CN104845927A - Gene substituted microbe and polyester preparation method using gene substituted microbe - Google Patents
Gene substituted microbe and polyester preparation method using gene substituted microbe Download PDFInfo
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Abstract
The invention aims at providing a recombined microbial strain capable of stably producing PHA (Polyhydroxybutyric Acid) at high property in a fermentation process for industry. The recombined microbial strain provided by the invention is obtained by substituting an exogenous polyhydroxybutyric acid synthetase gene for polyhydroxybutyric acid synthetase gene which exists on a microbial chromosome.
Description
The application is on July 21st, 2006 based on the applying date, application number is 200680055421.8 (PCT/JP2006/314498), and denomination of invention is the divisional application of the patent application of " gene replaces microorganism and uses the process for producing polyester of this microorganism ".
Technical field
The present invention relates to the microbial strain through improveing and utilize the PHA manufacture method of this microbial strain, the microbial strain of described improvement is useful in the commercial production of the poly (hydroxy alkanoate) (PHA) of microbiological deterioration polyester.
Background technology
Poly (hydroxy alkanoate) is the polyester type organic molecule polymkeric substance that can be produced by multiple-microorganism.These polymkeric substance have biological degradability, thermal plastic high polymer, and in addition, it can be produced by renewable resources, thus, carried out it can be used as harmonious environment shaped material or biological adaptation shaped material to carry out industrial production, be applied to the trial of each industry.
Form the general 3-hydroxyl alkane acid by name of monomer of this polyester, particularly, by making the 3-hydroxyl alkane acid of the lucky oxalic acid of 3-hydroxybutyrate, 3-hydroxyl, 3-hydroxycaproic acid, 3-Hydroxyoctanoic acid or more long alkyl chain overlap separately or copolymerization forms polymer molecule.(following as 3-hydroxybutyrate, referred to as 3HB) the poly-3-hydroxybutyrate of homopolymer (following, be called for short P (3HB)), P (3HB) is nineteen twenty-five Late Cambrian in Bacillus megatherium (Bacillus megaterium), but the crystallinity of this P (3HB) is high, its character is hard and crisp, and its range of application receives the restriction of practicality.Therefore, carried out studying to improve for the purpose of its character.
This wherein, disclose the interpolymer be made up of 3-hydroxybutyrate (3HB), the lucky oxalic acid (3HV) of 3-hydroxyl (following, be called for short P (3HB-co-3HV)) manufacture method (for example, referring to patent documentation 1, patent documentation 2).This P (3HB-co-3HV) is P (3HB) richer flexibility comparatively, therefore, can think that it has and apply widely.But, in fact, even if increase the molar ratio of 3HV, the physical property of P (3HB-co-3HV) also lacks consequential change, particularly flexibility does not improve, and therefore, it can only be applied in the hard formed body field such as handle of shampoo bottle, disposable razor.
In addition, the known carbonatoms by alkyl chain is the medium chain PHA that forms of 3-hydroxyl alkane acid of 6 ~ 16, compared with P (3HB), P (3HB-co-3HV) crystallinity low, be rich in elastic force (with reference to non-patent literature 1), its application in different field can be expected.In the manufacture research of medium chain PHA, carry out following research: by the pha synthesizing enzyme channel genes Rhodopseudomonas of Rhodopseudomonas (Pseudomonas), thunder Bordetella (Ralstonia), intestinal bacteria, but productivity is all low, be not suitable for industrial production (with reference to non-patent literature 2, non-patent literature 3, non-patent literature 4).
In recent years, carry out 3HB and 3-hydroxycaproic acid (following, be called for short 3HH) 2 composition copolyesters (hereinafter referred to as P (3HB-co-3HH)) and the research (with reference to patent documentation 3, patent documentation 4) of manufacture method.P (3HB-co-3HH) manufacture method in these reports is such: use isolated Aeromonas caviae (Aeromonas caviae) from soil, carry out fermentative production from the lipid acid such as oleic acid or olive wet goods grease.In addition, the property Quality Research (with reference to non-patent literature 5) about P (3HB-co-3HH) has also been carried out.In this report, with the lipid acid of carbonatoms more than 12 for sole carbon source cultivates Aeromonas caviae, fermentative production 3HH consists of the P (3HB-co-3HH) of 11 ~ 19mol%.Known: the increase that P (3HB-co-3HH) forms along with 3HH, transfer the soft character of display from the hard and crisp character of P (3HB) gradually, and demonstrate the flexibility more than P (3HB-co-3HV).Namely, by changing the composition of 3HH, P (3HB-copolymerization-3HH) can have the physical property applied of the wide scope from hard polyester to flexible polyester, is therefore expected to be applied to require that the product of hardness requires the wide field of the product of flexibility to such as film etc. from such as housing of TV set etc.But, in this manufacture method, production of polyester low (thalline turnout 4g/L, amount of polyester 30%), can't say that it is the practical production method towards this polyester fully, therefore, explore towards the practical method obtaining more high productivity.
Some effort have been carried out with the industrial production target of P (3HB-co-3HH).When using Aeromonas hydrophila (Aeromonas hydrophila) to cultivate, in the 43 hours feeding culture taking oleic acid as carbon source, produce that thalline output is 95.7g/L, amount of polyester is 45.2%, 3HH consist of 17% P (3HB-copolymerization-3HH) (with reference to non-patent literature 10).In addition, with glucose and lauric acid for carbon source cultivates Aeromonas hydrophila, can reach thalline output is 50g/L, and amount of polyester is 50%, and molecular weight is 1,000,000 dalton (with reference to non-patent literatures 11).But Aeromonas hydrophila has pathogenicity bo (with reference to non-patent literature 12) to human body, and it is not be suitable for industrial bacterial classification.In addition, these cultivate the carbon source of producing and all using high price, therefore from the view point of production cost, should seek to utilize more cheap carbon source.
Therefore, with the production in safe host and improve productivity for target, carried out some effort.Poly (hydroxy alkanoate) (PHA) synthase gene (with reference to patent documentation 5, non-patent literature 9) has been cloned from Aeromonas caviae.Use this channel genes Ralstonia eutropha (Ralstonia eutropha, be once called as Alcaligenes eutropha) and the transformant obtained carries out the production of P (3HB-co-3HH), result is: thalline productivity is 4g/L, amount of polyester is 30%.And use Vegetable oil lipoprotein to cultivate this transformant as carbon source, result achieves thalline turnout 4g/L, amount of polyester 80% (with reference to non-patent literature 10).And, cultural method being studied, by improving culture condition, bringing up to thalline turnout 45g/L, amount of polyester 62.5%, 3HH form 8.1% (patent documentation 6 reference).
In addition, although having constructed to take fructose as the Ralstonia eutropha that carbon source produces P (3HB-co-3HH), the production of polyester of this bacterial strain is low, is not talkatively applicable to actual production (with reference to non-patent literature 11).
Also constructing with intestinal bacteria is P (3HB-co-3HH) the production strain of host.The NADP-acetoacetyl Co-A reductase gene of the pha synthesizing enzyme gene of Aeromonas and Ralstonia eutropha etc. is imported intestinal bacteria and constructs bacterial strain.Be that carbon source is cultivated this colibacillary result and is with dodecane: productivity is when within 40.8 hours, cultivating, biomass 79g/L, amount of polyester 27.2%, 3HH composition 10.8% (with reference to non-patent literature 12).
To improve the productivity of P (3HB-co-3HH) and 3HH composition control for target, carry out the manually modified of pha synthesizing enzyme.Aeromonas caviae source pha synthesizing enzyme variant in, the variant enzyme that the amino acid of the 149th and l-asparagine are replaced by Serine, the 171st aspartic acid by the variant enzyme that glycine replaces demonstrate in intestinal bacteria, improve pha synthesizing enzyme activity, 3HH forms (non-patent literature 13 reference).In addition, have and report: the pha synthesizing enzyme of variant enzyme in intestinal bacteria that the α-amino-isovaleric acid of the variant enzyme that the phenylalanine of 518 of this enzyme is replaced by Isoleucine or 214 is replaced by glycine is active, amount of polyester increases (with reference to non-patent literature 14).But these methods use special intestinal bacteria as host, and amount of polyester is still low, therefore, the feature of these variant enzymes that are necessary to apply in a flexible way, carries out being more suitable for industrial improvement.
When the bacterial strain using application recombinant DNA technology to make, technical scale cultivation PHA, one of most important problem is the stability of the gene imported.Channel genes can utilize the method using plasmid, the method etc. being recombined into host chromosome.But, known plasmid can breed at recombinant bacterium, come off (with reference to patent documentation 7) when dividing.Therefore, the thalline that plasmid comes off loses PHA throughput, thus commercial production is low.Traditionally, general strategy is: in the cultivation of restructuring thalline, usually only optionally make plasmid possess thalline to breed and keep by adding antibiosis in the medium, but the cost brought by antibiotic use improves, the environmental influence etc. that caused by the residual antibiotic cultivated in waste liquid becomes problem.In addition, in order to make plasmid stabilisation, by the parB gene recombination of R1 plasmid origin in P (3HB) production of plasmid, recombination bacillus coli (with reference to non-patent literature 15) has been prepared.These intestinal bacteria substantially 100% maintain plasmid after cultivation 110-120 generation.But, still there is the danger come off in plasmid.
On the other hand, can think that restructuring gene is on chromosome stable, report the microorganism (reference patent documentation 8, non-patent literature 16, non-patent literature 17) that karyomit(e) of being recombinated to by PHA synthesis related gene obtain.
Patent documentation 8 discloses the e. coli strains by being prepared to escherichia coli chromosome by the encoding gene radom insertion of PHA GCMS computer enzyme, and this e. coli strains produces P (3HB) with the level of exceed cell dry weight 85%.But, in order to produce the good P of practical physical property (3HB-co-3HH) in intestinal bacteria, the gene that must also will coexist for providing matrix monomer, or expensive lipid acid etc. must be supplied in substratum, and, which hinders the realization of high efficiency.And, when random recombination on chromosome, depending on there is the site of restructuring, the expression of the gene on this site or around this site can be had influence on, there is the situation that can not give full play to ability as PHA production strain.
By exogenous pha synthesizing enzyme gene with during plasmid form importing host or when recombinating on chromosome, if use thunder Bordetella, Rhodopseudomonas etc. to produce the microbial species of PHA natively as host, then can utilize the bacterial strain cannot producing PHA natively.Such bacterial strain is prepared through mutation operation, therefore, compared with its parent plant, multiplication capacity, biological activity are in a disadvantageous position (with reference to non-patent literature 18, non-patent literature 19, non-patent literature 20 etc.), can't say played sufficient throughput as PHA production strain.
On the other hand, report the pha synthesizing enzyme gene becoming replacement Chromatium vinosum (Chromatium vinosum) to originate the pha synthesizing enzyme gene on the karyomit(e) of Ralstonia eutropha, have accumulated P (3HB) (non-patent literature 17) that exceed wild strain, that account for cell dry weight 91%.But biomass is low to moderate 1.8g/L, the polyester of production is also hard and crisp P (3HB), but not the P (3HB-co-3HH) that expectation is widely used, still have problems in the commercial production of polyester.
Patent documentation 1 JP 57-150393 publication
Patent documentation 2 JP 59-220192 publication
Patent documentation 3 Unexamined Patent 5-93049 publication
Patent documentation 4 Unexamined Patent 7-265065 publication
Patent documentation 5 Unexamined Patent 10-108682 publication
Patent documentation 6 JP 2001-340078 publication
Patent documentation 7 JP 59-205983 publication
Patent documentation 8 United States Patent (USP) No. 6593116 specification sheetss
Non-patent literature 1Madison etc., Microbiol.Mol.Biol.Rev., 63:21-53 (1999)
Non-patent literature 2Matsusaki etc., J.Bacteriol., 180:6459-6467 (1998)
Non-patent literature 3Matsusaki etc., Appl.Micrbiol.Biotechnol., 53:401-409 (2000)
Non-patent literature 4Langenbach etc., FEMS Microbiol.Lett., 150:303-309 (1997)
Non-patent literature 5Doi etc., Macromolecules, 28:4822-4828 (1995)
Non-patent literature 6Lee etc., Biotechnol.Bioeng., 67:240-244 (2000)
Non-patent literature 7Chen etc., Appl.Microbiol.Biotechnol., 57:50-55 (2001)
Security Administration not table 1 subordinate lists 1 (1999) such as non-patent literature 8 state-run infection disease institute pathogenic agent
Non-patent literature 9Fukui etc., J.Bacteriol., 179:4821-4830 (1997)
Non-patent literature 10Fukui etc., Appl.Microbiol.Biotecnol., 49:333-336 (1998)
Non-patent literature 11Fukui etc., Biomacromolecules, 3:618-624 (2002)
Non-patent literature 12Park etc., Biomacromolecules, 2:248-254 (2001)
Non-patent literature 13Kichise etc., Appl.Environ.Microbiol., 68:2411-2419 (2002)
Non-patent literature 14Amara etc., Appl.Microbiol.Biotechnol., 59:477-482 (2002)
Non-patent literature 15Lee etc., J.Biotechnol., 32:203-211 (1994)
Non-patent literature 16Kranz etc., Appl.Environ.Microbiol., 63:3003-3009 (1997)
Non-patent literature 17York etc., J.Bacteriol., 183:4217-4226 (2001)
Non-patent literature 18Schlegel etc., Arch.Mikrobiol., 71:283-294 (1970)
Non-patent literature 19Schubert etc., J.Bacteriol., 170:5837-5847 (1988)
Non-patent literature 20Peoples etc., J.Biol.Chem., 264:15298-15303 (1989)
Summary of the invention
Problem to be solved by this invention
As above-mentioned, in the commercial production of PHA, also there is many problems.
The object of the invention is: be provided in plant-scale culture process can expect to have extensive use, stably with the recombinant microorganism bacterial strain of high-level accumulation copolyester and provide the copolyester manufacture method utilizing this recombinant microorganism bacterial strain.
The method of dealing with problems
The present inventor conducts in-depth research for solving the problem, and found that: the poly (hydroxy alkanoate) synthase gene be present on karyomit(e) is substituted by exogenous poly (hydroxy alkanoate) synthase gene and the production of polyester microorganism that obtains can stably with high level accumulation PHA.
There is the synthesizing stable of prompting in order to make PHA, quiding gene is effective on chromosome, but, the effect when expression of the destruction and quiding gene of use wild strain, its pha synthesizing enzyme gene locus is substituted by specifically pha synthesizing enzyme gene, simultaneously carrying out existing gene carrys out accumulation copolyester is also not known, in addition, this can not predict simply from known technology.
As shown in the Examples, poly (hydroxy alkanoate) synthase gene on the karyomit(e) of Ralstonia eutropha H16 strain is replaced to the poly (hydroxy alkanoate) synthetic enzyme variant gene in Aeromonas caviae source and the microbial strain that obtains, in the cultivation of 48 hours, produce P (3HB-co-3HH) with thalline turnout 110.4g/L, amount of polyester 73.8wt%.The productivity of the P (3HB-co-3HH) that this productivity has been reported more has and significantly improves.In addition, also find: this bacterial strain all without the need to microbiotic or other any selective pressure, substantially all have accumulated P (3HB-co-3HH) in all cells at the end of cultivation in whole culturing engineering.
That is, the present invention is as follows.
1. produce the microorganism of copolyester for one kind, described copolyester is by being selected from 3-hydroxybutyrate, 3-hydroxycaproic acid, 3-hydroxyheptanoic acid, 3-Hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxy-dodecanoic acid, the acid of 3-hydroxy tridecyl, 3-hydroxyl tetradecane acid, two or more monomeric unit in 3-hydroxypentadecanoic acid and 3-hydroxyl cetane acid is formed, inherently poly (hydroxy alkanoate) synthase gene on the karyomit(e) of this microorganism, and now this poly (hydroxy alkanoate) synthase gene be replaced to exogenous poly (hydroxy alkanoate) synthase gene at least partially.
2. the microorganism described in 1, wherein, described copolyester is made up of 3-hydroxybutyrate and 3-hydroxycaproic acid monomeric unit.
3. the microorganism described in 1 or 2, wherein, on described karyomit(e), inherently the microorganism of poly (hydroxy alkanoate) synthase gene is Ralstonia eutropha (Ralstonia eutropha).
4. the microorganism described in 3, wherein, described Ralstonia eutropha is Ralstonia eutropha H16 strain.
5. the microorganism according to any one of 1 ~ 4, wherein, the enzyme that described exogenous poly (hydroxy alkanoate) synthase gene coding Aeromonas caviae (Aeromonas caviae) is originated or its variant.
6. the microorganism described in 5, wherein, described variant has at least carried out the aminoacid replacement of one of following (a), (b):
A amino acid that () is the 149th and l-asparagine are replaced to Serine,
B amino acid that () is the 171st and aspartic acid are replaced to glycine.
7. the microorganism according to any one of 1 ~ 4, wherein, the variant enzyme that described exogenous poly (hydroxy alkanoate) synthase gene coding is originated with the Aeromonas caviae that the aminoacid sequence of SEQ ID NO:9 represents, this variant enzyme is the amino acid of the 149th and l-asparagine is replaced to Serine and the amino acid of the 171st and aspartic acid are replaced to glycine.
8. a microorganism, it is KNK-005 strain.
9. employ the process for producing polyester of the microorganism according to any one of item 1 ~ 8.
Copolyester of the present invention be with following general formula represent with in the 3-hydroxyl alkane acid polymkeric substance that is monomeric unit, the copolymerized polymer that is made up of more than 2 kinds or the 3 kinds monomeric units be selected from 3-hydroxybutyrate, 3-hydroxycaproic acid, 3-hydroxyheptanoic acid, 3-Hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid, 3-hydroxy-dodecanoic acid, the acid of 3-hydroxy tridecyl, 3-hydroxyl tetradecane acid, 3-hydroxypentadecanoic acid and 3-hydroxyl cetane acid.
Chemical formula 1
(in formula, R
1and R
2represent that carbonatoms is the alkyl group of more than 1, they in the polymer can be identical, also can be different.M and n represents the monomeric unit number of this polymkeric substance, is more than 1.)。
As above-mentioned copolyester, be preferably made up of the monomeric unit of 3-hydroxybutyrate and 3-hydroxycaproic acid.
The microorganism used in the present invention, as long as belong to microorganism chromosomal DNA originally with the species of poly (hydroxy alkanoate) synthase gene, not restriction is used to it, comprises modified and the microorganism of other carbon source, modified and can synthesize or take in the microorganism of matrix monomer or modified and microorganism that is productivity raising can be utilized.Such as, the bacteriums such as the Rhodopseudomonass such as Alkaligenes, Pseudomonas aeruginosa, pseudomonas putida (Pseudomonas) such as the Aeromonass such as the Ralstonia such as Ralstonia eutropha (same with Wautersia eutropha, Cupriavidus necator on taxonomy) belong to, Aeromonas caviae, extensively Alcaligenes (Alcaligenes latus) are comprised.From security and productive viewpoint, preferred Ralstonia belongs to, more preferably Ralstonia eutropha, more preferably Ralstoniaeutropha H16 strain.
As long as the exogenous poly (hydroxy alkanoate) synthase gene used in the present invention, the gene making it possible to accumulation copolyester in the gene of various PHA accumulation biogenetic derivation, can be any gene.Have from such as Aeromonas caviae (non-patent literature 9) as such gene, nocardia corallina (Nocardia corallina) (GenBank accession number AF019964), Pseudomonas aeruginosa (Timm etc., Eur.J.Biochem., 209:15-30 (1992)), Pseudomonas oleovorans (Pseudomonasoleovorans) (Huisman etc., J.Biol.Chem., 266:2191-2198 (1991)), corydalis capsule sulphur bacterium (Thiocystis violaceae) (Liebergesell etc., Appl.Microbiol.Biotechnol., 38:493-501 (1993)) etc. isolated poly (hydroxy alkanoate) synthase gene.The enzyme in optimized encoding Aeromonas caviae source or the gene of its variant.
In addition, in the scope not losing object enzymic activity, a part of base sequence (thus aminoacid sequence is changed) of these genes is changed and the gene obtained also is operable.Preferred use is such as: that non-patent literature 13 is recorded, the 149th amino acid and l-asparagine have been replaced to the polyester synthase gene (N149S variant gene) in the Aeromonas caviae source of Serine, the amino acid of the 171st and aspartic acid have been replaced to the polyester synthase gene (D171G variant gene) in the Aeromonas caviae source of glycine, or be combined with above-mentioned 2 aminoacid replacement, polyester synthase gene etc. that the Aeromonas caviae that represents with the aminoacid sequence of SEQ ID NO:9 is originated.
Said gene can have promotor and/or ribosome bind site, but and inessential.
The replacement mode of polyester synthase gene, as long as this replacement to make on karyomit(e) the enzymic activity coded by polyester synthase gene (being substituted gene) originally existed lose and exogenous polyester synthase gene (substituting group because of) is expressed, and can be any replacement mode.After replacement, being substituted gene can disappear from karyomit(e) completely, still can there is a part, can also exist with the divided form opened.A kind of mode alternatively, at substituting group because having ribosome bind site, but when not having promotor, can select to make it be connected to and be substituted the substitute mode in the downstream of the promotor of gene.In addition, alternate manner alternatively, when substituting group is not because having promotor and ribosome bind site, can select to make it be connected to and be substituted the replacement mode in the downstream of the ribosome bind site of gene.After can selecting to make replacement, expressed polyester synthetase albumen is the replacement mode of the single albumen replacing genes encoding, and also can select to make to replace rear expressed polyester synthetase albumen is the replacement mode with the fusion rotein of the albumen being substituted genes encoding.The replacement mode of the adoptable polyester synthase gene of the present invention is not limited to above example, preferably such: by be substituted gene be replaced to from initiator codon to terminator codon substituting group because of from initiator codon to terminator codon.
The method replaced specifically the gene locus on karyomit(e) well known to a person skilled in the art.Representatively property method has: the method (Ohman etc. utilizing transposon and homologous recombination machinery, J.Bacteriol., 162:1068-1074 (1985)) or the Site-specific recombinase that causes with homologous recombination machinery and based on subordinate phase homologous recombination come off for the method (Noti etc. of principle, MethodsEnzymol., 154:197-217 (1987)) etc., in addition, such method can also be utilized: the sacB gene that Bacillus subtilus is originated and substituting group are because coexisting, come off sacB gene because of the homologous recombination of subordinate phase (Schweizer, Mol.Microbiol., 6:1195-1204 (1992), Lenz etc., J.Bacteriol., 176:4385-4393 (1994)) microbial strain add the strain of substratum patience as sucrose and easily separate, as long as can gene on substituted dyeing body, method used is not particularly limited.
Below, to the pha synthesizing enzyme gene (phaC by Ralstonia eutropha
re) be substituted by the pha synthesizing enzyme gene (phaC of Aeromonas caviae
ac) time substitution technique do example more specifically.
First, preparation replaces fragment.Replacing fragment is following form: at phaC
reencoding sequence (CDS: comprise from initiator codon to terminator codon) next-door neighbour (straight before) upstream sequence on connect phaC
accDS, connecting phaC thereafter
renext-door neighbour's (directly after) downstream sequence of CDS.That is, replacing fragment is that only CDS is substituted by phaC
acthe phaC of CDS
redNA fragmentation.Upstream sequence and downstream sequence are in order to the gene generation homologous recombination on karyomit(e) and required homologous sequence, and in general, the longer recombination frequency of its length is higher, as long as but homologous recombination can be caused, can set arbitrarily its length.
Replacement fragment can be attached to the gene as selective marker when gene replaces.As the gene of selective marker, can use such as: the antibiotic resistant gene such as kantlex, paraxin, Streptomycin sulphate, Ampicillin Trihydrate, or with the gene etc. of various auxotrophy complementation.When utilizing Ralstonia eutropha to carry out, preferred kalamycin resistance gene.
And in addition, can also add following gene, described gene is used for the microbial strain easily selecting the region containing selectable marker gene to come off because of the homologous recombination of subordinate phase.As such gene, the sacB gene (Schweizer, Mol.Microbiol., 6:1195-1204 (1992)) in Bacillus subtilus source can be listed.The microbial strain of known this gene of expression containing growth reproduction on the substratum of sucrose, by containing the growth reproduction in sucrose medium, can not easily select the bacterial strain that this gene is lost because coming off.
Gene replacement plasmid is prepared by the replacement formed like this fragment being connected on the carrier that do not copy in host microorganism strain.In such carrier that can utilize in thunder Bordetella, the Rhodopseudomonas etc., can list such as: pUC carrier, pBluescript carrier, pBR322 carrier or there is the carrier etc. of identical copy starting point with them.And the DNA sequence dnas such as mob, oriT can also be made to coexist, and above-mentioned DNA sequence dna makes it possible to carry out conjugal transfer.
The gene replacement plasmid DNA be prepared into such formation, can adopt the known method such as electroporation, conjugal transfer method to import the microorganisms such as Ralstonia eutropha, can carry out homologous recombination.
Then, Select gene replacement plasmid DNA inserts the bacterial strain on karyomit(e) because of homologous recombination.Selection can be undertaken by following method, and described method make use of the selection gene coexisted in gene replacement plasmid DNA.When using kalamycin resistance gene, can select containing the bacterial strain that the substratum of kantlex grows.
In next stage, select following bacterial strain, the region comprising selectable marker gene in described bacterial strain comes off from karyomit(e) because of second time homologous recombination.Can based on the selection gene utilized when inserting, select such as containing can not the bacterial strain of growth reproduction on the foster base of kantlex, but when sacB gene coexists on gene replacement plasmid, easily can select and contain the bacterial strain that sucrose medium grows.In order to confirm that the bacterial strain obtained like this is that desired gene replaces bacterial strain, the known methods such as PCR method, South hybrid method or DNA base sequence mensuration can be adopted.As aforesaid operations, the pha synthesizing enzyme gene (phaC of Ralstonia eutropha can be obtained
re) by the pha synthesizing enzyme gene (phaC of Aeromonas caviae
ac) bacterial strain that replaces.As such bacterial strain, the KNK-005 strain etc. prepared in following examples can be listed.
In the present invention, by carbon source exist under breed microorganism of the present invention, can in microbe accumulation copolyester.As carbon source, sugar, grease or lipid acid can be used.As the nutrition source beyond carbon source, nitrogenous source, inorganic salts, other organotrophy source can be used arbitrarily.
Carbohydrate such as has the carbohydrate such as glucose, fructose.Grease comprises and is rich in the grease that carbonatoms is the saturated unsaturated fatty acids of more than 10, such as, have Oleum Cocois, plam oil, palm-kernel wet goods.Fatty acids is if any the derivative of fatty acid such as fat or salt of caproic acid, sad, the saturated unsaturated fatty acids such as capric acid, lauric acid, oleic acid, palmitinic acid, linolic acid, linolenic acid, tetradecanoic acid or these lipid acid.
As nitrogenous source, such as, have the ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, other can also enumerate peptone, meat extract, yeast extract etc.
As inorganic salts, such as, there are potassium primary phosphate, dipotassium hydrogen phosphate, trimagnesium phosphate, magnesium sulfate, sodium-chlor etc.
As other organotrophy source, such as, there are the amino acid such as glycine, L-Ala, Serine, Threonine, proline(Pro); The VITAMIN such as VITMAIN B1, vitamin B12, vitamins C.
Culture temperature, as long as this bacterial strain can the temperature of growth reproduction, but preferably 20 DEG C to 40 DEG C.There is no particular limitation for incubation time, can be about 1-10 day.
The recovery of polyester, can adopt such as following method to carry out.After cultivation terminates, with the separating thallus from nutrient solution such as centrifuge separator, the thalline distilled water of separation and methanol cleaning, and dry.Use organic solvent extracting polyester from these dry thalline such as chloroform.Adopt the methods such as filtration to remove drive member from containing the organic solvent solution of this polyester, in this filtrate, add the poor solvent such as methyl alcohol or hexane with precipitated polyester.Then, the method removing supernatant liquors such as filtration or centrifugation can be adopted, dry, Pillar recovery.
Confirm that the simple and easy method producing polyester can adopt Nile red staining.That is, the nutrient agar of recombinant bacterium growth reproduction adds Nile red, cultivate recombinant bacterium 1-7 days, observe its recombinant bacterium and whether redden, be confirmed whether to create polyester with this.
The effect of invention
The invention provides following recombinant microorganism strain, described recombinant microorganism strain stably can produce to high-performance poly (hydroxy alkanoate) (PHA) in industry fermenting process, and the present invention can be easy, in a large number and manufacture highly purified poly (hydroxy alkanoate) (PHA) at an easy rate.
Accompanying drawing explanation
Fig. 1 is the gene replacement plasmid pBlue-phaC built in display embodiment 1
re:: the schematic diagram of the formation of N149S/D171G-KmSAC.
Fig. 2 is the figure of the program schematically showing the gene replacement carried out in embodiment 2.
the embodiment of invention
Below, by embodiment, the present invention is described, but the present invention is not by the restriction of these embodiments.
The preparation of (embodiment 1) gene replacement plasmid
With the supply source that the thalline of Ralstonia eutropha H16 strain is template DNA, use SEQ IDNO:1 and the primer shown in SEQ ID NO:2 to carry out PCR reaction, obtain containing poly (hydroxy alkanoate) synthase gene (phaC
re) the DNA fragmentation of structure gene.PCR condition is as follows: (1) 94 DEG C 2 minutes, (2) 94 DEG C 30 seconds, (3) 45 DEG C 30 seconds, (4) 72 DEG C 3 minutes, the circulation of 25, (2) ~ (4), (5) 72 DEG C 5 minutes; Use TaKaRa LA Taq (manufacture of TaKaRaBio company) as polysaccharase.The DNA fragmentation of PCR gained restriction enzyme BamHI is cut, be then subcloned into same enzyme cut vector pBluescriptIIKS (-) (manufactures of TOYOBO company) and formation site on (pBlue-phaC
re).
Prepare the N149S/D171G variant of the polyester synthetic enzyme variant gene as Aeromonas caviae source as follows.First, by pBluescriptIIKS (-) (manufacture of TOYOBO company) PstI process, use DNA Blunting Kit (manufacture of TaKaRaBio company) carry out flat end and connect, thus make PstI site deletion.Then, this plasmid is carried out XhoI process, use DNA BluntingKit (manufacture of TaKaRaBio company) carry out flat end and connect, make XhoI site deletion, thus prepared plasmid pBlue-New.The EcoRI site of this plasmid has been cloned the d13 fragment (pBlue-d13) that use same enzyme cuts out from pJRD215-EE32d13 (patent documentation 5).Then, the plasmid (non-patent literature 13) of originating with the clone E2-50 obtained from RIKEN is for template, the combination of primer of use described in SEQ IDNO:3 and 4 and the combination of the primer described in SEQ ID NO:5 and 6, increase respectively by PCR method, obtain 2 fragments.PCR condition be (1) 94 DEG C 2 minutes, (2) 94 DEG C 30 seconds, (3) 55 DEG C 30 seconds, (4) 72 DEG C 2 minutes, the circulation of 25, (2) ~ (4), (5) 72 DEG C 5 minutes.By mole mixing such as 2 fragments amplifying, then carry out PCR reaction, this 2 fragments are combined.PCR condition be (1) 96 DEG C 5 minutes, (2) 95 DEG C 2 minutes, (3) 72 DEG C 1 minute, the circulation of 12, (2) ~ (3), use Pyrobest polysaccharase (manufacture of TaKaRaBio company) as polysaccharase.Cut out from Normal Agarose Gel by the DNA fragmentation of object size, with PstI and XhoI process, the form of replacing with fragment is cloned in the pBlue-d13 with same enzyme process (pBlue-N149S/D171G).Base sequence measures the DNA sequencer 310Genetic Analyzer using PERKIN ELMER APPLIED BIOSYSTEMS company to manufacture to carry out, confirm to obtain mutant gene, wherein, the amino acid of the 149th of pha synthesizing enzyme and l-asparagine are substituted by Serine, the amino acid of the 171st and aspartic acid and are substituted by glycine.This aminoacid sequence is shown in SEQ ID NO:9.
Take pBlue-N149S/D171G as template, use SEQ ID NO:7 and the primer shown in SEQ ID NO:8 to carry out PCR reaction, the structure gene DNA of the N149S/D171G variant that increased.PCR condition be (1) 94 DEG C 2 minutes, (2) 94 DEG C 30 seconds, (3) 45 DEG C 30 seconds, (4) 72 DEG C 2 minutes, the circulation of 25, (2) ~ (4), (5) 72 DEG C 5 minutes, use TaKaRa LA Taq (manufacture of TaKaRaBio company) as polysaccharase.Then, by pBlue-phaC
rewith restriction enzyme SbfI and Csp45I process, to replace phaC with the above-mentioned amplification of DNA fragments of same enzyme process
rethe form of structure gene has carried out cloning (pBlue-phaC
re:: N149S/D171G).
Then, by plasmid pJRD215 (ATCC37533) restriction enzyme XhoI and DraI process, after isolating the DNA fragmentation containing the about 1.3kb of kalamycin resistance gene, use DNA BluntingKit (manufacture of TaKaRaBio company) to end smoothingization, be inserted into and cut pBlue-phaC with restriction enzyme SalI
re:: carry out flat end equally after N149S/D171G and (pBlue-phaC on the site formed
re:: N149S/D171G-Km).
Then, plasmid pMT5071 (Tsuda, GENE, 207:33-41 (1998)), with restriction enzyme NotI process, is isolated the DNA fragmentation of the about 8kb comprising sacB gene, is inserted into and cuts pBlue-phaC by same enzyme
re:: N149S/D171G-Km and on the site formed, thus prepared gene replacement plasmid pBlue-phaC
re:: N149S/D171G-KmSAC.
The gene replacement plasmid pBlue-phaC built in the present embodiment
re:: the structural representation of N149S/D171G-KmSAC is as Fig. 1.
(embodiment 2) gene replaces the preparation of strain
With gene replacement plasmid pBlue-phaC
re:: N149S/D171G-KmSAC transformation of E. coli S17-1 strain (ATCC47005), in the upper mixed culture of Nutrient Agar substratum (Difco company manufactures) together with Ralstonia eutropha H16 strain, carry out conjugal transfer.Select the bacterial strain gone out at the upper growth reproduction of Simmons nutrient agar (Trisodium Citrate 2g/L, sodium-chlor 5g/L, bitter salt 0.2g/L, primary ammonium phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, agar 15g/L, pH6.8) containing 250mg/L kantlex, thus obtain the bacterial strain (homologous recombination of first stage) that plasmid recombinates on the karyomit(e) of Ralstonia eutropha H16 strain.This bacterial strain Nutrient Broth substratum (manufacture of Difco company) was cultivated for 2 generations, then, dilution spread is on the Nutrient Agar substratum containing 15% sucrose, the bacterial strain that growth selection is bred out, obtains the bacterial strain (homologous recombination of subordinate phase) that the region containing selectable marker gene has come off.And, by the analysis of PCR-based, isolate phaC
regene has been replaced to the bacterial strain of N149S/D171G variant gene.This gene is replaced strain called after KNK-005 strain, the DNA sequencer 310Genetic Analyzer using PERKIN ELMER APPLIED BIOSYSTEMS company to manufacture carries out base sequence mensuration, confirms: in the bacterial strain of acquisition, the phaC on karyomit(e)
regene be replaced to from initiator codon to terminator codon N149S/D171G variant gene from initiator codon to terminator codon.
The program that the gene carried out in the present embodiment replaces schematically is shown in Fig. 2.By the homologous recombination of first stage, gene replacement plasmid is inserted in karyomit(e), and by the homologous recombination of subordinate phase, the part being equivalent to this plasmid again becomes ring and departs from from karyomit(e).When the homologous recombination of subordinate phase, depending on there is the position of homologous recombination, there are two kinds of situations---with originally identical substituting group because of the (phaC in figure
re:: the situation (Δ 1) N149S/D171G) departed from together, and, and be substituted the gene (phaC in figure
re) situation (Δ 2) that departs from together, KNK-005 strain of the present invention, is the homologous recombination by carrying out subordinate phase in the position of Δ 2 and the bacterial strain obtained.
The production of (embodiment 3) polyester and purifying
Seed culture medium consist of 1w/v% meat extract, 1w/v% Bacto Tryptone (Bacto-Trypton), 0.2w/v% yeast extract, 0.9w/v%Na
2pO
412H
2o, 0.15w/v%KH
2pO
4, pH6.8.
Front culture medium consist of 1.1w/v%Na
2pO
412H
2o, 0.19w/v%KH
2pO
4, 1.29w/v% (NH
4)
2sO
4, 0.1w/v%MgSO
47H
2o, 2.5w/v%Palm W olein oil, 0.5v/v% trace metal salts solution (dissolve 1.6w/v%FeCl in 0.1N hydrochloric acid
36H
2o, 1w/v%CaCl
22H
2o, 0.02w/v%CoCl
26H
2o, 0.016w/v%CuSO
45H
2o, 0.012w/v%NiCl
26H
2o and obtain).
Production of polyester substratum consist of 0.385w/v%Na
2pO
412H
2o, 0.067w/v%KH
2pO
4, 0291w/v% (NH
4)
2sO
4, 0.1w/v%MgSO
47H
2o, 0.5v/v% trace metal salts solution (dissolves 1.6w/v%FeCl in 0.1N hydrochloric acid
36H
2o, 1w/v%CaCl
22H
2o, 0.02w/v%CoCl
26H
2o, 0.016w/v%CuSO
45H
2o, 0.012w/v%NiCl
26H
2o and obtain), 0.05w/v%BIOSPUREX200K (defoamer: Cognis Japan company manufacture).In carbon source, the low melting point fraction of use palm-kernel oil classification and palm-kernel oil olein are as single carbon source, in whole culturing process, stream adds, and making than matrix feed speed is 0.08-0.1 (grease (g)) × (clean dry thalline weight (g))
-1× (h)
-1.
The glycerol stocks (50 μ l) of inoculation KNK-005 strain in seed culture medium (10ml), cultivate 24 hours, be inoculated in the 3L small-sized fermentation tank (ball water chestnut BIOENG produces, MDL-300 type) that substratum before 1.8L is housed by 1.0v/v%.Operational conditions is culture temperature 30 DEG C, stirring velocity 500rpm, air flow 1.8L/min, and control ph, between 6.7-6.8, is cultivated 28 hours.PH value controls use 7% ammonium hydroxide aqueous solution.
Production of polyester cultivate be front cultivation seed is inoculated in by 5.0v/v% 6L productive culture base is housed 10L small-sized fermentation tank (ball water chestnut BIOENG produces, MDL-1000 type) in.Operating condition is culture temperature 28 DEG C, stirring velocity 400rpm, air flow 3.6L/min, and control ph is between 6.7-6.8.PH value controls use 7% ammonium hydroxide aqueous solution.Cultivation carries out about 48 hours, and cultivate after terminating and reclaim thalline by centrifugation, with methanol cleaning and lyophilize, measuring dry thalline weight is 110.4g/L.
100ml chloroform is added in the dry thalline of about 1g gained, in stirring at room temperature diel, the endobacillary polyester of extracting.After elimination thalline residue, being concentrated into cubic capacity with vaporizer is about 30ml, then slowly adds the hexane of about 90ml, places 1 hour under slow stirring.After the polyester of precipitation is leached, in 50 DEG C of vacuum-dryings 3 hours.Measure the quality of dry polyester, calculate endobacillary amount of polyester.Consequently, the content of the polyester of KNK-005 strain generation in 48 hours is up to 73.8 (wt%).
The estimation of stability of (embodiment 4) production of polyester ability
Cultivation bacterium liquid at the end of the production of polyester cultivation of dilution embodiment 3, is inoculated in NutrientAgar substratum, and the colony inoculation gone out by growth reproduction is in containing Nile red (Nilered) substratum (Sodium phosphate dibasic 12H
2o 9g, potassium primary phosphate 1.5g, ammonium chloride 0.05g, magnesium sulfate 7H
2o 0.02g, fructose 0.5g, cobalt chloride 6H
2o 0.25ppm, iron(ic) chloride (III) 6H
2o 16ppm, calcium chloride 2H
2o10.3ppm, nickelous chloride 6H
2o 0.12ppm, copper sulfate 5H
2o 0.16ppm, Nilered 0.5mg, agar 15g/1L).30 DEG C, cultivate the bacterium colony taken on a red color after 3 days and can be judged to producing polyester.Investigated 100 bacterium colonies, result is: all bacterium colony all keeps production of polyester ability.
Industrial applicibility
The invention provides following recombinant microorganism strain, described recombinant microorganism strain stably can produce to high-performance poly (hydroxy alkanoate) (PHA) in industry fermenting process, and the present invention can be easy, in a large number and manufacture highly purified poly (hydroxy alkanoate) (PHA) at an easy rate.
Claims (6)
1. produce the thunder Bordetella microorganism of copolyester for one kind, described copolyester is made up of the monomeric unit of 3-hydroxybutyrate and 3-hydroxycaproic acid, inherently poly (hydroxy alkanoate) synthase gene on the karyomit(e) of this microorganism, and this poly (hydroxy alkanoate) synthase gene be replaced to exogenous poly (hydroxy alkanoate) synthase gene at least partially, described replacement makes the loss of activity of the enzyme coded by poly (hydroxy alkanoate) synthase gene on karyomit(e), and described exogenous poly (hydroxy alkanoate) synthase gene is expressed, described exogenous poly (hydroxy alkanoate) synthase gene coding derives from enzyme or its variant of Aeromonas caviae (Aeromonas caviae).
2. microorganism according to claim 1, wherein, on described karyomit(e), inherently the microorganism of poly (hydroxy alkanoate) synthase gene is Ralstonia eutropha (Ralstonia eutropha).
3. microorganism according to claim 2, wherein, described Ralstonia eutropha is Ralstonia eutropha H16 strain.
4. microorganism according to claim 1, wherein, described variant has at least carried out the aminoacid replacement of one of following (a), (b):
A amino acid that () is the 149th and l-asparagine are replaced to Serine,
B amino acid that () is the 171st and aspartic acid are replaced to glycine.
5. the microorganism according to any one of claims 1 to 3, wherein, the variant enzyme that described exogenous poly (hydroxy alkanoate) synthase gene coding is originated with the Aeromonas caviae that the aminoacid sequence of SEQ ID NO:9 represents, this variant enzyme is the amino acid of the 149th and l-asparagine is replaced to Serine and the amino acid of the 171st and aspartic acid are replaced to glycine.
6. employ the process for producing polyester of the microorganism according to any one of Claims 1 to 5.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116376856A (en) * | 2022-04-06 | 2023-07-04 | 深圳蓝晶生物科技有限公司 | Engineered microorganisms expressing acetoacetyl-CoA reductase variants and methods of increasing PHA production |
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| WO2005098001A1 (en) * | 2004-04-09 | 2005-10-20 | Kaneka Corporation | Novel transformant |
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2006
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| CN1703515A (en) * | 2002-10-10 | 2005-11-30 | 株式会社钟化 | Process for producing copolyester |
| CN1791673A (en) * | 2003-05-15 | 2006-06-21 | 株式会社钟化 | Improved transformant and process for producing polyester using the same |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116376856A (en) * | 2022-04-06 | 2023-07-04 | 深圳蓝晶生物科技有限公司 | Engineered microorganisms expressing acetoacetyl-CoA reductase variants and methods of increasing PHA production |
| CN116376856B (en) * | 2022-04-06 | 2023-11-03 | 深圳蓝晶生物科技有限公司 | Engineered microorganisms expressing acetoacetyl-CoA reductase variants and methods of increasing PHA production |
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