-scale, m colony Gene bacterial strain processed, as lucky blue sugar synthesis is carried out by example 7 above.According to the bacterium colony deriving them, these are large-scale, M colony is referred to as PDD-3 and PDD-6, and analyzes PHB and S-657 generation.PDD-3 and PDD-6 bacterial strain is preserved in American TypeCulture Collection i.e. American Type culture collecting center, numbering be respectively ATCC_ _ _ _ _ and ATCC_ _ _ _ _.Table 5 below indicates the gum production that PDD-6 provides higher than its ancestors' NPD-6 bacterial strain, the most qualitative See and do not generate PHB.

Embodiment 10: concentration of potassium hydroxide is on light transmittance and the impact of gel strength

Caustic agent potassium hydroxide (" KOH ") is evaluated dense in the defecation method including step that above-outlined is first scheme The impact of degree.To comprise the lucky blue sugar fermentation meat soup pretreatment lacking PHB mutant, and mix 15 points with the KOH of variable concentrations Clock, is followed by reaching as the 1000ppm Calgon of chelating agen 1 hour, is 22ppm lysozyme and 220ppm albumen the most successively Enzyme is each 2 hours in 55 DEG C.The KOH concentration of test changes between about 0.0g/L and about 0.45g/L.As shown in Figure 13, When KOH concentration increases to 0.45g/L, light transmittance adds nearly 20% (31% increases relatively).

TA-TX2Texture Analyzer(Texture Technologies company, Scarsdale, NY) is with two kinds Index i.e. makes the product rupturing required Puncture force and distance of the gel surface of preparation measure with pressure-sensing plunger Gel strength data.Measurement Puncture force when rupturing of gel surface is detected at load cell, and with the percentage of height change Ratio measures Puncture force.As shown in Figure 14, in the identical KOH scope of test, gel strength reduces for Puncture force 280g or 32%, this may give the credit to the part deacylated tRNA base of lucky blue sugar.But, gel strength with regard to distance percentage ratio for seemingly Do not have significantly affected, only the change of reflection 1.5%.

2x2 little factor research is carried out according to first scheme defecation method.The lucky blue sugar fermentation lacking PHB mutant will be comprised Meat soup pretreatment, and mix 15 minutes with the KOH of variable concentrations, it is followed by reaching 1 as the 2000ppm Calgon of chelating agen little Time, it is followed by 22ppm lysozyme and 220ppm protease successively each 2 hours in 55 DEG C.Research is studied light transmittance percentage at this Ratio, Puncture force and distance percentage ratio, as it is assumed that they are relevant to kinetics.KOH concentration about 0.225g/L with about Change between 0.45g/L, and temperature changes between about 55 DEG C and about 60 DEG C, and obtain following table (display transparent rate percentage Than, Puncture force and distance percentage result) result that shows, to evaluate KOH concentration and the temperature shadow to lucky blue sugar transparency Ring.

As shown in Table, after increasing KOH concentration or temperature respectively, light transmittance change is little.But, when KOH concentration and When temperature all increases, light transmittance adds about 6%, this instruction the two parameter to realize light transmittance increase it is critical that And superposition.Similar, gel strength displays that synergistic effect.After increasing temperature or KOH concentration respectively, Puncture force reduces About 130g-about 190g；But, when temperature and KOH concentration all increase, Puncture force reduces about 326g, thus Illustrate that gel strength is easily changed by both temperature and KOH concentration to be affected.

Embodiment 11: the sodium hexameta phosphate impact on lucky blue sugar characteristic

The defecation method described according to example 10 above, evaluates sodium hexameta phosphate (" SHMP ", also referred to as Calgon) right Light transmittance, Puncture force and the impact of distance percentage ratio.The lucky blue sugar fermentation meat soup pretreatment lacking PHB mutant will be comprised, And mix 15 minutes with 0.45g/L KOH, the Calgon being followed by variable concentrations reaches 1 hour, is followed by 22ppm lysozyme successively Each 2 hours in 55 DEG C with 220ppm protease.SHMP concentration changes between about 1000ppm and about 3000ppm, Er Qiezheng As shown in figure 15, in this range, there is linear correlation between SHMP concentration and light transmittance.SHMP increases about 1000ppm to be led Transmittance increase about 5%.As shown in figure 16, SHMP seems not affect gel strength, because in the range of test, and power of punching Spend and do not affected with distance relative not increased by SHMP concentration of percentage ratio.

Other clarification order of embodiment 12:SHMP

On native gellan broth, two kinds of variants of defecation method are carried out 2 liters of scales.According to manufacturer The information that Genencor International company (Rochester, New York) provides, MultifectLysozyme acid- Neutral pH level is stable, and can inactivate at short notice at alkaline pH.0.45g/L is being added according to defecation method After KOH, pH is usually more than pH8, and this is not optimal for lysozyme, and protease is it is said that play under these conditions very Good.Thus, according to alternative plan revise defecation method, lysozyme process after add KOH (sequence summary: lysozyme, then KOH, then protease).Before or after lysozyme processes, either add KOH, all observe the relative standard error of 5.5% Difference (" RSD "), the most as shown in the table.

* * * refers to unmeasured data

RSD refers to the relative standard deviation relative to meansigma methods percentage ratio

Embodiment 13: confectionery formulations

This embodiment demonstrates the formula that can be used for generating the elastic chewy confectionery showing excellent clarity and stability.

In adding heat container, mix the composition comprising part A and be heated to 40 DEG C.

Mix the composition of part B in the dry state, pour into add in heat container and quickly mix, and be heated to boiling.Will be mixed Compound is down to 72% solid.The composition of mixing part C, adds flavoring agent and pigment, and is mixed into homogenizing.

Material is placed in container and is filled in prefabricated starch mold.Then the starch mold that will be filled with is stored in 30 DEG C and 35% relative humidity reach 3-4 days, until solid level reaches about 82%-85%.By material release, wax, and preserve In sealing in bag.

Extra water can be added so that completing the hydration of hydrophilic colloid in Part A ingredients.

Embodiment 14: dessert gel formulations

This formula can be used for generating the elastic dessert gel with excellent clarity and stability.

With the flavoring agent of drying regime combination drying and pigment and the composition of part A, spread in part B and mix； It is heated to 90 DEG C.Then mixture is poured in suitable container, and be allowed to solidify in room temperature.

Embodiment 15: jelly prescription

This formula provides to be had excellent clarity, storage stability, taste release and smears the fruit jelly of ability.

Composition	Percentage ratio
		Part A
Concord Sucus Vitis viniferae	45.69
		High fructose corn syrup	30.46
Water	22.85
		Part B
The high acyl group Ji orchid sugar of clarification	0.18
		Sodium citrate	0.10
SHMP	0.10
		Potassium sorbate	0.09
Part C
		Citric acid, 50% solution	0.53

The composition of mixing part A.At the composition of drying regime mixing part B, and it is distributed to the composition of part A when mixing In.When mixing, the mixture of generation is heated to boiling, and under fluidized state, keeps about 1 minute-about 3 minutes, this Time the composition of part C is stirred in mixture.Then in the container after mixture being poured into sterilizing and seal.

The most by it is now recognized that preferred embodiment describe the present invention, it should be understood that on the invention is not restricted to Literary composition is described.On the contrary, this invention is intended to cover and be included in the various amendments within spirit and scope of the appended claims and equivalent Arrange.

The invention still further relates to following project:

1. project 1: the mutant strain of Sphingomonas (Sphingomonas), wherein coding participates in poly butyric At least one gene of the protein of synthesis is by selective mutation or disappearance so that mutant strain generates sphingans but do not generates Poly butyric.

2. project 2: according to the mutant strain of project 1, wherein mutant strain generates the sphingans selected from lower group: S-7, S- 60, S-88, S-130, S-194, S-198, S-657, NW-11 and B16.

3. project 3: according to the mutant strain of project 1, wherein coding participates in the protein of poly butyric synthesis at least A kind of gene is selected from lower group: phaA, phaB and phaC.

4. project 4: according to the mutant strain of project 3, wherein phaC is by selective mutation or disappearance.

5. project 5: according to the mutant strain of project 4, wherein sphingans is S-60.

6. project 6: according to the mutant strain of project 4, wherein sphingans is S-657.

7. project 7: the shortage prepared according to the method for the Sphingomonas mutant strain of project 1 by fermentation The sphingans of PHB.

8. project 8: from the isolated DNA sequence of many natural ponds Sphingol single-cell (Sphingomonas elodea), wherein should DNA sequence encoding has the PHB synthase of nucleotide sequence listed by SEQ ID NO:7.

9. project 9: for the method preparing the sphingan solutions of clarification, comprise the following steps:

A) aqueous sphingan solution is heated to clarifying temp about 30 DEG C-about 70 DEG C；

B) with selected from lower group at least one clarifier process aqueous sphingan solution: chelating agen, escharotic, oxidant and Its mixture；And

C) ferment treatment aqueous sphingan solution is used.

10. project 10: according to the method for sphingan solutions of the preparation clarification of project 9, carry out the most simultaneously step a) and b)。

11. projects 11: according to the method for project 9, wherein process aqueous sphingan solution with at least one chelating agen.

12. projects 12: according to the method for project 9, wherein process aqueous sphingan solution with at least one escharotic.

13. projects 13: according to the method for project 9, wherein with selected from least one oxidizer treatment sphingans water of lower group Solution: sodium hypochlorite or other hypochlorite, chlorine dioxide, hydrogen peroxide, peracetic acid and ozone.

14. projects 14: according to the method for project 9, wherein with at least one escharotic and at least one surfactant Reason aqueous sphingan solution.

15. projects 15: according to the method for project 9, wherein carry out ferment treatment at pH about 5-about 9.

16. projects 16: according to the method for project 9, at least one of which chelating agen is selected from lower group: ethylenediaminetetraacetic acid, phosphorus Acid, Metaphosphoric acid, carbonic acid, citric acid, tartaric acid, gluconic acid, glutamic acid, pyrophosphoric acid, polyphosphoric acid, Metaphosphoric acid, glucosaccharic acid, second Glycol two (beta-amino ether)-N, N, N ', N '-tetraacethyl (EGTA), ethylenediamine, 2,3-diaminobutane, 1,2-diamino basic ring Hexane, triamido triethylamine or their salt.

17. projects 17: according to the method for project 9, at least one of which chelating agen is selected from lower group: ethylenediaminetetraacetic acid two Sodium, ethylenediamine tetraacetic acid,dipotassium salt, tetrasodium ethylenediamine tetraacetate, ethylenediaminetetraacetic acid four potassium, trisodium citrate, potassium citrate, Sodium hexameta phosphate, hexa metaphosphoric acid potassium, sodium polyphosphate, potassium polyphposphate, sodium pyrophosphate, potassium pyrophosphate, mono phosphoric acid ester sodium, mono potasssium, Disodium hydrogen phosphate, dikalium phosphate, tertiary sodium phosphate, tripotassium phosphate, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, cation are handed over Change resin, dihydrochloride, diacetate, lithium salt, ethylenediamine two hydroiodic acid and mixture thereof.

18. projects 18: according to the method for project 9, wherein escharotic is selected from lower group: potassium hydroxide, sodium hydroxide and phosphoric acid Trisodium.

19. projects 19: by the clarification prepared by the method for clarified sphingan solution separation sphingans according to project 9 Sphingans.

20. projects 20: according to the sphingans of the clarification of project 19, wherein sphingans is by the saltant type of Sphingol single-cell Lacking what PHB bacterial strain generated, wherein coding participates at least one gene of the protein that poly butyric synthesizes by selectivity Sudden change or disappearance.

21. projects 21: according to the sphingans of the clarification of project 20, wherein phaC gene is by selective mutation or disappearance.

22. projects 22: according to the sphingans of the clarification of project 21, wherein sphingans is high acyl group S-60.

23. projects 23: according to the method for project 9, wherein process sphingans in step b) further with surfactant Aqueous solution.

24. projects 24: according to the method for project 23, wherein process sheath with a kind of chelating agen and at least one surfactant D-glucosamine aqueous solution.

25. projects 25: according to the method for project 23, at least one of which surfactant selected from lower group: SDS, polyoxy second Alkene sorbitan monooleate, lecithin, monoglyceride, tartaric esters of monoglycerides, phosphated monoglyceride, lactylated list are sweet Grease, acetylated monoglyceride, succinylated monoglycerides, ethoxylated monoglycerides, sorbitan ester, poly-sorbic acid Ester, polyglycerin ester, sucrose ester, sodium stearoyl lactate and propylene glycol ester.

26. projects 26: for the method preparing the sphingan solutions of clarification, comprise the following steps:

B) process with chelating agen；

C) process with lysozyme；

D) process with escharotic or oxidant；And

E) process with protease.

27. projects 27: according to the method for project 26, wherein carry out lysozyme process at pH about 3-about 7.5.

28. projects 28: according to the method for project 26, wherein carry out Protease Treatment at pH about 6.5-about 9.

29. projects 29: by according to project 26 prepared by the method for clarified sphingan solution separation sphingans clear Clear sphingans.

30. projects 30: according to the sphingans of the clarification of project 29, wherein sphingans is by the saltant type of Sphingol single-cell Lacking what PHB bacterial strain generated, wherein coding participates at least one gene of the protein that poly butyric synthesizes by selectivity Sudden change or disappearance.

31. projects 31: according to the sphingans of the clarification of project 30, wherein phaC gene is by selective mutation or disappearance.

32. projects 32: according to the sphingans of the clarification of project 31, wherein sphingans is high acyl group S-60.

33. projects 33: according to project 20 or the sphingans of the clarification of project 30, wherein aqueous sphingan solution has at least The light transmittance of 60%.

34. projects 34: comprise the food product of clarification of sphingans according to project 33.

35. projects 35: according to the food product of project 34, the wherein phaC base of the shortage PHB bacterial strain of Sphingol single-cell Because of by selective mutation or disappearance.

36. projects 36: according to the food product of project 35, wherein sphingans is high acyl group S-60.

37. projects 37: comprise the industrial products of clarification of sphingans according to project 33.

38. projects 38: comprise the industrial products of the shortage PHB sphingans according to project 7.

39. projects 39: according to the industrial products of project 38, the wherein phaC base of the shortage PHB bacterial strain of Sphingol single-cell Because of by selective mutation or disappearance.

40. projects 40: according to the industrial products of project 39, wherein sphingans is S-657.

41. projects 41: from the isolated DNA sequence of Sphingol single-cell ATCC 53159, wherein this DNA sequence encoding tool There are PHB synthase and the flanking region of nucleotide sequence listed by SEQ ID NO:13.

Sequence table

<110>Bower, Stan

Burke, Ellen

Harding, Nancy E.

Patel, Yamini N.

Schneider, J.Carrie

Meissner, Dagmar

Morrison, Neil

Bezanson, Ralph

<120>poly butyric generates the Sphingomonas mutant bacterial strains of defect

Method with clarification of sphingans and combinations thereof thing

<130>2047.144

<140>

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<151>2000-03-02

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Asp Pro Arg Arg Thr Leu Glu Ala Gln Thr His Leu Leu Gly Ser Phe

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Phe Asp Met Trp Ser Arg Thr Leu Gln Arg Met Ala Ala Asp Ala Val

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Val Thr Ser Asp Trp Ala Glu Arg Met Val Lys Asp Ala Glu Gly Leu

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Asp Asp His Thr Arg His Lys Ala Ala Phe Tyr Val Arg Gln Ile Ala

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Ser Ala Leu Ser Pro Thr Asn Phe Ile Thr Thr Asn Pro Gln Leu Tyr

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Arg Glu Thr Val Ala Ser Ser Gly Ala Asn Leu Val Lys Gly Met Gln

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Met Leu Ala Glu Asp Ile Ala Ala Gly Arg Gly Glu Leu Arg Leu Arg

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Gln Thr Asp Thr Ser Lys Phe Ala Ile Gly Glu Asn Ile Ala Ile Thr

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Pro Gly Lys Val Ile Ala Gln Asn Asp Val Cys Gln Val Leu Gln Tyr

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Pro Trp Ile Asn Lys Phe Tyr Val Leu Asp Leu Asn Pro Glu Lys Ser

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Pro Glu Ser Ser Leu Val Arg His Val Val Glu Gln Gly His Thr Val

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Arg Asp Ile Ser Gly Gln Asp Lys Ile Asn Val Leu Gly Phe Cys Val

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Ser Leu Gly Lys Asp Leu Ala Tyr Thr Pro Gly Ala Val Val Phe Glu

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Tyr Gln Thr Pro Ile Leu Val Val Pro Pro Phe Ile Asn Lys Tyr Tyr

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Val Leu Asp Leu Arg Glu Gln Asn Ser Leu Val Asn Trp Leu Arg Gln

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Gln Gly His Thr Val Phe Leu Met Ser Trp Arg Asn Pro Asn Ala Glu

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Gln Lys Glu Leu Thr Phe Ala Asp Leu Ile Thr Gln Gly Ser Val Glu

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Ala Leu Arg Val Ile Glu Glu Ile Thr Gly Glu Lys Glu Ala Asn Cys

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Ile Gly Tyr Cys Ile Gly Gly Thr Leu Leu Ala Ala Thr Gln Ala Tyr

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Tyr Val Ala Lys Arg Leu Lys Asn His Val Lys Ser Ala Thr Tyr Met

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Asn Glu Pro Val Val Ser Gly Leu Glu Asn Leu Asn Asn Gln Leu Gly

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Tyr Phe Asp Gly Arg Gln Leu Ala Val Thr Phe Ser Leu Leu Arg Glu

385 390 395 400

Asn Thr Leu Tyr Trp Asn Tyr Tyr Ile Asp Asn Tyr Leu Lys Gly Lys

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Ile Pro Ala Lys Ile His Asn Phe Leu Leu Arg Asn Leu Tyr Leu Asn

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Asn Glu Leu Ile Ser Pro Asn Ala Val Lys Val Asn Gly Val Gly Leu

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Asn Leu Ser Arg Val Lys Thr Pro Ser Phe Phe Ile Ala Thr Gln Glu

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Ala Lys Phe Glu Asn Thr Lys Gln Trp Leu Asp Gly Ala Glu Tyr His

530 535 540

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545 550 555 560

Gly Glu Gln Val Pro Ala Arg Asn Leu Gly Asn Ala Gln Tyr Pro Ser

565 570 575

Ile Glu Ala Ala Pro Gly Arg Tyr Val Leu Val Asn Leu Phe

580 585 590

<210>4

<211>601

<212>PRT

<213>hydrogenlike silicon ion (Rhodobacter sphaeroides)

<400>4

Met Ala Thr Glu Glu Gln Ser Pro Gly Ser Gly Arg Asp Ala Gln Phe

1 5 10 15

Glu Arg Leu Asn Ala Asn Leu Thr Arg Ile Asp Glu Leu Ser Lys Arg

20 25 30

Leu Thr Ala Ala Leu Thr Lys Arg Lys Leu Ser Asp Pro Ala Leu His

35 40 45

Gly Pro Ser Gly Asp Val Phe Leu Lys Ala Met Thr Ala Tyr Met Ala

50 55 60

Glu Met Met Gln Asn Pro Ala Lys Ile Leu Glu His Gln Ile Ser Phe

65 70 75 80

Trp Gly Lys Ser Leu Lys His Tyr Val Glu Ala Gln His Gln Leu Val

85 90 95

Lys Gly Glu Leu Lys Pro Pro Pro Asp Val Thr Pro Lys Asp Arg Arg

100 105 110

Phe Ser Asn Pro Leu Trp Gln Thr His Pro Phe Phe Asn Tyr Leu Lys

115 120 125

Gln Gln Tyr Leu Met Asn Ala Glu Ala Val Asn Gln Ala Val Glu Gly

130 135 140

Leu Glu His Ile Glu Pro Ser Asp Lys Lys Arg Val Glu Tyr Phe Ser

145 150 155 160

Arg Gln Ile Val Asp Leu Phe Ser Pro Thr Asn Phe Phe Gly Thr Asn

165 170 175

Pro Asp Ala Leu Glu Arg Ala Ile Ala Thr Asp Gly Glu Ser Leu Val

180 185 190

Gln Gly Leu Glu Asn Leu Val Arg Asp Ile Glu Ala Asn Asn Gly Asp

195 200 205

Leu Leu Val Thr Leu Ala Asp Pro Glu Ala Phe Gln Val Gly Gln Asn

210 215 220

Leu Ala Thr Thr Glu Gly Ser Val Val Tyr Arg Asn Arg Met Phe Glu

225 230 235 240

Leu Ile Gln Tyr Lys Pro Thr Thr Glu Thr Val His Glu Thr Pro Leu

245 250 255

Leu Ile Phe Pro Pro Trp Ile Asn Lys Phe Tyr Ile Leu Asp Leu Lys

260 265 270

Pro Gln Asn Ser Leu Leu Lys Trp Leu Val Asp Gln Gly Phe Thr Val

275 280 285

Phe Val Val Ser Trp Val Asn Pro Asp Lys Ser Tyr Ala Gly Ile Gly

290 295 300

Met Asp Asp Tyr Ile Arg Glu Gly Tyr Met Arg Ala Met Ala Glu Val

305 310 315 320

Arg Ser Ile Thr Arg Gln Lys Gln Ile Asn Ala Val Gly Tyr Cys Ile

325 330 335

Ala Gly Thr Thr Leu Thr Leu Thr Leu Ala His Leu Gln Lys Ala Gly

340 345 350

Asp Pro Ser Val Arg Ser Ala Thr Phe Phe Thr Thr Leu Thr Asp Phe

355 360 365

Ser Asp Pro Gly Glu Val Gly Val Phe Leu Asn Asp Asp Phe Val Asp

370 375 380

Gly Ile Glu Arg Gln Val Ala Val Asp Gly Ile Leu Asp Lys Thr Phe

385 390 395 400

Met Ser Arg Thr Phe Ser Tyr Leu Arg Ser Asn Asp Leu Ile Tyr Gln

405 410 415

Pro Ala Ile Lys Ser Tyr Met Met Gly Glu Ala Pro Pro Ala Phe Asp

420 425 430

Leu Leu Tyr Trp Asn Gly Asp Gly Thr Asn Leu Pro Ala Gln Met Ala

435 440 445

Val Glu Tyr Leu Arg Gly Leu Cys Gln Gln Asp Arg Leu Ala Gly Gly

450 455 460

Thr Phe Pro Val Leu Gly Ser Pro Val Gly Leu Lys Asp Val Thr Leu

465 470 475 480

Pro Val Cys Ala Ile Ala Cys Glu Thr Asp His Ile Ala Pro Trp Lys

485 490 495

Ser Ser Phe Asn Gly Phe Arg Gln Phe Gly Ser Thr Asp Lys Thr Phe

500 505 510

Ile Leu Ser Gln Ser Gly His Val Ala Gly Ile Val Asn Pro Pro Ser

515 520 525

Arg Asn Lys Tyr Gly His Tyr Thr Asn Glu Gly Pro Ala Gly Thr Pro

530 535 540

Glu Ser Phe Arg Glu Gly Ala Glu Phe His Ala Gly Ser Trp Trp Pro

545 550 555 560

Arg Trp Gly Ala Trp Leu Ala Glu Arg Ser Gly Lys Gln Val Pro Ala

565 570 575

Arg Gln Pro Gly Asp Ser Lys His Pro Glu Leu Ala Pro Ala Pro Gly

580 585 590

Ser Tyr Val Ala Ala Val Gly Gly Ala

595 600

<210>5

<211>605

<212>PRT

<213>demethylation bacillus (Methylobacterium extorquens) is turned round

<400>5

Met Gly Thr Glu Arg Thr Asn Pro Ala Ala Pro Asp Phe Glu Thr Ile

1 5 10 15

Ala Arg Asn Ala Asn Gln Leu Ala Glu Val Phe Arg Gln Ser Ala Ala

20 25 30

Ala Ser Leu Lys Pro Phe Glu Pro Ala Gly Gln Gly Ala Leu Leu Pro

35 40 45

Gly Ala Asn Leu Gln Gly Ala Ser Glu Ile Asp Glu Met Thr Arg Thr

50 55 60

Leu Thr Arg Val Ala Glu Thr Trp Leu Lys Asp Pro Glu Lys Ala Leu

65 70 75 80

Gln Ala Gln Thr Lys Leu Gly Gln Ser Phe Ala Ala Leu Trp Ala Ser

85 90 95

Thr Leu Thr Arg Met Gln Gly Ala Val Thr Glu Pro Val Val Gln Pro

100 105 110

Pro Pro Thr Asp Lys Arg Phe Ala His Ala Asp Trp Ser Ala Asn Pro

115 120 125

Val Phe Asp Leu Ile Lys Gln Ser Tyr Leu Leu Leu Gly Arg Trp Ala

130 135 140

Glu Glu Met Val Glu Thr Ala Glu Gly Ile Asp Glu His Thr Arg His

145 150 155 160

Lys Ala Glu Phe Tyr Leu Arg Gln Leu Leu Ser Ala Tyr Ser Pro Ser

165 170 175

Asn Phe Val Met Thr Asn Pro Glu Leu Leu Arg Gln Thr Leu Glu Glu

180 185 190

Gly Gly Ala Asn Leu Met Arg Gly Met Lys Met Leu Gln Glu Asp Leu

195 200 205

Glu Ala Gly Gly Gly Gln Leu Arg Val Arg Gln Thr Asp Leu Ser Ala

210 215 220

Phe Thr Phe Gly Lys Asp Val Ala Val Thr Pro Gly Glu Val Ile Phe

225 230 235 240

Arg Asn Asp Leu Met Glu Leu Ile Gln Tyr Ala Pro Thr Thr Glu Thr

245 250 255

Val Leu Lys Arg Pro Leu Leu Ile Val Pro Pro Trp Ile Asn Lys Phe

260 265 270

Tyr Ile Leu Asp Leu Asn Pro Gln Lys Ser Leu Ile Gly Trp Met Val

275 280 285

Ser Gln Gly Ile Thr Val Phe Val Ile Ser Trp Val Asn Pro Asp Glu

290 295 300

Arg His Arg Asp Lys Asp Phe Glu Ser Tyr Met Arg Glu Gly Ile Glu

305 310 315 320

Thr Ala Ile Asp Met Ile Gly Val Ala Thr Gly Glu Thr Asp Val Ala

325 330 335

Ala Ala Gly Tyr Cys Val Gly Gly Thr Leu Leu Ala Val Thr Leu Ala

340 345 350

Tyr Gln Ala Ala Thr Gly Asn Arg Arg Ile Lys Ser Ala Thr Phe Leu

355 360 365

Thr Thr Gln Val Asp Phe Thr His Ala Gly Asp Leu Lys Val Phe Ala

370 375 380

Asp Glu Gly Gln Ile Lys Ala Ile Glu Glu Arg Met Ala Glu His Gly

385 390 395 400

Tyr Leu Glu Gly Ala Arg Met Ala Asn Ala Phe Asn Met Leu Arg Pro

405 410 415

Asn Asp Leu Ile Trp Ser Tyr Val Val Asn Asn Tyr Val Arg Gly Lys

420 425 430

Ala Pro Ala Ala Phe Asp Leu Leu Tyr Trp Asn Ala Asp Ala Thr Arg

435 440 445

Met Pro Ala Ala Asn His Ser Phe Tyr Leu Arg Asn Cys Tyr Leu Asn

450 455 460

Asn Thr Leu Ala Lys Gly Gln Met Val Leu Gly Asn Val Arg Leu Asp

465 470 475 480

Leu Lys Lys Val Lys Val Pro Val Phe Asn Leu Ala Thr Arg Glu Asp

485 490 495

His Ile Ala Pro Ala Leu Ser Val Phe Glu Gly Ser Ala Lys Phe Gly

500 505 510

Gly Lys Val Asp Tyr Val Leu Ala Gly Ser Gly His Ile Ala Gly Val

515 520 525

Val Ala Pro Pro Gly Pro Lys Ala Lys Tyr Gly Phe Arg Thr Gly Gly

530 535 540

Pro Ala Arg Gly Arg Phe Glu Asp Trp Val Ala Ala Ala Thr Glu His

545 550 555 560

Pro Gly Ser Trp Trp Pro Tyr Trp Tyr Lys Trp Leu Glu Glu Gln Ala

565 570 575

Pro Glu Arg Val Pro Ala Arg Ile Pro Gly Thr Gly Ala Leu Pro Ser

580 585 590

Leu Ala Pro Ala Pro Gly Thr Tyr Val Arg Met Lys Ala

595 600 605

<210>6

<211>408

<212>DNA

<213>many natural ponds Sphingol single-cell (Sphingomonas elodea)

<400>6

tctagattcg atctcctcta ctggaattcg gacgtcacca acctgccggc gacctggcac 60

ctcagctacc tgaccgacct ctaccgcgac aacaagctga tcgcgcccgg cgcgctcagc 120

atcggcggta ccccgatcga cctgtcgaag gtagaaacgc cgtcctatat ccaggccggg 180

cgcgaagatc acatcgcacc gccccgcagc gtctggaaga tgacggagca tttccgcggg 240

ccgcacaagt tcgtgctggc cggttccggc catatcgccg gcgtaatcaa tccgccttcg 300

gcaaagaaat accaatactg gaccaatgcc gggccggccg agtcgctcga atcctttgtc 360

gaaaacgcga cggaacatgc cggcagctgg tggcccccct ggactaga 408

<210>7

<211>1925

<212>DNA

<213>many natural ponds Sphingol single-cell (Sphingomonas elodea)

<400>7

ctgcaggaca tggccaaggg ccagatgacg cagaccgccg ccggcgcgtt cgagctcggc 60

cgcaacctgg cgatgacgcc gggcaaggtg gtgaagcgca cgccgctgta cgaactgatc 120

cagtattcgc cgacgacgga cacggtgctg gaaacgccgc tgatcatctt cccgccctgg 180

atcaaccgct tctacattct cgacctgacg ccggagaaga gcttcatccg ctgggcggtg 240

gcgcagggga tcaccgtgtt cgtcgtgtcg tggcgctcgg ccgatgcgag catgaaggac 300

gtggtgtggg acgattatgt cgagcgcggc cagatcgacg cgatcgacac cgtgcgcgag 360

ctgctcggcg tggaaagcgt ccacacgatc ggctattgcg tggcgggcac cacgctggcg 420

gcgacgctgg cggtgctcgc ggcgcgcggg gaggcggcga aggtggcgag cgcgaccttc 480

ttcaccgccc aggtcgactt caccgaggcg ggcgacctgc gcgtgttcgt cgacgacgac 540

cagctggcga tgatccgcag cctcggcgcc gacgggttcc tcgacgggcg ctacatggcg 600

gcgacgttca acctgctgcg cgggcgcgac ctgatctgga actacgtcac caacaactat 660

ctgatggggc aggaatatgc gccgttcgac ctgctccact ggaactcgga cgtcaccaac 720

ctgccggcgr cctggcacct cagctacctg accgacctct accgcgacaa caagctgatc 780

gcgcctctag acggcgcgct cagcatcggc ggtaccccga tcgacctgtc gaaggtagaa 840

acgccgtcct atatccaggc cgggcgcgaa gatcacatcg caccgccccg cagcgtctgg 900

aagatgacgg agcatttccg cgggccgcac aagttcgtgc tggccggttc cggccatatc 960

gccggcgtaa tcaatccgcc ttcggcaaag aaataccaat actggaccaa tgccgggccg 1020

gccgagtcgc tcgaatcctt tgtcgaaaac gcgacggaac atgccggaag ctggtggccg 1080

gactgggtgg actggttggt tgcgttgaac agtgcaaagg ttgcgacgaa aggtgcgcgg 1140

cttcccggca gtggaaacct ttgtgcaatc gccgacgcgc ccggcgaata tgttagaatg 1200

cgctgacggg aaggccgaat tttcgcgggt ttgacgattt ttgtgcactg cacaatggcg 1260

ccttgcaaaa tggccgtcga gcctttatat gttgcagcca gcaattggca gggaaagcta 1320

gtcacatggc cagcaaagga cctaagacga cggccaaacc ggcggcacgc ggtgctacca 1380

agcccgcgac tctggccgaa gctgccgcgg cgaagccgac gcctgcaccc gcccttgccg 1440

agacgatcgt cccggcagcg gcgccggtgc cggcgcctgc cgaagccgct gcaccgcagg 1500

acgtgaagac caacatcgaa gaggcgatca ccgccccggt ggaaacggca gccgccgtca 1560

ccgagcaggc gatcgaagcc gcagagaccg tcgcgccggc ggtcaccacc agcaccgcga 1620

aggaaacgac tatcatggct accactttcg aaaacgcgac tacccaggcc cagaccgttt 1680

tcgccgacct gaatgagcgc accaaggccg ccgtcgagaa gtcgaccaag ctggtcgagg 1740

aagccaacga gttcgccaag ggcaacatcg aagccctggt cgaatcgggc cgcatcgccg 1800

ccaagggctt cgagagcctg ggccaggaag ctgccgatta cagccgccgc tcgttcgaga 1860

gcgcgaccgc cgcgctgaag ggcctgtcgt cggtcaagtc gccgaccgaa ttcttcaagc 1920

tgcag1925

<210>8

<211>399

<212>PRT

<213>many natural ponds Sphingol single-cell (Sphingomonas elodea)

<400>8

Leu Gln Asp Met Ala Lys Gly Gln Met Thr Gln Thr Ala Ala Gly Ala

1 5 10 15

Phe Glu Leu Gly Arg Ash Leu Ala Met Thr Pro Gly Lys Val Val Lys

20 25 30

Arg Thr Pro Leu Tyr Glu Leu Ile Gln Tyr Ser Pro Thr Thr Asp Thr

35 40 45

Val Leu Glu Thr Pro Leu Ile Ile Phe Pro Pro Trp Ile Asn Arg Phe

50 55 60

Tyr Ile Leu Asp Leu Thr Pro Glu Lys Ser Phe Ile Arg Trp Ala Val

65 70 75 80

Ala Gln Gly Ile Thr Val Phe Val Val Ser Trp Arg Ser Ala Asp Ala

85 90 95

Ser Met Lys Asp Val Val Trp Asp Asp Tyr Val Glu Arg Gly Gln Ile

100 105 110

Asp Ala Ile Asp Thr Val Arg Glu Leu Leu Gly Val Glu Ser Val His

115 120 125

Thr Ile Gly Tyr Cys Val Ala Gly Thr Thr Leu Ala Ala Thr Leu Ala

130 135 140

Val Leu Ala Ala Arg Gly Glu Ala Ala Lys Val Ala Ser Ala Thr Phe

145 150 155 160

Phe Thr Ala Gln Val Asp Phe Thr Glu Ala Gly Asp Leu Arg Val Phe

165 170 175

Val Asp Asp Asp Gln Leu Ala Met Ile Arg Ser Leu Gly Ala Asp Gly

180 185 190

Phe Leu Asp Gly Arg Tyr Met Ala Ala Thr Phe Asn Leu Leu Arg Gly

195 200 205

Arg Asp Leu Ile Trp Asn Tyr Val Thr Asn Asn Tyr Leu Met Gly Gln

210 215 220

Glu Tyr Ala Pro Phe Asp Leu Leu His Trp Asn Ser Asp Val Thr Asn

225 230 235 240

Leu Pro Ala Xaa Trp His Leu Ser Tyr Leu Thr Asp Leu Tyr Arg Asp

245 250 255

Asn Lys Leu Ile Ala Pro Gly Ala Leu Ser Ile Gly Gly Thr Pro Ile

260 265 270

Asp Leu Ser Lys Val Glu Thr Pro Ser Tyr Ile Gln Ala Gly Arg Glu

275 280 285

Asp His Ile Ala Pro Pro Arg Ser Val Trp Lys Met Thr Glu His Phe

290 295 300

Arg Gly Pro His Lys Phe Val Leu Ala Gly Ser Gly His Ile Ala Gly

305 310 315 320

Val Ile Asn Pro Pro Ser Ala Lys Lys Tyr Gln Tyr Trp Thr Asn Ala

325 330 335

Gly Pro Ala Glu Ser Leu Glu Ser Phe Val Glu Asn Ala Thr Glu His

340 345 350

Ala Gly Ser Trp Trp Pro Asp Trp Val Asp Trp Leu Val Ala Leu Asn

355 360 365

Ser Ala Lys Val Ala Thr Lys Gly Ala Arg Leu Pro Gly Ser Gly Asn

370 375 380

Leu Cys Ala Ile Ala Asp Ala Pro Gly Glu Tyr Val Arg Met Arg

385 390 395

<210>9

<211>28

<212>DNA

<213>artificial sequence

<220>

<223>description of artificial sequence: with PCR primer PHADG5 of many natural ponds Sphingol single-cell phaC genetic fragment annealing

<400>9

agtttctaga ttcgayctst aytggaay 28

<210>10

<211>31

<212>DNA

<213>artificial sequence

<220>

<223>description of artificial sequence: with PCR primer PHADG7 of many natural ponds Sphingol single-cell phaC genetic fragment annealing

<400>10

gtatactagt ccaiiisggc caccagctgc c 31

<210>11

<211>30

<212>DNA

<213>artificial sequence

<220>

<223>description of artificial sequence: with PCR primer PHAC12 of many natural ponds Sphingol single-cell phaC genetic fragment annealing

<400>11

gttctctaga ggcgcgatca gcttgttgtc 30

<210>12

<211>31

<212>DNA

<213>artificial sequence

<220>

<223>description of artificial sequence: with PCR primer PHAC11 of many natural ponds Sphingol single-cell phaC genetic fragment annealing

<400>12

gttctctaga gagtcgctcg aatcctttgt c 31

<210>13

<211>3180

<212>DNA

<213>Sphingol single-cell (Sphingomonas) ATCC 53159

<400>13

gatccacacc ttgttctcgc gcgcccaggc gacgaggcgc tcgtagaagg cgaggtccac 60

cgtctccgcc gtcgggttcg acggatagtt gacgacgagg atcgacgggc gcggcacggt 120

gaagttcatt gcccgctcga ggctttcgaa ataggcgtcg tcgggcgtgg tcggcaccgc 180

gcggatcgtc gcgccggcga tgatgaagcc gaaggtgtgg atcgggtagc tggggttggg 240

cgcgagcacc acgtcgcccg gcgcggtgat cgcggtggcg aggctggcaa ggccctcctt 300

cgagcccatc gtgacgacga cctcggtctc gggatcgagc tcgacgccga atcggcggcc 360

ataataattg gcctgggcgc ggcgcaggcc cggaatgccc ttggactgcg aatagccgtg 420

cgcgtcgggc ttgcgcgcca cttcgcacag tttctcgatc acatggtcgg gcggcggcag 480

gtccggattg cccatgccga ggtcgataat gtcctctccg cccgcgcgtg ccgctgcccg 540

catcgcgttc acttcggcga tgacataggg aggcaagcgc ttgatgcggt agaattcttc 600

ggacatttcc tcgactttca agggttttga cacgcgacac aaaattgtgt cgtgcgcgcg 660

ttctacgcca taatcgcgca tccgggaatg acgcattgct ccgcctgcgc taagccgggg 720

cgaaggagag gaccgaatgg ccgatacgct cacgccgacc ctgccccgac tggaagacct 780

gcagcattgg acctgggtgc tgggccgcgc gcagcagatg atgctggagc atgggctgga 840

cctgatggag catgtgcccg ccgcgccccc cttcggcatg ctgctcgatc cgaccccggc 900

aatgcgggcg agcgcggacc tctgggcgga cacgatgcag ctgtggcagc gcttcctcga 960

tcccgcccat gccgagccgt tcgtcgaatc gcccgagcag gcgcgcgaca agcgcttcaa 1020

ggcgccgcaa tggcgcgagg agccggtgtt cgatttcctg cggcagagct atttcgtgat 1080

cgccgaccac atgctcaggc aggtcgaggc gctcgagcat gtcgacgagc ggcagcggga 1140

ccagatccgc ttcgccacca agggcttcat cgacgcgatc agccccacca acttccccgc 1200

caccaatccg caggtgatcg agaagatcgt cgagaccaag ggggaaagcc tgctcaaggg 1260

cctgcagcat atgctgcagg acatggccaa gggccagatg acgcagaccg ccgccggtgc 1320

gttcgagctc ggccgcaacc tggcgatgac gcccggcaag gtggtgaagc gcaccccgct 1380

ctacgaactg atccagtatt cgccgaccac cgagaccgtg ctggaaacgc cgctgatcat 1440

cttcccgccc tggatcaacc gcttctacat cctcgacctg acgcccgaga aaagcttcat 1500

ccgctgggcg gtggagcagg ggatcaccgt gttcgtcgtc tcctggcgct cggccgatgc 1560

gagcatgaag gacgtggtgt gggacgatta tgtcgagcgc ggccagatcg acgcgatcga 1620

cacggtgcgc gcgctgctcg gcgtcgagag cgtccatacc atcggctatt gcgtggcggg 1680

caccacgctg gcggcgacgc tggcggtgct cgccgcgcgc gggcaggcgg cgaaggtggc 1740

gagcgcgacc ttcttcaccg cgcaggtcga tttcaccgag gcgggcgacc tgcgcgtgtt 1800

cgtcgatgac gaccagctgg cgatgatccg cagcctcagc gccgacggct tcctcgacgg 1860

gcgctacatg gcggcgacct tcaacctgct gcgcggccgc gacctgatct ggaactacgt 1920

caccaacaac tatctgatgg ggcaggaata tgcgccgttc gacctgctcc actggaactc 1980

ggacgtcacc aacctgccgg cgacctggca tctcagctac ctgaccgacc tgtaccgcga 2040

caacaagctg atcgcgcccg gcgcgctgcg catcggcggc accccggtcg acctttcgaa 2100

ggtcgaaacg ccgtcctaca tccaggccgg ccgcgaagat catatcgcgc cgccgcaaag 2160

cgtctggaag atcaccgagc atttccgcgg gccgcacaag ttcgtgctgg cgggttccgg 2220

gcatatcgca ggtgtaataa accccccggc ggcgaagaaa taccaatact ggaccaatac 2280

agggcctgcc gagtcgctcg actcctttat cgaaaccgcg acggaacatg cgggaagttg 2340

gtggccggat tggctggatt gggtccgtgc gctgaacggt gcaaaggttg cgacgagcgg 2400

tgcgcgggtg ccggggggtg gtaacctttg tgcagttgcg gaagcgcccg gcgactatgt 2460

tagaatgcgc tgacaaagag gcagaatttc gtgggtttct ggcgtttttg tgcactgcac 2520

aatgatcgct tgcaaaagca gcgccaagtc tttatatgct gcagtgcagc aatagccagg 2580

gaaagctagt cacatggcca gcaaaggacc caagacgacg gccaaacccg ccgcaaaatc 2640

agcggctcgc ggtgctatca agcccgcgat tctggccgaa gctgccgcgg cgacgccggc 2700

gtctgtacct cccgttgccg agacgatcgt cccggccgcg gcgttggtgc ctgcgccgga 2760

cgaagccgct gcaacgcagg aagtgacgac tcacatcaaa gacacggtcg acgttgcggc 2820

ggaaacggta aaggccgtcg ccgaacacgc gatcgaagcc gcagagaccg tcgcgccggc 2880

ggtcaccacc agcaccgcga aggaaccgac tatcatggcc accactttcg agaacgcgac 2940

cacccaggcc cagactgttt tcgccgacct caacgagcgc accaaggccg ccgtcgaaaa 3000

gtcgaccaag ctggtcgagg aagccaacga gttcgccaag ggcaacatcg aggcgctggt 3060

cgaatccggc cgcatcgctg ccaagggctt cgagacgctg ggccaggaag ccgccgatta 3120

cagccgtcgc tcgttcgaga acgccacgac cacgctgaag agcctgtcgt cggtgaagtc 3180

<210>14

<211>32

<212>DNA

<213>artificial sequence

<220>

<223>description of artificial sequence: with PCR primer 1Xba of Sphingol single-cell ATCC 53159 genetic fragment annealing

<400>14

attctagaga tgatgaagcc gaaggtgtgg at 32

<210>15

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>description of artificial sequence: with PCR primer 4Xba of Sphingol single-cell ATCC 53159 genetic fragment annealing

<400>15

attctagatg gtgcgctcgt tgagg 25

<210>16

<211>34

<212>DNA

<213>Sphingomonas sp.ATCC 531559

<400>16

gaaattctgc ctctttgtcg gtcctctcct tcgc 34

Claims

1. more than natural pond Sphingol single-cell (Sphingomonas elodea) or the mutant strain of ATCC53159, wherein encode participation The phaC gene of protein of poly butyric synthesis is by selective mutation or disappearance so that mutant strain generate sphingans but Do not generate poly butyric.

2., according to the mutant strain of claim 1, wherein mutant strain generates the sphingans selected from lower group: S-60 and S-657.

3. according to the mutant strain of claim 1, the wherein phaC of many natural ponds Sphingol single-cell after selective mutation or disappearance Gene is missing from the SEQ ID NO:7 of nucleotide 792 to 1023, or wherein ATCC53159 after selective mutation or disappearance PhaC gene is missing from the SEQ ID NO:13 of nucleotide 737 to 2473.

4. the method for the sphingans of the shortage PHB of preparation clarification, including fermentation according to the mutant bacteria any one of claim 1-3 Strain and the step of the sphingans by fermentation broth clarification shortage PHB.