Method for producing PHA (polyhydroxyalkanoate) by culturing halophilic bacteria in low-salt culture medium
阅读说明：本技术 一种以低盐培养基培养嗜盐菌生产pha的方法 (Method for producing PHA (polyhydroxyalkanoate) by culturing halophilic bacteria in low-salt culture medium ) 是由 陈国强 张李湛 叶健文 黄悟哲 吴赴清 兰宇轩 于 2021-09-28 设计创作，主要内容包括：本发明提供了一种嗜盐菌,其保藏编号为：CGMCC No.22795,保藏日期为2021年06月28日,本发明还提供了该嗜盐菌的制备方法,以及以低盐培养基培养嗜盐菌生产PHA的方法及应用。在本方法中,使用的嗜盐菌是可以通过诱变筛选、分子改造的育种手段获得,所述的低盐培养基中实现添加非相关碳源(与微生物聚羟基脂肪酸脂PHA结构没有直接关系的底物,如葡萄糖)生产包含多种单体PHA方法,且根据碳源的种类调节各单体的比例。本发明可以克服传统嗜盐菌培养分离步骤繁杂、下游高盐污水难以处理等问题,来进行开放发酵生产PHA。(The invention provides halophilic bacteria, which have the preservation numbers as follows: CGMCC No.22795, preservation date of 2021, 06 months and 28 days, the invention also provides a preparation method of the halophilic bacteria, and a method for producing PHA by culturing the halophilic bacteria in a low-salt culture medium and application thereof. In the method, the halophilic bacteria can be obtained by a breeding means of mutagenic screening and molecular modification, a method for producing PHA (polyhydroxyalkanoate) containing various monomers by adding a non-related carbon source (a substrate which is not directly related to the PHA structure of the microbial polyhydroxyalkanoate, such as glucose) in a low-salt culture medium is realized, and the ratio of each monomer is adjusted according to the type of the carbon source. The method can solve the problems of complicated steps of culturing and separating the traditional halophilic bacteria, difficult treatment of downstream high-salinity sewage and the like, and can produce PHA through open fermentation.)
1. A halophilic bacterium, which is characterized in thatHalomonas bluephagenesisThe preservation number is: CGMCC No.22795, with preservation date of 2021, 06 months and 28 days.
2. A halophilic bacterium, wherein the halophilic bacterium is any one of:
A) editing polyhydroxyalkanoate PHA synthase gene on the genome of the halophilic bacteria of claim 1phaCObtainingHalomonas bluephagenesis TD01AB；
B) Editing a 4-hydroxybutyryl-coa transferase gene on the genome of the halophile of claim 1orfZObtainingHalomonas bluephagenesis TD68AB；
C) Editing succinic semialdehyde dehydrogenase gene on the genome of halophilic bacteria according to claim 1sucD4 hydroxybutyric acid dehydrogenase gene4hbdAnd 4-hydroxybutyryl-CoA transferase geneorfZObtainingHalomonas bluephagenesisTD68-194AB；
D) Knock-out of the genome of halophilic bacteria according to claim 1acyl-CoA degrading geneprpCSimultaneous overexpression of succinyl-CoA transferase GenescpABObtainingHalomonas bluephagenesis TY194AB；
E) Overexpression of PHA synthase gene on the genome of halophilic bacteria according to claim 1phaCSimultaneous knock-out of fatty acid degradation genesfadAAndfadBobtainingHalomonas bluephagenesisTDHxAB; or the like, or, alternatively,
F) overexpression of a diol degradation gene on the genome of halophilic bacteria of claim 1pcsHydroxyaldehyde reductasealdDHydroxy acid reductasedhaTThree genes obtainedHalomonas bluephagenesis TDp129AB。
3. A process for producing the halophilic bacteria according to claim 1 or 2, which comprises subjecting a microorganism to the reactionHalomonas bluephagenesis、Halomonas campaniensisOrHalomonas aydingkolgenesisPerforming ARTP mutagenesis and/or low salt pressure screening.
4. A method for producing PHA, comprising using the halophilic bacteria of any one of claims 1 to 2 to produce PHA.
5. The method as claimed in claim 4, which comprises culturing the halophilic bacteria of claim 1 or 2 in a low salt medium containing 5-20g/L salt to produce PHA.
6. The method as claimed in claim 5, wherein the salt in the low-salt medium is selected from one or more of sodium chloride, sodium sulfate, sodium phosphate, sodium nitrate, sodium carbonate, sodium acetate, sodium citrate, and sodium gluconate.
7. The method of claim 5, wherein the low salt medium comprises a non-related carbon source selected from one or a combination of two or more of glucose, gluconic acid, gluconate, starch, glycerol, acetic acid, hexanoic acid, xylose, cellulose hydrolysate, lactose, and fructose, and/or a related carbon source selected from γ -butyrolactone, oleic acid, and/or propionic acid.
8. The method as claimed in claim 4, wherein the PHA is selected from the group consisting of 3-hydroxybutyrate homopolymer PHB, 3-hydroxybutyrate and 4-hydroxybutyrate bipolymer P3HB4HB, 3-hydroxybutyrate, 4-hydroxybutyrate and 3-hydroxyvalerate bipolymer PHBV4HB, a homopolymer or copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate bipolymer PHBHHx, 3-hydroxypropionic acid, the homopolymer of 3-hydroxypropionic acid is P3HP, and the copolymer of 3-hydroxypropionic acid is P (3HB-co-3HP) or PHBHP.
9. The method according to any one of claims 4 to 8,
A) glucose is used as a carbon source, the salt concentration is 5-15g/L, and the product is PHB;
B) gamma-butyrolactone is used as a carbon source, the salt concentration is 5-15g/L, and the mole percentage of 4HB in the product P3HB4HB is 3-20;
C) gamma-butyrolactone and propionic acid are used as carbon sources, the salt concentration is 5-15g/L, the mole percentage of 4HB in the product P (3HB-co-4HB-co-3HV) is 1-20, and the mole percentage of 3HV is 1-20;
D) oleic acid is used as a carbon source, the salt concentration is 5-15g/L, and the mole percentage of HHx in the product PHBHHx is 1-20;
E) propionic acid is used as a carbon source, the salt concentration is 5-15g/L, and the mole percentage of 3HP in the product P (3HB-co-3HP) is 5-50.
Technical Field
The invention relates to the technical field of biochemistry, in particular to a novel halophilic bacteria (preservation number is CGMCC No.22795, and the classification is named as:Halomonas bluephagenesisand the preservation date is 28/06/2021), and culturing halophilic bacteria in a low-salt medium by using a non-relevant carbon source (such as glucose and the like) to produce PHB, P3HB4HB, P (3HB-co-4HB-co-3HV), PHBHHx, a homopolymer P3HP containing 3HP or a copolymer P (3HB-co-3HP) (or PHBHP), and a method for regulating monomer components of various PHAs.
Background
Polyhydroxyalkanoates (PHAs) are degradable polyesters that can be accumulated by various bacteria, and are classified into short-chain and/or medium-long-chain PHAs, as well as homopolymers and copolymers, depending on their monomer composition. PHA is an environment-friendly material, has wide application prospect, and can be used as an environment-friendly substitute for the traditional petroleum-based plastics. Currently, the industry often uses biofermentation methods to obtain PHA-enriched bacteria, followed by complex purification work.
PHA is produced by Eubacterium reuteri (R) ((R))Ralstonia eutropha) Genetically modified Escherichia coli (A)E. coli) And halophilic bacteria. Although the thallus in the fermentation liquor is rich in PHA, the extraction, purification and impurity removal processes of PHA are complex, and the waste water after PHA is extracted by using halophilic bacteria has high salt content, so that the waste water treatment is very complicated, and the further improvement is realizedThe production cost of PHA is reduced.
The current process for producing PHA by using halophilic bacteria usually uses seawater or culture medium with salt content of 3-6% (w/v) (30 g/L) for fermentation Production, which can be referred to as the Pilot Scale-up of Poly (3-hydroxybutyric-co-4-hydroxybutyric) Production byHalomonas bluephagenesisvia Cell Growth adaptation optimization Process (Ye J, Huang W, Wang D, et al, Biotechnology Journal, 2018, 13). CN111117906A discloses an improved microbial cultivation method, which adds sodium salt and carbon source in the basic culture medium to maintain the high salinity environment of halophilic bacteria, wherein, the concentration of sodium salt is 5g/L-100 g/L. Due to the high salt concentration of the wastewater, the treatment costs are high, which increases the production costs of PHA.
Physical rays, such as art p technology, is a short for normal temperature and pressure plasma technology, and the technology uses the radio frequency glow discharge principle to generate high-energy plasma under normal temperature and pressure, and the high-energy chemical active particles rich in the plasma can generate high-strength genetic material damage to strains/plants/animal cells, and further uses the cell-initiated SOS high fault tolerance repair mechanism to generate various mismatched sites, and finally forms a mutant diverse library through stable inheritance, which can be referred to in literature, Atmospheric and room temperature plasma (art p) as a new powerfull reagent (Zhang X, Li H, et al, applied microbial biotechnology, 2014, 98). .
At present, a means combining genetic engineering and plasma irradiation (ARTP) mutation screening breeding is one of novel means for transforming PHA engineering production bacteria. The microbial low-salt fermentation production of binary or multi-element PHA (polyhydroxyalkanoate) such as PHB (polyhydroxybutyrate), P3HB4HB and the like is developed, and the method has important significance for reducing the cost and controlling the process.
Disclosure of Invention
In a first aspect of the invention, a method for producing PHA is provided, comprising producing PHA using a halophilic bacteria.
Preferably, the method comprises culturing the halophile in a low salt medium to produce PHA.
The content of salt in the low-salt culture medium is 5-20 g/L.
Preferably, the salt content in the low-salt culture medium is 5-15 g/L.
In one embodiment of the invention, the low salt medium has a salt content of 5-10 g/L.
Preferably, the salt in the low-salt medium is one or a combination of more than two of sodium chloride, sodium sulfate, sodium phosphate, sodium nitrate, sodium carbonate, sodium acetate, sodium citrate and sodium gluconate.
In one embodiment of the invention, the salt is selected from sodium chloride and/or sodium sulphate.
Preferably, the low salt medium comprises a non-related carbon source and/or a related carbon source.
The non-related carbon source is selected from one or more of glucose, gluconic acid, gluconate (preferably sodium gluconate, potassium gluconate or calcium gluconate, etc.), gluconate, starch, glycerol, acetic acid, caproic acid, xylose, cellulose hydrolysate, lactose and fructose.
The relevant carbon source is selected from one or more of gamma-butyrolactone, oleic acid and/or propionic acid, and other medium-long chain fatty acids.
The low salt medium can be prepared by adding a carbon source related or unrelated to product synthesis to the basal medium, for example, the final medium can comprise glucose, gluconic acid, gluconate or a combination thereof as a carbon source for halophilic bacteria. Preferably, the medium comprises glucose as a carbon source. Optionally, the medium may or may not also contain a carbon source other than glucose.
The concentration of the non-relevant carbon source or the relevant carbon source as the carbon source may be appropriately adjusted by those skilled in the art according to the culture conditions and halophilic bacteria used, and may be, for example, in the range of about 1 to 100g/L, about 1 to 90g/L, about 1 to 80g/L, about 1 to 70g/L, or about 1 to 60 g/L; preferably, the concentration may be in the range of about 3-60g/L, about 3-50g/L, or about 3-40 g/L; more preferably in the range of about 5-60g/L, about 10-60g/L, about 20-40g/L, including for example about 25.5-34.5 g/L. It is to be understood that the above concentration ranges are not exhaustive, but may be appropriately adjusted by those skilled in the art through experiments according to the conditions of the fermentation system, and are included in the scope of the present invention as long as they do not adversely affect the object of the present invention.
The salt in the low-salt medium may be added to the basal medium before the start of the halophilic bacteria culture. Alternatively, it may be added to the medium once, in batches or in streams during the cultivation of the halophilic bacteria. Further alternatively, it may be added to the medium at the same time as the halophilic bacteria are inoculated to the medium.
Preferably, the low salt medium may also contain a conditioning substance, such as acetic acid, acetate salt, acetate ester or other acetic acid derivative, which may be added to the basal medium before the start of the microbial culture. Alternatively, acetic acid, acetate or other acetic acid derivatives may be added to the medium once, in portions or in streams during the cultivation of the microorganism. Alternatively, the acetic acid, acetate ester or other acetic acid derivative may be added to the culture medium at the same time as the microorganism is inoculated into the culture medium. The acetic acid, acetate salt, acetate ester or other acetic acid derivative may be added at a concentration, for example, in the range of about 1-12g/L, about 1-11g/L, about 1-10g/L, about 1-9g/L, or about 1-8 g/L; preferably about 1-8g/L, for example, in the range of about 2-8g/L, about 2-7g/L, about 2-6g/L, about 2-5g/L, about 2-4g/L, or 2-3 g/L; more preferably 2-6g/L, for example in the range of about 3-6g/L or about 3-5 g/L. These concentrations may be appropriately adjusted depending on factors such as the composition of the medium and the culture conditions on the basis of the present invention as long as they do not affect the effects of the present invention.
The PHA is homopolymerized PHA and/or copolymerized PHA. Preferably, the PHA is selected from the group consisting of 3-hydroxybutyrate (3 HB) homopolymer PHB, 3-hydroxybutyrate (3 HB) and 4-hydroxybutyrate (4HB) bipolymer P3HB4HB, 3-hydroxybutyrate (3 HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate tripolymer P (3HB-co-4HB-co-3HV), homopolymers or copolymers of 3-hydroxybutyrate (3 HB) and 3-hydroxyhexanoate bipolymer PHBHHx, 3-hydroxypropionic acid (3 HP),
preferably, the homopolymer of 3-hydroxypropionic acid (3 HP) is P3HP,
preferably, the copolymer of the 3-hydroxypropionic acid (3 HP) is P (3HB-co-3HP) or PHBHP.
The halophilic bacteria may be wild-type (i.e., in its native form under suitable culture conditions capable of synthesizing a polymer, such as PHA, using an appropriate substrate), or recombinant, artificially engineered, including but not limited to, bacteria obtained by mutagenesis, genetic engineering, and the like. For example, in the case of recombinant type, the halophilic bacteria may be those which have been artificially transformed (for example, by introducing a gene involved in PHA synthesis) into a microorganism incapable of synthesizing PHA by itself, and which are capable of synthesizing a polymer using an appropriate substrate under appropriate culture conditions, for example, those obtained by introducing the synthetic gene phaC gene of polyhydroxybutyrate into Escherichia coli. It may also be in a wild-type microorganism capable of synthesizing PHA (e.g.in a wild-type microorganismHalomonas bluephagenesis TD01) by random mutagenesis and directed screening of the entire genome of the strain, more particularly, for example, by screening in the absence of a suitable carrierHalomonas bluephagenesis(preferably TD01, accession number CGMCC No. 4353),Halomonas campaniensis(preferably LS21, accession number CGMCC No. 6593),Halomonas aydingkolgenesis(preferably M1, preservation number CGMCC NO. 19880) or Pseudomonas putida (Pseudomonas putida) The bacteria thus obtained are mutagenized and screened on the basis of (1). For example, the halophilic bacteria involved in the method of the present invention may beHalomonas bluephagenesis TD01 (preferably with preservation number of CGMCCNo.4353), or atHalomonas bluephagenesis The engineered bacteria obtained by mutagenesis, genetic engineering, etc. based on TD01 can be obtained by plasma irradiation (ARTP), other physical methods, chemical mutagens, and molecular engineering gene recombination techniques. As another embodiment of the present invention, the halophilic bacteria may beHalomonas bluephagenesis TDH4-ΔphaPOr is atHalomonas bluephagenesis TDH4-ΔphaPOn the basis of the method of mutagenesis, genetic engineering modification and the likeThe resulting engineered bacteria are obtained, for example, by plasma irradiation (ARTP), other physical methods, and chemical mutagens and molecular engineering gene recombination techniques. As another embodiment of the present invention, the halophilic bacteria may beHalomonas bluephagenesisTDH4ABP (preservation number CGMCC No. 22795), orHalomonas bluephagenesisEngineering bacteria obtained by mutagenesis, genetic engineering modification and other methods on the basis of TDH4ABP (preservation number of CGMCC No. 22795) are obtained by plasma irradiation (ARTP), other physical methods, chemical mutagens and molecular modification gene recombination technology.
Preferably, the mutagenesis, genetic engineering is to modulate the expression of genes that regulate the anabolic pathways of multiple PHA products, wherein the genes includesucD、ogdA、4hbD、orfZ、gabD、phaA、phaB、phaCAnd one or more or all of the beta oxidation pathways of fatty acids. The enzymes encoded by them are succinate semialdehyde synthase, alpha ketoglutarate decarboxylase, 4-hydroxybutyrate dehydrogenase, CoA transferase, succinate semialdehyde degrading enzyme, beta-ketothiolase, NADH type acetoacetyl-CoA reductase and PHA synthase, respectively. Wherein the content of the first and second substances, sucD、ogdA、4hbD、orfZ、phaA、phaBandphaCgenes and the like are overexpressed, andgabDdeletion or attenuation of expression of a Gene in beta oxidation pathway。sucD、ogdA、4hbD、orfZ、 phaA、phaBAndphaCgenes and the like may be overexpressed on plasmids and/or chromosomes, and the expression may be constitutive or inducible. Wherein the content of the first and second substances,phaCthe gene codes polyhydroxyalkanoate PHA synthase;orfZa gene encoding 4-hydroxybutyryl-coa transferase;sucDa gene encoding succinic semialdehyde dehydrogenase;4hbdencoding 4-hydroxybutyrate dehydrogenase;prpCencoding propionyl-CoA degradation protein.
In one embodiment of the invention, the halophilic bacteria are subjected to ARTP mutagenesis and/or low salt pressure screening.
Preferably, the halophilic bacteria is halophilic bacteriaHalomonasMore preferablyHalomonas bluephagenesis TD01 (Collection edition)Number: CGMCC No. 4353), Halomonas campaniensisLS21 (accession number: CGMCC No. 6593) andHalomonas aydingkolgenesis m1 (accession number: CGMCC NO. 19880).
In one embodiment of the present inventionHalomonas bluephagenesis TD01、Halomonas bluephagenesis TD68、Halomonas bluephagenesis TD68-194、Halomonas bluephagenesis TDH 4-delta phaP (TDH 4P for short)、Halomonas bluephagenesis TY194、Halomonas bluephagenesis TDHx orHalomonas bluephagenesis TDp129。
In one embodiment of the invention, the composition will beHalomonas bluephagenesis TD01、Halomonas bluephagenesis TD68、Halomonas bluephagenesis TD68-194、Halomonas bluephagenesis TDH4-ΔphaP、Halomonas bluephagenesis TY194、Halomonas bluephagenesis TDHx orHalomonas bluephagenesisTDp129 was subjected to ARTP mutagenesis and low salt pressure screening, and the strains selected were named separatelyHalomonas bluephagenesis TD01AB、Halomonas bluephagenesis TD68AB、Halomonas bluephagenesis TD68-194AB、Halomonas bluephagenesis TDH4-ΔphaPAB、Halomonas bluephagenesis TY194AB、Halomonas bluephagenesis TDHxAB orHalomonas bluephagenesis TDp129AB。
In one embodiment of the present invention, the halophilic bacteria isHalomonas bluephagenesis TDH4-ΔphaPAB(TDH 4ABP, taxonomic name:Halomonas bluephagenesisthe preservation number is: CGMCC No.22795, wherein the preservation date is 28 days 06 months 2021, the preservation unit is China general microbiological culture Collection center (CGMCC) of culture Collection of microorganisms, and the address is No. 3 Siro No.1 of Xilu, North Kyoto area of Chaoyang, Beijing), or the halophilic bacteria are strains derived by taking TDH4ABP as a chassis, and specifically comprise:
A) editing polyhydroxyalkanoate PHA synthase gene on TDH4ABP genomephaCObtainingHalomonas bluephagenesis TD01AB；
B) Editing 4-hydroxybutyryl-coa transferase gene on TDH4ABP genomeorfZObtainingHalomonas bluephagenesis TD68AB；
C) Editing of 4-hydroxybutyrate dehydrogenase Gene on TDH4ABP genome4hbd、Succinic semialdehyde dehydrogenase genesucDAnd 4-hydroxybutyryl-CoA transferase geneorfZObtainingHalomonas bluephagenesis TD68-194AB；
D) Knock-out of propionyl coenzyme A degradation gene on TDH4ABP genomeprpCSimultaneous overexpression of succinyl-CoA transferase GenescpABObtainingHalomonas bluephagenesis TY194AB；
E) Over-expressing PHA synthase gene phaC on TDH4ABP genome and knocking out fatty acid degradation genefadAAndfadBobtainingHalomonas bluephagenesis TDHxAB; or the like, or, alternatively,
F) overexpression of diol degradation genes on TDH4ABP genomepcs、Hydroxy aldehyde reductasealdD、Hydroxy acid reductasedhaTThree genes obtainedHalomonas bluephagenesis TDp129AB。
The halophilic bacteria may be cultured under appropriate culture conditions (temperature, rotation speed, dissolved oxygen, pH, etc.) as long as the culture enables synthesis of the desired PHA polymer. For example, the temperature and the rotation speed during the culture can be appropriately set by those skilled in the art according to the characteristics of the microorganism or selected by routine optimization experiments.
In one embodiment of the invention, glucose (preferably at a concentration of 3-10g/L, in one embodiment of the invention at a concentration of 5 g/L) is used as carbon source, the salt concentration is 5-15g/L (preferably 8-10 g/L) and the product is PHB. The formula of the low-salt culture medium is as follows: 0.1-2 ‰ (NH)4)2SO4Or urea, 0.1-1 MgSO4，5‰-10‰Na2HPO4·12H2O，0.5‰-2‰KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH4)2SO4Or 0.2% urea, 0.02% MgSO4，1.0％Na2HPO4·12H2O，0.15％KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2O) (pH adjusted to about 9.0), and glucose 3%, NaCl 1%.
In one embodiment of the invention, gamma-butyrolactone (preferably at a concentration of 3 to 10g/L, in one embodiment of the invention at a concentration of 5 g/L) is used as the carbon source, the salt concentration is 5 to 15g/L (preferably 8 to 10 g/L), and the molar percentage of 4HB in the product P3HB4HB is 3 to 20 (preferably 10 to 15). The formula of the low-salt culture medium is as follows: 0.1-2 ‰ (NH)4)2SO4Or urea, 0.1-1 MgSO4，5‰-10‰Na2HPO4·12H2O，0.5‰-2‰KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH4)2SO4Or 0.2% urea, 0.02% MgSO4，1.0％Na2HPO4·12H2O，0.15％KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2O) (pH adjusted to about 9.0), and glucose 2%, γ -butyrolactone 0.5%, NaCl 1%.
In one embodiment of the present invention, gamma-butyrolactone and propionic acid (preferably at a concentration of 3 to 10g/L, and in one embodiment of the present invention, at a concentration of 5 g/L) are used as carbon sources, the salt concentration is 5 to 15g/L (preferably 8 to 10 g/L), the molar percentage of 4HB in the product P (3HB-co-4HB-co-3HV) is 1 to 20 (preferably 2 to 7), and the molar percentage of 3HV is 1 to 20 (preferably 1 to 7). The formula of the low-salt culture medium is as follows: 0.1-2 ‰ (NH)4)2SO4Or urea, 0.1-1 MgSO4，5‰-10‰Na2HPO4·12H2O，0.5‰-2‰KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH4)2SO4Or 0.2% urea, 0.02% MgSO4，1.0％Na2HPO4·12H2O，0.15％KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2O) (pH adjusted to about 9.0), and glucose 2%, γ -butyrolactone 0.5%, propionic acid 0.5%, NaCl 1%.
In one embodiment of the invention, oleic acid (preferably at a concentration of 3 to 10g/L, in one embodiment of the invention, concentratedDegree of 5 g/L) as carbon source, salt concentration of 5-15g/L (preferably 8-10 g/L), and mole percentage of HHx in product PHBHHx of 1-20 (preferably 2-7). The formula of the low-salt culture medium is as follows: 0.1-2 ‰ (NH)4)2SO4Or urea, 0.1-1 MgSO4，5‰-10‰Na2HPO4·12H2O，0.5‰-2‰KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH4)2SO4Or 0.2% urea, 0.02% MgSO4，1.0％Na2HPO4·12H2O，0.15％KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2O) (pH adjusted to about 9.0), and oleic acid 1%, NaCl 1%.
In one embodiment of the invention, propionic acid (preferably at a concentration of 3 to 10g/L, in one embodiment of the invention at a concentration of 5 g/L) is used as the carbon source, the salt concentration is 5 to 15g/L (preferably 8 to 10 g/L), and the molar percentage of 3HP in the product P (3HB-co-3HP) is 5 to 50 (preferably 12 to 40). The formula of the low-salt culture medium is as follows: 0.1-2 ‰ (NH)4)2SO4Or urea, 0.1-1 MgSO4，5‰-10‰Na2HPO4·12H2O，0.5‰-2‰KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH4)2SO4Or 0.2% urea, 0.02% MgSO4，1.0％Na2HPO4·12H2O，0.15％KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH)4-Citrate，CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2O) (pH adjusted to about 9.0), and propionic acid 2%, NaCl 1%.
In a second aspect of the invention, there is provided a PHA obtained by the above method.
In a third aspect of the invention, there is provided the use of a PHA as described above for the preparation of a new biodegradable material. Preferably in the development of medical devices, medical microspheres, surgical sutures, patches, disposable packaging materials or textile fibres.
According to the fourth aspect of the invention, the recombinant bacterium is prepared by subjecting halophilic bacteria to ARTP mutagenesis and/or low-salt pressure screening.
Preferably, the halophilic bacteria is halophilic bacteriaHalomonasMore preferablyHalomonas bluephagenesis TD01 (preservation number: CGMCC. number 4353),Halomonas campaniensisLS21 (accession number: CGMCC No. 6593) andHalomonas aydingkolgenesis m1 (accession number: CGMCC NO. 19880).
In one embodiment of the present inventionHalomonas bluephagenesis TD01、Halomonas bluephagenesis TD68、Halomonas bluephagenesis TD68-194、Halomonas bluephagenesis TDH4-ΔphaP、Halomonas bluephagenesis TY194、Halomonas bluephagenesis TDHx orHalomonas bluephagenesis TDp129。
In one embodiment of the invention, the composition will beHalomonas bluephagenesis TD01、Halomonas bluephagenesis TD68、Halomonas bluephagenesis TD68-194、Halomonas bluephagenesis TDH4-ΔphaP、Halomonas bluephagenesis TY194、Halomonas bluephagenesis TDHx orHalomonas bluephagenesisTDp129 was subjected to ARTP mutagenesis and low salt pressure screening, and the strains selected were named separatelyHalomonas bluephagenesis TD01AB、Halomonas bluephagenesis TD68AB、Halomonas bluephagenesis TD68-194AB、Halomonas bluephagenesis TDH4-ΔphaPAB(TDH 4ABP, preservation number: CGMCC No.22795, preservation date: 2021, 06 months and 28 days, preservation unit is China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center, address: Beijing City, Chaoyang district, Beichen Xilu No.1 Hospital No. 3)、Halomonas bluephagenesis TY194AB、Halomonas bluephagenesis TDHxAB orHalomonas bluephagenesis TDp129AB。
In a fifth aspect of the present invention, there is provided a halophilic bacterium, which is classified and named as:Halomonas bluephagenesisthe preservation number of the halophilic bacteria is as follows: CGMCC No.22795, with preservation date of 28/06/2021, the preservation unit is China general microbiological culture Collection center (CGMCC), and the address is No. 3 Xilu No.1 North Chen of the sunward area in Beijing.
In a sixth aspect of the present invention, there is provided a method for culturing PHA-producing halophilic bacteria in a low-salt culture medium, said halophilic bacteria being classified and named as:Halomonas bluephagenesisthe halophilic bacteria isHalomonas bluephagenesisTDH 4-delta phaPAB (TDH 4ABP for short), wherein the preservation number of the halophilic bacteria is as follows: CGMCC No.22795, with preservation date of 28/06/2021, the preservation unit is China general microbiological culture Collection center (CGMCC), and the address is No. 3 Xilu No.1 North Chen of the sunward area in Beijing.
In a seventh aspect of the present invention, there is provided a halophilic bacterium, wherein said halophilic bacterium isHalomonas bluephagenesisThe halophilic bacteria is any one of the following bacteria:
A) editing polyhydroxyalkanoate PHA synthase gene on TDH4ABP genomephaCObtainingHalomonas bluephagenesis TD01AB；
B) Editing 4-hydroxybutyryl-coa transferase gene on TDH4ABP genomeorfZObtainingHalomonas bluephagenesis TD68AB；
C) Editing of 4-hydroxybutyrate dehydrogenase Gene on TDH4ABP genome4hbd、Succinic semialdehyde dehydrogenase genesucDAnd 4-hydroxybutyryl-CoA transferase geneorfZObtainingHalomonas bluephagenesis TD68-194AB；
D) Knock-out of propionyl coenzyme A degradation gene on TDH4ABP genomeprpCSimultaneous overexpression of succinyl-CoA transferase GenescpABObtainingHalomonas bluephagenesis TY194AB；
E) Over-expressing PHA synthase gene phaC on TDH4ABP genome and knocking out fatty acid degradation genefadAAndfadBobtainingHalomonas bluephagenesisTDHxAB; or the like, or, alternatively,
F) overexpression of diol degradation genes on TDH4ABP genomepcs、Hydroxy aldehyde reductasealdD、Hydroxy acid reductasedhaTThree genes obtainedHalomonas bluephagenesis TDp129AB。
The eighth aspect of the present invention provides a method for preparing the halophilic bacteria, comprising the steps ofHalomonas bluephagenesis、Halomonas campaniensisOrHalomonas aydingkolgenesisPerforming ARTP mutagenesis and/or low salt pressure screening.
The ninth aspect of the invention provides an application of the halophilic bacteria or recombinant bacteria in PHA production.
In a tenth aspect, the invention relates to a method for the regulation of monomer composition in PHA production, said method comprising fermenting under suitable conditions a source of non-related and/or related carbon as a source of carbon for the production of PHA.
The method of the present invention uses non-related carbon source (such as glucose) or its mixture with related carbon source as carbon source, and produces PHA through fermentation in low salt culture medium (salt content is 5-20 g/L). At the same time, the ratio of the monomers in PHA is freely adjusted by the specific carbon source used and the difference in the ratio. The method has the advantages of mild process, low cost, low requirement on equipment and simple production environment, and most importantly, the salt content is low, the specific gravity difference between water and thalli is large, the downstream wastewater treatment cost is extremely low, the equipment is simple, a better separation effect can be realized under the same condition, and large-scale industrial production can be realized.
PHA as referred to herein means polyhydroxyalkanoate, which can be classified into homopolymers and copolymers according to monomer composition. Depending on the number of carbon atoms of the monomer, the PHA of the present invention may be a short chain PHA (i.e., a hydroxy fatty acid monomer C3-C5) or a medium-long chain PHA (i.e., a hydroxy fatty acid monomer C6-C18), but is not limited thereto. The PHA synthesized in the methods of the invention can be a homopolymer, a copolymer, or a combination thereof. In some embodiments of the invention, the PHA may be a homopolymer, including but not limited to polyhydroxypropionate, Polyhydroxybutyrate (PHB), polyhydroxyvalerate, and the like, for example, poly-3-hydroxybutyrate (PHB), poly-4-hydroxybutyrate (P4 HB), poly-3-hydroxypropionate (P3 HP), or poly-3-hydroxyvalerate (P3 HV), and the like. In some embodiments of the invention, the PHA may be a copolymer, which may be a dimer, a trimer, but is not limited thereto, for example, the copolymer may be a copolymer of a hydroxypropionate ester and a hydroxybutyrate ester; copolymers of a hydroxy propionate and a hydroxy valerate; a copolymer of hydroxybutyrate and hydroxyvalerate; hydroxy propionate, hydroxy butyrate, hydroxy valerate, and the like. More specifically, it may be poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), poly (3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV), or the like.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: the metabolic pathways involved in various PHA products.
Detailed Description
The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art.
The chemicals used in the following reactions are all commercially available products unless otherwise indicated.
The product purity, such as the content (wt%) of P3HB4HB, referred to herein is the mass of P3HB4HB as a percentage of the mass of the ice-dried cells participating in the esterification, where the mass of P3HB4HB is the total mass of 3HB and 4HB obtained after esterification.
The product recovery rate mentioned here is the ratio of the quality of the freeze-dried thallus after wall breaking, purification and collection to the thallus after the direct freeze-drying of the fermentation product.
The term "plasmid" is used herein to denote a plasmid vector overexpressed gene, and the term "chromosome" is used herein to denote a genome integration overexpressed gene.
Halomonas bluephagenesisTD01 (TD 01) is a gene editing polyhydroxyalkanoate PHA synthase on TDH4 genomephaCAnd (4) obtaining the product. TD01AB is TD01 which is subjected to two rounds of ARTP mutagenesis and low salt pressure screening to obtain a strain which can grow and accumulate PHA in a low salt medium.
Halomonas bluephagenesisTD68 (TD 68) is a gene obtained by editing 4-hydroxybutyryl-CoA transferase on TDH4 genomeorfZAnd (4) obtaining the product. TD68AB is a strain obtained by two rounds of ARTP mutagenesis and low salt pressure screening of TD68, which can grow and accumulate PHA in low salt medium.
Halomonas bluephagenesisTD68-194 (TD 68-194) is a gene editing succinic semialdehyde dehydrogenase on TDH4 genomesucD4 hydroxybutyric acid dehydrogenase gene4hbdAnd 4-hydroxybutyryl-CoA transferase geneorfZAnd (4) obtaining the product. TD68-194AB is a strain obtained by two rounds of ARTP mutagenesis and low salt pressure selection of TD68-194, and capable of growing and accumulating PHA in low salt medium。
Halomonas bluephagenesis TY194 (TY 194 for short) is modified on the basis of TDH4, the content of 3HB is increased, and P (3HB-co-4HB-co-3HV) can be synthesized after 3HV and 4HB related carbon sources are added. Particularly inHalomonas bluephagenesis TDH4-ΔphaPGenome knockoutprpCGenes, at the same time over-expressed on the genomescpABA gene. TY194AB is a strain that was obtained after two rounds of ARTP mutagenesis and low salt pressure selection on TY194 and that could grow and accumulate PHA in low salt medium. The function is similar to TY194, and P (3HB-co-4HB-co-3HV) can be synthesized after adding 3HV and 4HB related carbon sources. Reference may be made to the Chromosome engineering of the TCA cycle inHalomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV)（Chen Y, Chen X, Ye J, et al., Metabolic Engineering, 2019, 54，69-82.）
Halomonas bluephagenesis TDHx (TDHx for short) is modified on the basis of TDH4, the content of 3HB is increased, and PHBHHx can be synthesized after HHx related carbon source is added. Particularly inHalomonas bluephagenesis TDH4-ΔphaPOver-expression on genomephaCGene with deletion of fatty acid degradation genefadAAndfadB. PHBHHx can be synthesized after adding HHx-related carbon source. TDHxAB is a strain which can grow and accumulate PHA in a low-salt culture medium after TDHx is subjected to two rounds of ARTP mutagenesis and low-salt pressure screening. The function of the PHBHHx is similar to that of TDHx, and the PHBHHx can be synthesized after the relevant carbon source of HHx is added. Reference may be made to Engineering of the diversity of polyesters (Meng D, Shen R, Yao H, et al, Current Opinion in Biotechnology, 2014, 29, 24-33).
Halomonas bluephagenesis TDp129 (TDp 129 for short) is modified on the basis of TDH4, the content of 3HB is increased, and P (3HB-co-3HP) can be synthesized after 3HP related carbon sources are added. Particularly inHalomonas bluephagenesis TDH4-ΔphaPOver-expression on genomepcs、aldD、dhaTThree 3HP synthesis related genes, and P (3HB-co-3HP) with the 3HP molar ratio of 40% can be synthesized. TDp129AB is TDp129 passing two rounds AAfter RTP mutagenesis and low salt pressure screening, strains which can grow and accumulate PHA in a low salt medium are obtained. The function of the product is similar to that of TDp129, and P (3HB-co-3HP) can be synthesized after a 3HP related carbon source is added. Reference may be made to the Hyperproduction of 3-hydroxyproprionate byHalomonas bluephagenesis（Jiang X, Yan X,Yu L, et al., Nature Communications, 2021, 12，1513-1526）。
All the strains post AB represent low-salt versions corresponding to the original strains, i.e., strains which can grow and accumulate PHA in a low-salt medium after two rounds of ARTP mutagenesis and low-salt pressure screening.
The culture medium mentioned herein:
basic culture medium: refers to a medium suitable for culturing a microorganism and for the microorganism to synthesize polyhydroxyalkanoate using a carbon source added to the medium, such as MM medium, LB medium, mineral medium, and the like, but is not limited thereto. The formulation of these media is routinely known to those skilled in the art, and those skilled in the art can routinely make appropriate adjustments to their components or component concentrations. In this context, unless otherwise specified, the medium or basal medium used for culturing the microorganism to synthesize the desired product is referred to as liquid medium.
The general formulation of LB liquid medium is: 4-6g/L yeast extract, 8-12g/L peptone, 8-12g/L NaCl, and the balance of distilled water (pH adjusted to 7.0-7.2); preferably: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl, and the balance distilled water (pH adjusted to 7.0-7.2).
The general formulation of MM liquid medium is: 0.1-2 ‰ (NH4)2SO4Or urea, 0.1-1 MgSO4，5‰-10‰Na2HPO4·12H2O，0.5‰-2‰KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH4-Citrate, CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH4)2SO4Or 0.2% urea, 0.02% MgSO4，1.0％Na2HPO4·12H2O，0.15％KH2PO4Not more than 0.1% of other trace elements (Fe (III) -NH4-Citrate, CaCl2·2H2O，ZnSO4·7H2O，MnCl2·4H2O，H3BO3，CoCl2·6H2O，CuSO4·5H2O，NiCl2·6H2O，NaMoO4·2H2O) (pH adjusted to about 9.0).
10LB medium: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl and the balance of distilled water; adjusting the pH value to 7.0-7.2; then autoclaved.
10MMG medium: preparing NaCl solution of yeast extract, wherein the concentration of the yeast extract is 1g/L, and the concentration of NaCl is 10 g/L; after dissolution, autoclaving; after cooling, 1mL of component I was added per 50mL of solution (to 10g (NH4)2SO4And 2g MgSO4Adding distilled water to a volume of 200mL, followed by autoclaving) and 1mL of component II (to 96.5g Na)2HPO4·12H2O and 15g KH2PO4Adding distilled water to a constant volume of 200mL, and then sterilizing by high-pressure steam); finally, the pH of the system was adjusted to about 9.0 with 5M aqueous NaOH.
60LB medium: 5g/L of yeast extract, 10g/L of peptone, 60g/L of NaCl and the balance of distilled water; adjusting the pH value to 7.0-7.2; then autoclaved.
60MMG medium: preparing NaCl solution of yeast extract, wherein the concentration of the yeast extract is 1g/L, and the concentration of NaCl is 60 g/L; after dissolution, autoclaving; after cooling, 1mL of component I was added per 50mL of solution (to 10g (NH4)2SO4And 2g MgSO4Adding distilled water to a volume of 200mL, followed by autoclaving) and 1mL of component II (to 96.5g Na)2HPO4·12H2O and 15g KH2PO4Adding distilled water to a constant volume of 200mL, and then sterilizing by high-pressure steam); finally, the pH of the system was adjusted to about 9.0 with 5M aqueous NaOH.
ARTP mutagenesis and low salt pressure screening referred to herein: the mutagenesis carrier gas is helium, the air input is 1 slm, the machine working power is 115w, and the irradiation is carried out for 1-10 min. Setting four salt concentration gradients of 0, 5, 7 and 10 at low salt, and only drying under the pressure condition of 10g/L to obtain the target strain.
Example 1: culturing in low-salt culture medium to produce PHB-containing halophilic bacteria, and extracting purified PHA from fermentation liquor
The present invention is characterized by adding glucose as carbon source into halophilic bacteria (see Table 1), and PHA synthase (in the cells themselves)phaC) Under the action, the PHB is polymerized into PHB by self-metabolism to generate 3 HB-CoA.
TD01, TD01AB, TDH4P, TDH4ABP, TD68, TD68AB, TD68-194 and TD68-194AB were cultured in 10LB and TD01, TDH4P, TD68 and TD68-194 in 60LB medium at 37 ℃ and 200rpm for 12 hours, respectively, and then inoculated in 50mL of 10MMG and 60MMG media at 1% concentration, and at the same time, 30g/L of glucose was added as a carbon source to culture for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 1 below:
TABLE 1 variousHalomonas bluephagenesisSynthesis of PHA (PHB) by low-salt recombinant bacteria
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, and meanwhile, the cell dry weight and the PHA content of the product are greatly improved.
Example 2: culturing in low-salt culture medium to produce halophilic bacteria thallus containing P3HB4HB, and extracting purified PHA from fermentation liquor
The invention is realized byHalomonas bluephagenesisTD incorporates 4-hydroxybutyryl-CoA transferase (orfZ) PHA synthase(s) to synthesize 4HB-CoA and to the cells themselvesphaC) Under the action, the copolymer is copolymerized with 3HB-CoA produced by self metabolism into P (3HB-co-4 HB).
TD01, TD01AB, TDH4P, TDH4ABP, TD68, TD68AB, TD68-194 and TD68-194AB are respectively cultured in 10LB, TD01, TDH4P, TD68 and TD68-194 are respectively cultured in 60LB culture medium at 37 ℃ and 200rpm for 12 hours, and then the culture medium is inoculated into 50mL of 10MMG and 60MMG culture medium according to 1 percent, and simultaneously 5g/L of gamma-butyrolactone is added as a carbon source for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 2 below:
TABLE 2 variousHalomonas bluephagenesisCase of synthesizing P3HB4HB by low-salt recombinant bacteria
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, meanwhile, the cell dry weight and the PHA content of the product are both greatly improved, and more importantly, the content of the 4HB monomer in the polymer is improved.
Example 3: the content and the dry weight of the halophilic bacteria fermentation liquor P3HB4HB are improved in the low-salt culture medium. (toHalomonas bluephagenesis TDH4P series as an example)
The invention is realized byHalomonas bluephagenesisTD01 incorporates 4-hydroxybutyryl-CoA transferase (orfZ) PHA synthase(s) to synthesize 4HB-CoA and to the cells themselvesphaC) Under the action, the copolymer is copolymerized with 3HB-CoA produced by self metabolism into P (3HB-co-4 HB). On the basis, ARTP mutagenesis and low-salt pressure screening are carried out, and PHA synthase (F) derived from Eutrophobacter rolfsii is overexpressed on plasmids and genomes respectivelyphaC) Beta-ketothiolase (a)phaA) NADH type acetoacetyl-CoA reductase (b) (ii)phaB) And finally, the success in obtaining strains that can grow and accumulate large amounts of PHA in low-salt media. Both dry weight and PHA content were increased.
TDH4P, TDH4ABP + phaCAB (plasmid) and TDH4ABP + phaCAB (chromosome) were cultured in 10LB and TDH4P in 60LB medium at 37 ℃ and 200rpm for 12 hours, and then inoculated to 50mL of 10MMG and 60MMG medium at 1%, respectively, while adding 5g/L of gamma-butyrolactone as a carbon source, and culturing was carried out for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 3 below:
TABLE 3 variousHalomonas bluephagenesis Synthesis of P3HB4HB by TDH4P low-salt recombinant bacteria
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, meanwhile, the cell dry weight and the PHA content of the product are both greatly improved, and more importantly, the content of the 4HB monomer in the polymer is improved.
Example 4: the halophilic bacteria thallus containing P (3HB-co-4HB-co-3HV) is cultured and produced in a low-salt culture medium, and PHA is extracted and purified from the fermentation liquor.
The invention is realized byHalomonas bluephagenesisTD01 incorporates 4-hydroxybutyryl-CoA transferase (orfZ) PHA synthase(s) to synthesize 4HB-CoA and to the cells themselvesphaC) Under the action, the copolymer is copolymerized with 3HB-CoA produced by self metabolism into P (3HB-co-4 HB). In addition, methylmalonyl-CoA translocase is introducedscpAAndscpB、succinate dehydrogenase constitutive factorsdhAAndsdhE. On the basis of the previous work, ARTP mutagenesis and low-salt pressure screening were carried out, and finally a strain that can grow and accumulate a large amount of PHA in a low-salt medium was obtained.
TY194 and TY194AB were cultured in 10LB together with TY194 and TY194AB in 60LB medium at 37 ℃ and 200rpm for 12 hours, and then inoculated at 1% into 50mL of 10MMG and 60MMG media, respectively, while adding 5g/L of gamma-butyrolactone and 5g/L of propionic acid as carbon sources, and cultured for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 4 below:
TABLE 4 variousHalomonas bluephagenesisCase of synthesizing P (3HB-co-4HB-co-3HV) by TY194 recombinant bacteria
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, the cell dry weight and the PHA content of the product are greatly improved, and more importantly, the contents of 4HB and 3HV monomers in the polymer are improved.
Example 5: culturing in low-salt culture medium to produce halophilic bacteria containing PHBHHx, and extracting and purifying PHA from the fermented liquid.
The invention is realized byHalomonas bluephagenesisPHA synthase (TD 01) in which HHx-CoA is synthesized by introducing enoyl-CoA hydratase, trans-enoyl-CoA reductase, and 3-hydroxybutyryl-CoA dehydrogenase into cells of cells: (phaC) Under the action, the copolymer is copolymerized with 3HB-CoA produced by self metabolism into P (3 HB-co-HHx). On the basis of the previous work, ARTP mutagenesis and low-salt pressure screening were carried out, and finally a strain that can grow and accumulate a large amount of PHA in a low-salt medium was obtained.
TDHx and TDHxAB are cultured in 10LB, TDHxAB and TDHxAB in 60LB culture medium at 37 ℃ and 200rpm for 12 hours, then inoculated into 50mL 10MMG and 60MMG culture medium according to 1 percent respectively, and simultaneously 5g/L oleic acid is added as HHx related carbon source for culturing for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 5 below:
TABLE 5 variousHalomonas bluephagenesis Condition of synthesizing PHBHHx by TDHx recombinant bacteria
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, meanwhile, the cell dry weight and the PHA content of the product are both greatly improved, and more importantly, the content of the HHx monomer in the polymer is improved.
Example 6: culturing in low-salt culture medium to produce halophilic bacteria thallus containing P (3HB-co-3HP), and extracting and purifying PHA from the fermentation liquor.
The invention is realized byHalomonas bluephagenesisTD01 incorporates 3-hydroxypropionyl-CoA transferase (C)Pcs) To synthesize 3HP monomer, and PHA synthase (B) in the cell itselfphaC) Under the action of the copolymer, the copolymer is copolymerized with 3HB-CoA produced by self metabolism into P (3HB-co-3 HP). On the basis of the previous work, ARTP mutagenesis and low-salt pressure screening were carried out, and finally a strain that can grow and accumulate a large amount of PHA in a low-salt medium was obtained.
After culturing TDp129 and TDp129AB in 10LB at 37 ℃ and 200rpm in 60LB medium together with TDp129 and TDp129AB at 12 hours, 50mL of 10MMG medium and 60MMG medium were inoculated with 1% of each of them, and simultaneously 5g/L of propionic acid was added as a 3 HP-related carbon source, and the mixture was cultured for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 6 below:
TABLE 6 variousHalomonas bluephagenesisCase of synthesizing P (3HB-co-3HP) by TDp129 recombinant bacteria
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, meanwhile, the cell dry weight and the PHA content of the product are both greatly improved, and more importantly, the 3HP monomer content in the polymer is improved.
It will be appreciated by persons skilled in the art that although the invention has been described with reference to specific embodiments thereof, the invention is not limited to these specific embodiments. Based on the teaching of the present invention and the technical solutions, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and thus the resulting equivalent embodiments are within the scope of the present invention.
Example 7: low salt culture medium cultureHalomonas campaniensis LS21 CGMCC No.6593 PHB production
The invention is realized byHalomonas campaniensis LS21 CGMCC No.6593The strain LS21p was obtained by mutagenesis to grow in a low salt medium and to produce PHA synthase (S) in the cells themselvesphaC) Polymerized with 3HB-CoA produced by self metabolism to form PHB under the action of the PHB. On the basis of the previous work, ARTP mutagenesis and low-salt pressure screening were carried out, and finally a strain that can grow and accumulate a large amount of PHA in a low-salt medium was obtained.
LS21 and LS21p were cultured in 10LB and LS21 and LS21p in 60LB medium at 37 ℃ and 200rpm for 12 hours, and then inoculated at 1% concentration into 50mL of 10MMG and 60MMG media, respectively, and cultured for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 7 below:
TABLE 7 variousHalomonas campaniensisCase of recombinant bacterium synthesizing P (3HB-co-3HP)
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, and meanwhile, the cell dry weight and the PHA content of the product are greatly improved.
Example 8: low salt culture medium cultureHalomonas aydingkolgenesis M1 CGMCC NO.19880 for producing PHBV
The invention is realized byHalomonas aydingkolgenesis M1 CGMCC No.19880, M1p strain obtained by mutagenesis, to grow in low salt medium and to have PHA synthase (in the cell itselfphaC) Polymerized with 3HB-CoA produced by self metabolism to form PHB under the action of the PHB. On the basis of the previous work, ARTP mutagenesis and low-salt pressure screening were carried out, and finally a strain that can grow and accumulate a large amount of PHA in a low-salt medium was obtained.
After culturing M1, M1p in 10LB and M1, M1p in 60LB medium at 37 ℃ and 200rpm for 12 hours, the mixture was inoculated at 1% concentration into 50mL of 10MMG and 60MMG media, respectively, and cultured for 48 hours. And collecting thalli after 48 hours, detecting the dry weight of cells and the content of PHA, setting three parallel samples in each group of experiment, and taking the average value of the results. The results are shown in table 8 below:
TABLE 8 variousHalomonas aydingkolgenesisCase of recombinant bacterium synthesizing P (3HB-co-3HP)
The results show that the strains subjected to ARTP mutagenesis and low-salt pressure screening can better adapt to the environment of a low-salt culture medium, and meanwhile, the cell dry weight and the PHA content of the product are greatly improved.
It will be appreciated by persons skilled in the art that although the invention has been described with reference to specific embodiments thereof, the invention is not limited to these specific embodiments. Based on the teaching of the present invention and the technical solutions, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and thus the resulting equivalent embodiments are within the scope of the present invention.