Method for producing polyhydroxyalkanoate

Abstract

The present invention provides a method for producing a polyhydroxyalkanoate (PHA). The method comprises: using valeric acid, a valerate salt, enanthic acid, or an enanthate salt to culture Halomonas bluephagenesis. The Halomonas bluephagenesis can be used for producing a PHA containing a 3-hydroxyvalerate (3HV) monomer at high yield by using a C5 or C7 compound as a substrate. The molar ratio of the 3HV monomer in the PHA is further controlled by adjusting the ratio of the valeric acid, valerate salt, enanthic acid, or enanthate salt to glucose. In practical application, PHAs with different 3HV molar ratios can be customized according to needs, and diversified materials can also meet more application needs. Therefore, the method provided by the present application has important practical significance in improving the product competitiveness, large-scale production benefit and the like of PHAs containing a 3HV monomer.

Description

A method for producing polyhydroxyalkanoate Technical Field

[0001] The present invention relates to the technical fields of microbial metabolic engineering, fermentation engineering and synthetic biology, and in particular to a recombinant halomonas bacteria for producing polyhydroxyalkanoate and a preparation method thereof. Background Art

[0002] Polyhydroxyalkanoate (PHA) is a class of environmentally friendly biopolyesters synthesized by a variety of bacteria and represents a promising alternative to petroleum-based products. PHAs also have broad application prospects in medical implants, biodegradable packaging, food-grade coatings, and animal feed. Traditional fermentation processes suffer from limitations such as high energy and freshwater consumption during sterilization and susceptibility to bacterial contamination, severely hindering the rapid development of modern industrial biotechnology. To address these deficiencies, the Next Generation Industrial Biotechnology (NGIB) system, developed based on extremely halophilic microorganisms, allows for open, continuous fermentation without sterilization, resulting in a simple engineering process and significantly improved fermentation stability. Halomonas bluephagenesis, a halophilic bacterium isolated by Chen Guoqiang's research group at Tsinghua University from Aiding Lake in Xinjiang, exhibits broad environmental adaptability and salt and alkali tolerance, making it a key base strain for the NGIB process. Wild-type Halomonas bluephagenesis can accumulate more than 80% of the cell dry weight of P3HB (poly 3-hydroxybutyrate) under non-sterile conditions with glucose as the sole carbon source.

[0003] PHA polymers can be divided into homopolymer PHA and copolymer PHA according to the composition of monomers. Homopolymer PHA is composed of one monomer and has relatively simple material properties. Copolymer PHA is composed of two or more monomers, and the material properties can be controlled by changing the molar ratio of the monomers to meet different application requirements. Poly (3-hydroxybutyrate-co-3-hydroxyvalerate), abbreviated as P (3HB-co-3HV) or P3HB3HV, is a copolymer PHA formed by the polymerization of short-chain monomers (C4) and medium-chain monomers (C5). Its material properties can be controlled by adjusting the ratio of 3HV monomers. It has broad application prospects and huge market demand.

[0004] Currently, CN101845414B discloses the production of PHA using Pseudomonas putida, but only involves homopolymers of PHA containing 3HV monomers, and does not involve homopolymers containing 3HV and other monomers.

[0005] Patent CN114134096B discloses that recombinant Halomonas bluephagenesis can produce P3HB3HV using glucose as the sole carbon source, or using glucose and propionate as a mixed carbon source. The molar ratio of the 3HV monomer can be further controlled by adjusting the amount of propionate added. However, the resulting P3HB3HV material has a low molar ratio of the 3HV monomer, a narrow adjustable range of the 3HV monomer, and relatively limited material properties.

[0006] Therefore, in the face of the increasing market demand and diversified application scenarios of bio-based degradable materials, the development of a method for producing P3HB3HV with adjustable 3HV monomer ratio, sustainable and efficient production based on the Halomonas chassis strain and NGIB process will play a very important role in further reducing production costs and broadening the material properties of P3HB3HV.

[0007] Summary of the Invention

[0008] Based on the current existing technology, the production of PHA containing 3HV monomers requires the introduction of genes related to the synthesis of 3HV into the starting strain for modification before production, and the regulation range of the molar amount of 3HV is also limited. The present application provides a production method for PHA containing 3HV monomers that is applicable to both wild-type Halomonas and modified Halomonas. The method has a large adjustable range of 3HV, and the molar amount of 3HV can also be increased by genetic modification. Specifically, the culture of Halomonas is regulated by using a specific carbon source and the ratio between different carbon sources, and then the Halomonas is modified in combination with gene editing. It is possible to obtain PHA with a 3HV molar ratio adjusted in the range of 0-98%, thereby regulating the material properties of PHA containing 3HV monomers. In addition, it has also been confirmed that the molar ratio of 3HV monomers in PHA can be significantly increased by weakening the key genes (such as fadB) of the endogenous β-oxidation cycle pathway of Halomonas. The specific scheme is as follows:

[0009] In a first aspect, the present invention provides a method for producing polyhydroxyalkanoates or a method for regulating the molar ratio of 3-hydroxyvaleric acid monomer in polyhydroxyalkanoates produced by Halomonas, wherein the method comprises culturing Halomonas with a substrate, wherein the substrate comprises one or more of valeric acid, valerate, heptanoic acid or heptanoate.

[0010] The substrate also includes a conventional carbon source, such as one or more of glucose, gluconate, gluconate ester, sucrose, fructose, organic acid or organic acid salt. The organic acid is one or more of acetic acid, propionic acid, butyric acid, caproic acid or medium-chain fatty acids (such as lauric acid, palm oil or oleic acid), and the organic acid salt is one or more of acetate, propionate, butyrate, caproate or medium-chain fatty acid salt.

[0011] The salt may be a potassium salt, a calcium salt, a sodium salt, a magnesium salt, an aluminum salt, a zinc salt or an iron salt, etc. For example, the valerate may be potassium valerate, calcium valerate, sodium valerate, magnesium valerate, aluminum valerate, zinc valerate or iron valerate, etc. For example, the heptanoate may be potassium heptanoate, calcium heptanoate, sodium heptanoate, magnesium heptanoate, aluminum heptanoate, zinc heptanoate or iron heptanoate, etc.

[0012] The substrate is contained in the culture medium.

[0013] The culture medium can be a solid culture medium, a liquid culture medium or a semi-solid culture medium.

[0014] When the culture medium is a liquid culture medium, the substrate concentration is any value in the range of 0.0001-100 g / L, preferably any value in the range of 1-50 g / L, for example, 0.0001, 0.1, 1, 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50 g / L, etc.

[0015] The concentration of one or more of the valeric acid, valerate, heptanoic acid or heptanoate in the culture medium is any value in the range of 0.0001-10 g / L, preferably any value in the range of 1-7 g / L, for example, 0.0001, 0.1, 1, 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 g / L, etc.

[0016] In a specific embodiment of the present invention, the substrate comprises:

[0017] Component A: one or more of valeric acid, valerate, heptanoic acid or heptanoate;

[0018] Component B: glucose;

[0019] Among them, the mass ratio of component A to component B is (0.0001-7):(0-30), for example, (0.0001, 1, 2, 3, 4, 5, 6, 7):(0, 5, 10, 15, 20, 25, 30), preferably 7:(0-30), for example, 7:(0, 5, 10, 15, 20, 25, 30).

[0020] The culture medium also includes nitrogen sources, inorganic salts, trace elements, growth factors and other substances that are beneficial to bacterial growth and metabolism.

[0021] The nitrogen source may be an inorganic nitrogen source and / or an organic nitrogen source. Examples of the inorganic nitrogen source include one or more of nitrates, ammonium salts, nitrites, and ammonia. Examples of the organic nitrogen source include one or more of peanut meal, soybean meal, cottonseed meal, corn steep liquor, yeast powder, yeast extract, fish meal, silkworm pupa meal, peptone, tryptone, bran, and waste mycelium.

[0022] The inorganic salt is used to maintain the osmotic pressure required by the Halomonas bacteria. Examples include potassium salts and / or sodium salts. Potassium salts include, but are not limited to, one or more of potassium chloride, potassium sulfate, potassium phosphate, potassium citrate, potassium acetate, potassium gluconate, potassium nitrate, potassium carbonate, potassium bicarbonate, or potassium dihydrogen phosphate. Sodium salts include, but are not limited to, one or more of sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, sodium acetate, sodium gluconate, sodium nitrate, sodium carbonate, sodium bicarbonate, or dihydrogen phosphate.

[0023] In one embodiment of the present invention, bacterial growth, polyhydroxyalkanoate yield, and the molar ratio of 3-hydroxyvaleric acid in the polyhydroxyalkanoate can be adjusted by adjusting the substrate type or content or the ratio between different substrates. For example, the molar ratio of 3HV monomer in PHA can be adjusted by adjusting the ratio of component A to component B.

[0024] The polyhydroxyalkanoate is a homopolymer or copolymer composed of monomers, and the monomers include 3-hydroxyvaleric acid.

[0025] In a specific embodiment of the present invention, the polyhydroxyalkanoate (PHA) includes but is not limited to one or more of PHV, P3HB3HV, P3HB4HB3HV or P3HB3HV3HHx.

[0026] The molar ratio of the 3-hydroxyvaleric acid monomer in the polyhydroxyalkanoate is any value in the range of 0-98%, preferably any value in the range of 0.001-98%, more preferably any value in the range of 5-98% or 60-98%, for example, 0%, 0.001%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, etc.

[0027] The Halomonas bacteria express enoyl-CoA hydratase (PhaJ). Preferably, the PhaJ can be endogenous or exogenous.

[0028] Preferably, the enoyl-CoA hydratase is derived from Aeromonas.

[0029] More preferably, the enoyl-CoA hydratase is derived from Aeromonas caviae (further preferably Aeromonas caviae FA440) and / or Aeromonas hydriphila (further preferably Aeromonas hydriphila 4AK4).

[0030] Among them, the amino acid sequence of PhaJ derived from Aeromonas caviae FA440 comprises SEQ ID NO: 1 or an amino acid sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99.1%, at least 99.5%, or at least 99.9% homologous to SEQ ID NO: 1.

[0031] Furthermore, the Halomonas bacteria overexpress enoyl-CoA hydratase. This overexpression may include increasing the promoter strength of the enoyl-CoA hydratase gene (phaJ) and / or introducing the enoyl-CoA hydratase gene into the Halomonas bacteria. For example, overexpression may be achieved by introducing phaJ in addition to endogenous background expression. Another example is replacing endogenous phaJ with a phaJ that is more strongly expressed or more active than the endogenous phaJ, even if the endogenous phaJ is inactivated before introduction.

[0032] The introduced enoyl-CoA hydratase encoding gene is expressed on a plasmid or on a chromosome.

[0033] The introduction is carried out using an expression vector, which contains a PhaJ coding gene.

[0034] The PhaJ encoding gene is regulated by an inducible promoter and / or a constitutive promoter, and can also be regulated by transforming the constitutive promoter into a promoter with inducible properties.

[0035] The inducible promoter includes but is not limited to P lux Promoter, P lac promoter, phaP promoter or fadBA promoter, or two or more thereof.

[0036] The P luxThe promoter is an AHL (homoserine lactone) type inducible promoter. Preferably, the AHL induction concentration is 0-2mM, preferably any value in the range of 0.0001-0.01mM, such as 0, 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2mM. Preferably, P lux The nucleotide sequence of the promoter comprises SEQ ID NO: 5 or a variant thereof.

[0037] The P lac The promoter (SEQ ID NO: 6) is an IPTG (isopropyl-β-D-thiogalactopyranoside)-inducible promoter. Preferably, the IPTG induction concentration is 0-5 g / L, preferably any value between 0.02-2 g / L, for example, 0, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2, 3, 4, or 5 g / L.

[0038] The constitutive promoter includes but is not limited to wild-type P porin (SEQ ID NO: 7) or its mutant. Preferably, the P porin Mutants include but are not limited to mutant P porin58 (SEQ ID NO: 8), mutant P porin42 (SEQ ID NO: 9), mutant P porin68 (SEQ ID NO: 10), mutant P porin140 , mutant P porin278 (SEQ ID NO: 11), mutant P porin194 (SEQ ID NO: 12), mutant P porin211 , mutant P porin221 (SEQ ID NO: 13), mutant P porin203 (SEQ ID NO: 14).

[0039] The phaP promoter has a binding site for the PHA granule regulatory protein (PhaR), and the produced PHA is used as an inducer to regulate expression. Of course, the constitutive promoter in the prior art can also be modified to include a PhaR binding site and have promoter activity, and the produced PHA is used as an inducer to regulate expression (see the technology in patent application 2021116569347).

[0040] The fadBA promoter has a binding site for a protein that responds to oleic acid, and oleic acid is used as an inducer to regulate expression. Of course, the constitutive promoter in the prior art can also be modified to contain a binding site for a protein that responds to oleic acid and has promoter activity, and oleic acid is used as an inducer to regulate expression (refer to the technology in patent application 2022103805971).

[0041] The introduced enoyl-CoA hydratase is derived from Aeromonas.

[0042] Preferably, the introduced enoyl-CoA hydratase is derived from one or more of Aeromonas caviae (more preferably Aeromonas caviae FA440) and / or Aeromonas hydriphila (more preferably Aeromonas hydriphila 4AK4).

[0043] The Halomonas bacteria also express or overexpress PHA hydratase (PhaC). Preferably, the PhaC can be endogenous or exogenous.

[0044] Preferably, the PhaC is derived from Halomonas or Aeromonas; more preferably, the PhaC is derived from one or more of Halomonas bluephagenesis (further preferably Halomonas bluephagenesis TD01), Aeromonas caviae (further preferably Aeromonas caviae FA440) or Aeromonas hydriphila (further preferably Aeromonas hydriphila 4AK4).

[0045] Among them, the amino acid sequence of PhaC derived from Aeromonas caviae FA440 comprises SEQ ID NO: 18 or an amino acid sequence that is at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99.1%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 18.

[0046] The overexpression includes increasing the promoter strength of the endogenous PhaC encoding gene and / or introducing the PhaC encoding gene into Halomonas and / or replacing the endogenous PhaC with a PhaC with better enzyme activity, such as replacing it with a PhaC from Aeromonas caviae (preferably Aeromonas caviae FA440).

[0047] The introduced PhaC encoding gene is expressed on a plasmid or on a chromosome.

[0048] The PhaC encoding gene is regulated by an inducible promoter and / or a constitutive promoter, and can also be regulated by transforming the constitutive promoter into a promoter with inducible properties.

[0049] The method comprises weakening key genes of the endogenous β-oxidation cycle pathway of Halomonas, such as fadA, fadB, fadD, fadE or fadL, etc. Preferably, the method comprises inactivating key genes of the endogenous β-oxidation cycle pathway of Halomonas.

[0050] The inactivation includes: knocking out or knocking down all or part of the key genes of the β-oxidation cycle pathway, and / or mutating the key genes of the β-oxidation cycle pathway so that the gene cannot express protein normally or the activity of the expressed protein is weakened or inactive.

[0051] Preferably, the Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function.

[0052] The fatty acid oxidation complex subunit α is FadB (comprising SEQ ID NO: 3).

[0053] The method includes knocking out or knocking down the gene encoding fatty acid oxidation complex subunit alpha (fadB). Preferably, all or part of the bases of fadB are knocked out, or the gene is mutated so that it cannot normally express protein, or the expressed protein activity is weakened or inactive.

[0054] The method comprises knocking out or knocking down using an sgRNA targeting fadB, wherein the sgRNA comprises SEQ ID NO: 15 or 2.

[0055] In one embodiment of the present invention, the Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function, the Halomonas does not express endogenous PHA hydratase, and the Halomonas expresses a PHA hydratase derived from Halomonas or Aeromonas.

[0056] (Aeromonas), and expressing the enoyl-CoA hydratase from Aeromonas (Aeromonas).

[0057] Preferably, the Halomonas sp. expresses PHA hydratase derived from Halomonas bluephagenesis, Aeromonas caviae or Aeromonas hydriphila, and expresses enoyl-CoA hydratase derived from Aeromonas caviae or Aeromonas hydriphila.

[0058] Preferably, the Halomonas sp. expresses 4-hydroxybutyryl-CoA transferase.

[0059] Preferably, the Halomonas does not express lauroyl acyltransferase and / or lipid A biosynthesis myristoyltransferase, or the expressed lauroyl acyltransferase and / or lipid A biosynthesis myristoyltransferase is not functional.

[0060] Preferably, the Halomonas does not express PHA granule structural protein 1, or the expressed PHA granule structural protein 1 is non-functional.

[0061] The genome of the Halomonas bacteria contains phaCAB.

[0062] In one embodiment of the present invention, the Halomonas bacteria is characterized by integrating the 4HB-CoA transferase encoding gene into the genome, knocking out or knocking down the phaP1 gene, knocking out or knocking down the lpxL gene, knocking out or knocking down the lpxM gene, and expressing the phaCAB gene. Mmp1 The promoter was replaced by P porin promoter, and then overexpresses the additional phaCAB linked to the essential gene ompW.

[0063] The Halomonas is of the genus Halomonas, for example including Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives or Halomonas aydingkolgenesis or its derivatives.

[0064] In a specific embodiment of the present invention, the Halomonas is Halomonas bluephagenesis WZY278, which has an outer membrane defect, which is beneficial for downstream extraction, but is not suitable for the production of 3HV. The present application uses the bacterium to produce 3HV by adjusting the substrate, and the molar amount of 3HV is adjustable.

[0065] The culture conditions can be appropriately adjusted according to the specific Halomonas bacteria.

[0066] The culture includes seed culture, shake flask culture or fermentation tank culture.

[0067] The culture temperature is 30-40°C.

[0068] The culture process does not require sterilization.

[0069] The second aspect of the present invention provides a polyhydroxyalkanoate obtained by the above method.

[0070] The third aspect of the present invention provides a use of the polyhydroxyalkanoate prepared by the above method in the preparation of biodegradable materials, such as medical implant materials, degradable packaging materials, food-grade coating materials, and animal feed.

[0071] In a fourth aspect, the present invention provides a recombinant Halomonas bacteria for producing polyhydroxyalkanoates, wherein the recombinant Halomonas bacteria does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function.

[0072] The fatty acid oxidation complex subunit α is FadB.

[0073] The recombinant Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function, which is achieved by knocking out or knocking down the fatty acid oxidation complex subunit α encoding gene.

[0074] Preferably, knockout or knockdown is performed using an sgRNA targeting the gene encoding fatty acid oxidation complex subunit α, wherein the sgRNA comprises SEQ ID NO: 15 or 2.

[0075] The recombinant Halomonas bacteria expresses or overexpresses enoyl-CoA hydratase. The enoyl-CoA hydratase is derived from Aeromonas;

[0076] Preferably, the enoyl-CoA hydratase is derived from Aeromonas caviae (more preferably Aeromonas caviae FA440) and / or Aeromonas hydriphila (more preferably Aeromonas hydriphila 4AK4).

[0077] The Halomonas bacteria also express or overexpress PHA hydratase (PhaC). Preferably, the PhaC can be endogenous or exogenous.

[0078] Preferably, the PhaC is derived from Halomonas or Aeromonas;

[0079] More preferably, the PhaC is derived from one or more of Halomonas bluephagenesis (further preferably Halomonas bluephagenesis TD01), Aeromonas caviae (further preferably Aeromonas caviae FA440), or Aeromonas hydriphila (further preferably Aeromonas hydriphila 4AK4).

[0080] The polyhydroxyalkanoate is a homopolymer or copolymer composed of monomers, wherein the monomers include 3-hydroxyvaleric acid.

[0081] The recombinant Halomonas includes Halomonas bluephagenesis, Halomonas campaniensis or Halomonas aydingkolgenesis.

[0082] The recombinant Halomonas produces polyhydroxyalkanoate containing 3-hydroxyvaleric acid monomer using one or more of valeric acid, valerate, heptanoic acid or heptanoate as substrates.

[0083] In a fifth aspect, the present invention provides a recombinant Halomonas bacteria for producing polyhydroxyalkanoates, wherein the recombinant Halomonas bacteria overexpresses enoyl-CoA hydratase.

[0084] Preferably, the enoyl-CoA hydratase is derived from Aeromonas.

[0085] Further preferably, the enoyl-CoA hydratase is derived from Aeromonas caviae (preferably Aeromonas caviae FA440) and / or Aeromonas hydriphila (preferably Aeromonas hydriphila 4AK4).

[0086] Preferably, the recombinant Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function.

[0087] In a sixth aspect, the present invention provides a recombinant Halomonas, wherein the recombinant Halomonas does not express lauroyl acyltransferase and / or lipid A biosynthesis myristoyltransferase, or the expressed lauroyl acyltransferase and / or lipid A biosynthesis myristoyltransferase is non-functional.

[0088] Preferably, the Halomonas does not express PHA granule structural protein 1, or the expressed PHA granule structural protein 1 is non-functional.

[0089] Preferably, the Halomonas sp. expresses 4-hydroxybutyryl-CoA transferase.

[0090] The genome of the Halomonas bacteria contains phaCAB.

[0091] The genome of the Halomonas bacteria contains phaCAB.

[0092] In one embodiment of the present invention, the Halomonas bacteria is characterized by integrating the 4HB-CoA transferase encoding gene into the genome, knocking out or knocking down the phaP1 gene, knocking out or knocking down the lpxL gene, knocking out or knocking down the lpxM gene, and expressing the phaCAB gene. Mmp1 The promoter was replaced by P porin promoter, and then overexpresses the additional phaCAB linked to the essential gene ompW.

[0093] In a seventh aspect, the present invention provides a method for preparing a recombinant Halomonas bacteria for producing polyhydroxyalkanoates, wherein the recombinant Halomonas bacteria does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function.

[0094] The recombinant Halomonas expresses enoyl-CoA hydratase.

[0095] The polyhydroxyalkanoate is a homopolymer or copolymer composed of monomers, and the monomers include 3-hydroxyvaleric acid.

[0096] The preparation method comprises using an sgRNA targeting a gene encoding a fatty acid oxidation complex subunit α for knockout or knockdown, wherein the sgRNA comprises SEQ ID NO: 15 or 2.

[0097] The preparation method comprises:

[0098] 1) Knockout of the fadB gene of Halomonas, preferably, using the CRISPR / Cas9 genome editing method to knock out the fadB gene; preferably, the CRISPR / Cas9 genome editing method includes the use of sgRNA, and preferably the nucleotide sequence of the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 15 or 2.

[0099] 2) The fadB gene inactivation plasmid obtained in 1) was combined and transformed into Halomonas.

[0100] The preparation method comprises increasing the promoter strength of the enoyl-CoA hydratase encoding gene and / or introducing a heterologous enoyl-CoA hydratase encoding gene into the recombinant Halomonas.

[0101] The introduced enoyl-CoA hydratase encoding gene is expressed on a plasmid or on the chromosome.

[0102] The enoyl-CoA hydratase encoding gene is regulated by an inducible promoter and / or a constitutive promoter.

[0103] In one embodiment of the present invention, the preparation method comprises introducing any one or two or more of the following plasmids into Halomonas:

[0104] a)phaJ FA440 gene expression plasmids;

[0105] b) fadB gene inactivation plasmid. Preferably, the plasmid contains sgRNA, upstream and downstream homology arms, and / or a gene encoding the Cas9 protein. More preferably, the sgRNA targets the fadB gene, and the upstream and downstream homology arms are derived from the fadB gene.

[0106] In an eighth aspect, the present invention provides a recombinant Halomonas obtained by the above-mentioned preparation method.

[0107] The ninth aspect of the present invention provides a vector, wherein the vector is phaJ FA440 One or more of the following: gene expression plasmid, fadB gene inactivation plasmid or phaC-phaJ functional module integration plasmid.

[0108] The phaJ FA440 Gene expression plasmid containing phaJ FA440 The gene preferably also includes regulatory elements.

[0109] The phaC-phaJ functional module integration plasmid comprises the phaC gene and the phaJ gene, as well as upstream and downstream homology arms. Preferably, the sequence of the phaC-phaJ functional module integration plasmid comprises SEQ ID NO: 4 or a variant thereof.

[0110] Preferably, the vector further comprises a promoter, such as an inducible promoter and / or a constitutive promoter, or a constitutive promoter transformed into a promoter with inducible properties.

[0111] Preferably, the vector further comprises a ribosome binding site and a terminator. Further preferably, the ribosome binding site and / or terminator can be any ribosome binding site and / or terminator sequence in the prior art.

[0112] The tenth aspect of the present invention provides a cell comprising the above-mentioned vector.

[0113] The eleventh aspect of the present invention provides use of the above-mentioned vector, cell, Halomonas or recombinant Halomonas in the production of PHA (particularly P3HB3HV).

[0114] A twelfth aspect of the present invention provides a method for producing polyhydroxyalkanoates, which comprises culturing the above-mentioned recombinant Halomonas and / or the recombinant Halomonas obtained by the above-mentioned preparation method.

[0115] A thirteenth aspect of the present invention provides a fermentation method, which comprises fermenting and culturing the above-mentioned recombinant Halomonas and / or the recombinant Halomonas obtained by the above-mentioned preparation method.

[0116] Preferably, the culture medium used in the fermentation culture is a culture medium known in the prior art or the composition of the culture medium is appropriately adjusted to suit the survival of the microorganism and the production of the product. Alternatively, one or more of the above-mentioned valeric acid, valerate, heptanoic acid, or heptanoate can be used as a substrate.

[0117] In a fourteenth aspect of the present invention, a fermentation method is provided, comprising culturing Halomonas with one or more of valeric acid, valerate, heptanoic acid or heptanoate as substrates to produce PHA containing 3HV monomers.

[0118] Preferably, the fermentation method does not require sterilization.

[0119] Preferably, the fermentation product is P3HB3HV.

[0120] The fermentation conditions can be appropriately adjusted according to the specific recombinant Halomonas bacteria.

[0121] The fermentation equipment can be a shake flask, a small-scale fermentation tank, a pilot fermentation tank or a large-scale fermentation tank for industrial mass production.

[0122] In a fifteenth aspect of the present invention, a method for regulating the molar ratio of 3-hydroxyvaleric acid monomer in polyhydroxyalkanoates produced by Halomonas is provided, wherein the method comprises culturing the above-mentioned recombinant Halomonas and / or the recombinant Halomonas obtained by the above-mentioned preparation method.

[0123] The carbon source in the culture process includes one or more of valeric acid, valerate, heptanoic acid or heptanoate. Preferably, the carbon source also includes glucose.

[0124] In a specific embodiment of the present invention, the carbon source comprises:

[0125] Component A: one or more of valeric acid, valerate, heptanoic acid or heptanoate;

[0126] Component B: glucose;

[0127] Among them, the mass ratio of component A to component B is (0.0001-7): (0-30).

[0128] In the sixteenth aspect of the present invention, a method for producing 3-hydroxyvaleric acid or PHA containing 3-hydroxyvaleric acid monomer using a substrate catalyzed by Halomonas is provided, wherein the method comprises culturing Halomonas, and the substrate comprises one or more of valeric acid, valerate, heptanoic acid or heptanoate.

[0129] In a seventeenth aspect of the present invention, a culture medium for culturing Halomonas to produce PHA containing 3HV monomers is provided, wherein the culture medium contains a carbon source, wherein the carbon source comprises one or more of valeric acid, valerate, heptanoic acid, or heptanoate, and preferably further comprises glucose.

[0130] The Halomonas may be a wild type or a modified type, and the modified type is preferably the recombinant Halomonas described above.

[0131] The "β-oxidation cycle" described in this invention refers to the process of breaking down fatty acids into shorter fatty acids and acetyl-CoA, and the synthesis of longer fatty acids from acetyl-CoA and fatty acids. This includes fatty acid activation, which involves combining fatty acids with coenzyme A to form acyl-CoA; fatty acid transport, which involves transporting acyl-CoA into mitochondria; fatty acid oxidation, which involves breaking down acyl-CoA into shorter fatty acids and acetyl-CoA; and fatty acid synthesis, which involves synthesizing longer fatty acids from acetyl-CoA and other fatty acids. The "key genes for the β-oxidation cycle" include genes encoding all enzymes involved in the β-oxidation cycle, such as those required for activation, transport, oxidation, and synthesis, as well as genes that regulate the activity or expression of these enzymes.

[0132] "Overexpression" as used herein refers to upregulating gene expression to a level higher than its natural expression or causing a gene to be expressed that was previously unexpressed. "Upregulating expression" can be achieved by enhancing regulatory elements to increase gene expression, or by further introducing a certain number of copies of a coding sequence.

[0133] The "variant" described in the present invention can be a sequence with a certain range of homology obtained by substitution, deletion, mutation, insertion, etc., and the sequence still retains the original activity or has higher activity than the original or has other advantages, etc. The homology range is preferably any value between 60-100%, for example, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.1%, 99.5%, 99.9%, 100%, etc.

[0134] The terms "include" or "comprising" of the present invention are open-ended descriptions, including all the specified components or steps described, as well as other specified components or steps that will not substantially affect them; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0135] The term "and / or" in this invention includes all combinations of the items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0136] The English abbreviation and the full Chinese name of this application are compared in Table 1.

[0137] Table 1: Comparison of English abbreviations and Chinese full names

[0138] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0139] All patents and publications mentioned in this application are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications may be made to the present invention without departing from the spirit or scope of the present invention. The following examples further illustrate the present invention and are not to be construed as limiting the scope of the present invention or the specific methods described herein. DETAILED DESCRIPTION

[0140] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0141] The present invention is described in detail below by way of examples.

[0142] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0143] Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial sources.

[0144] The Halomonas bluephagenesis TD01 used in the examples is the (Halomonas sp.) TD01 described in patent application publication number CN102120973A; the public can obtain this bacterium from Tsinghua University.

[0145] Halomonas bluephagenesis WZY 278 used in the examples is a recombinant Halomonas bluephagenesis TD01 genome in which the 4HB-CoA transferase encoding gene is integrated into the genome, ΔphaP1, ΔlpxL, ΔlpxM, and the P expressing phaCAB is Mmp1 The promoter was replaced by P porin promoter, and then overexpressing an additional phaCAB associated with the essential gene ompW, as described in the article (Halomonas sp.) WZY278 in Ji, M., et al. PHB production from food waste hydrolysates by Halomonas bluephagenesis Harboring PHB operon linked with an essential gene. Metabolic Engineering, 2023, 77, 12-20. The public can obtain this bacterium from Tsinghua University.

[0146] In the examples, the sites of Halomonas, such as G3 and G4, refer to or follow the naming rules of the article: Low-cost industrial production of poly(3-hydroxybutyric acid-co-4-hydroxybutyrate) (Tsinghua University Doctoral Dissertation, Ye Jianwen, Chen Guoqiang), or the article: Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas (Ye JW et al, Metabolic Engineering, 2020). Specifically, G3 (guide RNA) is shown in SEQ ID NO: 16, and G4 (guide RNA) is shown in SEQ ID NO: 17.

[0147] Escherichia coli was grown in LB medium, and the medium composition configuration contained: 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast extract, and 0.1-50 g / L carbon source combination.

[0148] Unless otherwise specified, Halomonas were cultured in 60 LB medium. The 60 LB medium had the same ingredients as the LB medium except that the concentration of sodium chloride was adjusted to 60 g / L.

[0149] The growth temperature of Escherichia coli and Halomonas is 37℃.

[0150] The CRISPR / Cas9 technology in the Halomonas gene editing technology used in this application, including endogenous DNA knockout and heterologous DNA integration technology, can be found in the literature Qin et.al. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering. 47 (2018) 219-229.

[0151] P3HB3HV culture medium for shake flask fermentation:

[0152] 60LB fermentation medium: 60g / L sodium chloride, 5g / L yeast extract, 10g / L tryptone, 0.1-50g / L carbon source combination.

[0153] 60MM culture medium: 60g / L sodium chloride, 1.0g / L yeast extract, 0.6g / L urea, 1.5g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate, 3.81g / L sodium dihydrogen phosphate, 10.0ml / L component III, 1.0ml / L component IV. Add carbon source as needed.

[0154] Component III: 5 g / L ammonium ferric citrate, 2 g / L calcium chloride dihydrate, 41.7 ml of concentrated hydrochloric acid (12 mol / L), add water to make up to 1000 mL.

[0155] Component IV: 100 mg / L zinc sulfate heptahydrate, 30 mg / L manganese chloride tetrahydrate, 300 mg / L boric acid, 200 mg / L cobalt chloride hexahydrate, 10 mg / L anhydrous copper sulfate, 20 mg / L nickel chloride hexahydrate, 30 mg / L sodium molybdate dihydrate.

[0156] The above culture media can be prepared using standard preparation methods.

[0157] Cell dry weight determination method:

[0158] Weigh a 50-mL empty centrifuge tube; collect a certain volume of bacterial culture in a shake flask or fermenter using a centrifuge tube, centrifuge at 10,000 × g for 10 minutes, and discard the supernatant to collect the cells; resuspend the cells in an appropriate amount of deionized water and centrifuge at 10,000 × g for 10 minutes, discard the supernatant, and collect the cells again; freeze the resulting cell pellet at -80°C for at least 3 hours, then place it in a vacuum freeze dryer to a constant weight; weigh the total weight of the centrifuge tube and the dried cells in the tube; calculate the dry cell weight by the difference method.

[0159] P3HB3HV detection method:

[0160] 30-40 mg of dried bacterial cells or approximately 15 mg of standard sample were placed in an esterification tube, and 2 mL of esterification solution (3% (v / v) concentrated sulfuric acid and 0.5 g / L benzoic acid added to a chromatographic-grade methanol solution) and 2 mL of chloroform were added. The tube was sealed with a cap. The reaction was incubated at 100°C for 4 hours and then cooled to room temperature. 1 mL of deionized water was added to each tube, the mixture was shaken and mixed, and the mixture was allowed to stand until the liquid was completely separated. An appropriate amount of the lower chloroform sample was aspirated for GC analysis. The GC analysis procedure was as follows: the column temperature was increased from room temperature to 80°C and held for 90 seconds, then increased at a rate of 0.5°C / s to 140°C and held for 0 seconds, then increased at a rate of 0.7°C / s to 240°C and held for 120 seconds, and then cooled to room temperature for analysis. PHA was quantitatively analyzed based on the peak area value using the internal standard normalization method, and the mass ratio of PHA to cell dry weight and the molar ratio of 3HB and 3HV to PHA were calculated.

[0161] P3HB3HV content (wt%) = (mass of 3HB + mass of 3HV) ÷ mass of freeze-dried product × 100%;

[0162] 3HV content (mol%) = number of moles of 3HV ÷ (number of moles of 3HB + number of moles of 3HV) × 100%;

[0163] 3HB content (mol %) = number of moles of 3HB ÷ (number of moles of 3HB + number of moles of 3HV) × 100%.

[0164] The embodiments described in this application are detailed descriptions of the construction method and application of Halomonas for producing P3HB3HV. They are illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should be within the scope of protection of the present invention.

[0165] Example 1: Production of P3HB3HV by wild-type Halomonas using carbon sources

[0166] Halomonas was used to ferment and produce P3HB3HV in a shake flask using different carbon sources in 60MM medium.

[0167] The specific implementation process is as follows:

[0168] Halomonas bluephagenesis TD01 was inoculated into 20 mL of 60 LB medium, cultured for 10-12 h, and then transferred to new 20 mL of 60 LB medium at a volume ratio of 1%, and cultured for another 8-12 h to serve as the seed solution for shake flask fermentation.

[0169] A shake flask experiment was performed by inoculating 2.5 mL of fermentation seed liquid into 47.5 mL of 60 MM culture medium in a 500 mL Erlenmeyer flask. The carbon source concentration was 7 g / L, and the shaker temperature was 37°C at 200 rpm. After 48 hours of culture, cell dry weight, PHA content, and 3HV content were measured. Three replicates were performed for each experiment, and the average value was calculated. The results are shown in Table 2.

[0170] Table 2: P3HB3HV production by Halomonas bluephagenesis TD01 using different carbon sources

[0171] The results showed that Halomonas bluephagenesis TD01 could synthesize P3HB3HV using valeric acid or sodium valerate, heptanoic acid or sodium heptanoate as carbon sources, and the molar ratio of 3HV monomer was higher than 60%.

[0172] Example 2: Production of P3HB3HV by Halomonas WZY278 using carbon sources

[0173] Halomonas WZY278 was fermented in 60MM medium with different ratios of carbon sources as substrates in shake flasks to produce P3HB3HV.

[0174] The specific implementation process is as follows:

[0175] Halomonas bluephagenesis WZY278 was inoculated into 20 mL of 60 LB medium, cultured for 10-12 hours, and then transferred to new 20 mL of 60 LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as a seed solution for shake flask fermentation.

[0176] A shake flask experiment was performed by inoculating 2.5 mL of fermentation seed liquid into 47.5 mL of 60 MM culture medium in a 500 mL Erlenmeyer flask. The carbon source was a mixture of glucose and / or sodium valerate. The shaker was maintained at 37°C and 200 rpm. After 48 hours of culture, cell dry weight, PHA content, and 3HV content were measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 3.

[0177] Table 3: PHA production by Halomonas bluephagenesis WZY278 using different ratios of carbon sources

[0178] The results showed that Halomonas bluephagenesis WZY278 could synthesize P3HB3HV using mixed carbon sources, and the molar ratio of 3HV monomers could be adjusted by adjusting the ratio of the mixed carbon sources.

[0179] Example 3: Production of P3HB3HV by Recombinant Halomonas bluephagenesis G34 Using Carbon Source

[0180] In Example 1, wild-type Halomonas bluephagenesis TD01 can synthesize P3HB3HV using valeric acid or sodium valerate, heptanoic acid or sodium heptanoate as substrates. This example further genetically modified Halomonas bluephagenesis G34 strain to verify its ability to produce P3HB3HV using substrates.

[0181] Halomonas bluephagenesis G34 is a recombinant strain preferably obtained by knocking out the endogenous phaC gene and fadB gene and integrating the optimized phaC gene and phaJ gene functional expression modules into the genome of Halomonas bluephagenesis TD01 strain.

[0182] The specific steps for constructing the strain are as follows:

[0183] 1) The phaC gene was knocked out to obtain a PHA synthase (the amino acid sequence of PHA synthase is shown in SEQ ID NO: 19)-deficient halophilic bacterium Halomonas bluephagenesis, which was named TDC.

[0184] 2) Using CRISPR / Cas9 genome editing to knock out fadB. Specifically, a plasmid containing the sgRNA (SEQ ID NO: 2) and the recombination template was constructed by inserting the DNA fragments, including 1000 bp upstream and downstream homology arms and the sgRNA expression module, into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson Assembly method. The plasmid was arranged in the following order: sgRNA expression module, upstream homology arm, and downstream homology arm.

[0185] The pSEVA241 plasmid expressing the sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the corresponding Halomonas bacteria via conjugation in E. coli S17-1. The amino acid sequence of FadB is shown in SEQ ID NO: 3.

[0186] Primers were designed by colony PCR to screen for knockout fadB mutants, which were confirmed by gene sequencing. Colony PCR is a routine procedure. Furthermore, by continuously and multiple times passage the genome-edited strains in liquid culture and streaking them on spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains that had lost the CRISPR / Cas9 plasmid were identified to facilitate the next round of genome editing.

[0187] Finally, colony PCR and gene sequencing confirmed that the fadB gene had been knocked out in the corresponding Halomonas genome.

[0188] 3) Construction of phaC-phaJ functional module integration plasmid

[0189] The phaC-phaJ functional module includes a promoter, RBS, phaC derived from Aeromonas caviae FA440 (amino acid sequence shown in SEQ ID NO: 18), and a terminator, as well as a promoter, RBS, phaJ derived from Aeromonas caviae FA440 (SEQ ID NO: 1), and a terminator, namely, P porin58 -RBS1-phaC FA440 -T1-P porin68 -RBS2-phaJ FA440 -T2 (SEQ ID NO: 4). The CRISPR / Cas9 genome editing method is used to integrate the target DNA sequence. Specifically, the plasmid containing sgRNA and the recombination template is constructed as follows: DNA fragments such as 1000bp upstream and downstream homology arms, sgRNA expression module, phaC-phaJ functional module, etc. are inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) by the Gibson Assembly method. The order of plasmid arrangement is: sgRNA expression module-upstream homology arm-"phaC-phaJ functional module"-downstream homology arm.

[0190] P porin58 Sequence (SEQ ID NO: 8): TtgcgttcactggaatcccaATAtagagtttgacctgcgagca

[0191] P porin68Sequence (SEQ ID NO: 10): ttgcgttcactggaatcccaGACtagagtttgacctgcgagca

[0192] 4) The recombinant halophile obtained by knocking in the phaC-phaJ functional module at the G3 and G4 sites in the TDC genome was named Halomonas bluephagenesis G34.

[0193] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the halophilic bacteria TDC through Escherichia coli S17-1 conjugation.

[0194] Primers were designed through colony PCR to screen for knock-in mutants of the phaC-phaJ functional module, and these were confirmed by gene sequencing. Colony PCR is a routine procedure. Furthermore, by continuously and multiple times passage the genome-edited strain in liquid culture and streaking it onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains that had lost the CRISPR / Cas9 plasmid were identified to facilitate the next round of genome editing.

[0195] Finally, colony PCR and DNA sequencing confirmed that the phaC-phaJ functional module had been knocked into a specific site in the recombinant halophilic bacteria genome.

[0196] The specific implementation process of substrate verification is as follows:

[0197] Halomonas bluephagenesis G34 was inoculated into 20 mL of 60 LB medium, cultured for 10-12 hours, and then transferred to new 20 mL of 60 LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as a seed solution for shake flask fermentation.

[0198] Shake flask experiments were performed by inoculating 2.5 mL of fermentation seed liquid into 47.5 mL of 60 MM culture medium in a 500 mL Erlenmeyer flask. 7 g / L of sodium heptanoate or sodium valerate was added as a carbon source. The shaker was maintained at 37°C and 200 rpm. After 48 hours of culture, cell dry weight, P3HB3HV content, and 3HV content were measured. Three replicates were performed for each experiment, and the average value was calculated. The results are shown in Table 4.

[0199] Table 4: Recombinant Halomonas produces P3HB3HV using different carbon sources as substrates

[0200] The results showed that Halomonas bluephagenesis G34 could synthesize P3HB3HV using sodium heptanoate or sodium valerate as carbon sources, and the molar ratio of 3HV exceeded 90 mol%.

[0201] Example 4: Effect of overexpression of the phaJ gene on the production of P3HB3HV by recombinant Halomonas using different carbon sources

[0202] In this example, the overexpression of phaJ FA440 Effect of the gene (amino acid sequence comprising SEQ ID NO: 1) on the utilization of carbon sources by recombinant Halomonas to produce P3HB3HV.

[0203] The specific implementation process is as follows:

[0204] (1) Inducible phaJ FA440 Construction of gene expression plasmids

[0205] Using the genome of Halomonas bluephagenesis G34 as a template, specific primers were designed to PCR amplify phaJ FA440 Gene elements. Direct synthesis of AHL-inducible promoter elements (containing AHL promoter (P lux :Acctgtaggatcgtacaggtttacgcaagaaaatggtttgttactttcgaataaa, SEQ ID NO: 5) and regulatory module), ribosome binding site RBS, terminator. The expression module "P lux -RBS-phaJ FA440 -Terminator" was combined into the multiple restriction enzyme site of the low-copy plasmid pSEVA321. After confirmation by colony PCR and gene sequencing, the successfully constructed plasmid was named: p321-P lux -phaJ FA440 .

[0206] (2) Overexpression of phaJ FA440 Construction of recombinant Halomonas

[0207] The constructed plasmid p321-P lux -phaJ FA440 First, it was transformed into E. coli S17-1, and then the plasmid p321-P lux -phaJ FA440 The recombinant Halomonas strains were transformed into different types of Halomonas to obtain different recombinant Halomonas strains.

[0208] Specifically, plasmid p321-P lux -phaJ FA440 The recombinant Halomonas transformed into wild-type Halomonas bluephagenesis TD01 was named: TD-phaJ FA440 .

[0209] Specifically, plasmid p321-P lux -phaJ FA440 The recombinant Halomonas transformed into the recombinant Halomonas bluephagenesis G34 was named: G34-phaJ FA440 .

[0210] (3) Shake flask fermentation experiment

[0211] The above-mentioned recombinant Halomonas strains were inoculated into 20 mL of 60LB (containing 25 μg / mL chloramphenicol) medium, cultured for 10-12 h, and then transferred to new 20 mL of 60LB (containing 25 μg / mL chloramphenicol) medium at a volume ratio of 1%, and cultured for another 8-12 h as the seed liquid for shake flask fermentation.

[0212] A shake flask experiment was performed by inoculating 2.5 mL of the fermentation seed culture into a 500 mL conical flask containing 47.5 mL of 60 MM fermentation medium. The concentration of chloramphenicol was 25 μg / mL. The concentration of AHL was 0, 10×10 -4 mM, 100×10 -4 mM. The sodium valerate concentration was 7 g / L. The shaker temperature was 37°C and the rotation speed was 200 rpm. After 48 hours of culture, cell dry weight and PHA content were measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 5.

[0213] Table 5: Overexpression of phaJ FA440 Effects of Genes on the Production of P3HB3HV by Recombinant Halomonas

[0214] The results showed that by overexpressing phaJ FA440 The gene can further increase the molar ratio of 3HV monomers in the P3HB3HV copolymer. This also shows that by regulating phaJ FA440 The molar ratio of 3HV monomer in P3HB3HV copolymer was regulated by adjusting the expression level of the gene.

[0215] Example 5: Effect of inactivating the endogenous fadB gene on carbon source utilization by recombinant Halomonas to produce P3HB3HV

[0216] In Example 2, recombinant Halomonas bluephagenesis G34 was able to synthesize P3HB3HV using either five or seven carbon atoms as substrates, with a 3HV monomer molar ratio exceeding 90 mol%. This result suggests that the molar ratio of 3HV monomer in the P3HB3HV copolymer can be further increased by weakening the endogenous fadB gene in Halomonas. In this example, the effect of knocking out the endogenous fadB gene in wild-type Halomonas bluephagenesis TD01 on the synthesis of P3HB3HV was investigated.

[0217] The specific implementation process is as follows:

[0218] (1) Knockout of the fadB gene of Halomonas

[0219] The target gene was knocked out using CRISPR / Cas9 genome editing. Specifically, a plasmid containing the sgRNA (SEQ ID NO: 15) and recombination template was constructed by inserting DNA fragments, including 1000bp upstream and downstream homology arms and the sgRNA expression module, into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson Assembly method. The plasmids were arranged in the following order: sgRNA expression module, upstream homology arms, and downstream homology arms.

[0220] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transformed into the corresponding Halomonas through Escherichia coli S17-1 conjugation.

[0221] Primers were designed by colony PCR to screen for knockout fadB mutants, which were confirmed by gene sequencing. Colony PCR is a routine procedure. Furthermore, by continuously and multiple times passage the genome-edited strains in liquid culture and streaking them on spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, strains that had lost the CRISPR / Cas9 plasmid were identified to facilitate the next round of genome editing.

[0222] Finally, colony PCR and gene sequencing confirmed that the fadB gene had been knocked out in the corresponding Halomonas genome.

[0223] Specifically, the recombinant Halomonas obtained by knocking out the fadB gene in the genome of the halophilic bacterium Halomonas bluephagenesis TD01 was named Halomonas bluephagenesis TD01 (ΔfadB).

[0224] (2) Shake flask verification of the ability of recombinant Halomonas to produce P3HB3HV

[0225] Recombinant Halomonas bluephagenesis TD01 (ΔfadB) was inoculated into 20 mL of 60 LB medium, cultured for 10-12 hours, and then transferred to new 20 mL of 60 LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as fermentation seed liquid.

[0226] A shake flask experiment was performed by inoculating 2.5 mL of the fermentation seed culture into 47.5 mL of 60 MM culture medium in a 500 mL Erlenmeyer flask. The sodium valerate concentration was 7 g / L, and the shaker temperature was 37°C at 200 rpm. After 48 hours of incubation, cell dry weight and P3HB3HV content were measured. Wild-type Halomonas bluephagenesis TD01 served as a control group. Three replicates were performed for each experiment, and the results were averaged. The results are shown in Table 6.

[0227] Table 6: Effect of inactivation of fadB gene on the production of P3HB3HV by recombinant Halomonas

[0228] The results showed that weakening the expression of the endogenous fadB gene in Halomonas can indeed further increase the molar ratio of the 3HV monomer in the P3HB3HV copolymer. Knocking out the fadB gene in wild-type Halomonas can increase the molar ratio of the 3HV monomer in the P3HB3HV copolymer to 90 mol%.

[0229] Example 6: Production of P3HB3HV by Recombinant Halomonas Using Mixed Carbon Sources

[0230] The recombinant Halomonas bluephagenesis TD01 (ΔfadB) constructed in Example 5 was used as the fermentation strain, and P3HB3HV was produced in a shake flask using glucose and sodium valerate as a mixed carbon source.

[0231] The specific implementation process is as follows:

[0232] Recombinant Halomonas bluephagenesis TD01 (ΔfadB) was inoculated into 20 mL of 60 LB medium, cultured for 10-12 h, and then transferred to new 20 mL of 60 LB medium at a volume ratio of 1%, and cultured for another 8-12 h to serve as the seed solution for shake flask fermentation.

[0233] 2.5 mL of shake flask fermentation seed culture was inoculated into 47.5 mL of 60 MM fermentation medium in a 500 mL Erlenmeyer flask for culture. Final concentrations of sodium valerate and glucose were added as shown in Table 7. The shaker temperature was 37°C and the rotation speed was 200 rpm. After 48 hours of culture, cell dry weight and PHA content were measured. Three replicates were performed for each experiment, and the results were averaged.

[0234] Table 7: Recombinant Halomonas producing P3HB3HV using mixed carbon sources

[0235] The results showed that the molar ratio of 3HV in the P3HB3HV copolymer could be controlled by adjusting the ratio of glucose to sodium valerate in the mixed carbon source. Controlling the sodium valerate ratio in the mixed carbon source between 0 and 100% resulted in a corresponding controllable molar ratio of 3HV monomer in the P3HB3HV copolymer produced by recombinant Halomonas bluephagenesis TD01 (ΔfadB) between 0 and 93 mol%.

[0236] Example 7: Production of P3HB3HV by Recombinant Halomonas with Mixed Carbon Sources in a Fermenter

[0237] The recombinant Halomonas bluephagenesis TD01 (ΔfadB) constructed in Example 5 was used as the fermentation strain, and P3HB3HV was produced in a 7 L fermentor using glucose and sodium valerate as a mixed carbon source.

[0238] The specific implementation process is as follows:

[0239] Recombinant Halomonas bluephagenesis TD01 (ΔfadB) was inoculated into 20 ml of 60 LB medium and cultured for 12-16 h. The culture was then transferred to new 60 LB medium at a volume ratio of 1% and cultured for another 8-12 h. 300 mL of seed solution was prepared as the inoculation seed solution for a 7 L bioreactor (NBS Bioflo3000).

[0240] Prepare 2.7 L of base medium. The concentrations of the following ingredients are: glucose (60 g), sodium chloride (180 g), yeast extract (30 g), urea (9 g), disodium citrate (7.8 g), anhydrous magnesium sulfate (0.6 g), potassium dihydrogen phosphate (15.6 g), component III (30 mL), and component IV (3 mL).

[0241] Feed I medium was prepared to contain glucose (200 g), yeast extract (8 g) and urea (32 g).

[0242] Feed II medium was prepared to contain glucose (200 g), yeast extract (4 g) and urea (28 g).

[0243] During the fermentation process, the dissolved oxygen content was adjusted by stirring and aeration; the pH value of the culture medium was set to 8.5 and automatically adjusted by NaOH; and the temperature was set to 37°C and automatically controlled by the instrument.

[0244] After 8 h of fermentation, feed I and feed II were added sequentially. The residual glucose content in the fermenter was immediately detected by a blood glucose meter and its concentration was controlled between 5-10 g / L.

[0245] At the same time, sodium valerate was added after 8 h of fermentation. The concentration of sodium valerate was detected online by HPLC to control the concentration of sodium valerate in the tank to not exceed 3 g / L.

[0246] After 48 h of fermentation, the molar ratio of 3HV in P3HB3HV was about 18 mol%. This experimental result demonstrated that P3HB3HV can be produced using mixed carbon sources on a fermentation tank scale. Combined with the results of Example 6, the molar ratio of 3HV in P3HB3HV can also be controlled by the ratio of sodium valerate to glucose in the fermentation tank.

[0247] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A method for producing polyhydroxyalkanoate, characterized in that: The polyhydroxyalkanoate is a homopolymer or copolymer composed of monomers, wherein the monomers include 3-hydroxyvaleric acid, and the method includes culturing Halomonas with a substrate, wherein the substrate includes one or more of valeric acid, valerate, heptanoic acid or heptanoate.

2. The method according to claim 1, characterized in that The method comprises weakening the key genes of the endogenous β-oxidation cycle pathway of Halomonas.

3. The method according to claim 2, characterized in that The Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function; the fatty acid oxidation complex subunit α is FadB.

4. The method according to claim 3, characterized in that The method comprises knocking out or knocking down the gene encoding fatty acid oxidation complex subunit α.

5. The method according to claim 4, characterized in that The method comprises knocking out or knocking down using sgRNA targeting a gene encoding a fatty acid oxidation complex subunit alpha; Preferably, the sgRNA comprises SEQ ID NO: 15 or 2.

6. The method according to claim 1, characterized in that The Halomonas overexpresses enoyl-CoA hydratase; Preferably, the overexpression comprises increasing the promoter strength of the gene encoding enoyl-CoA hydratase and / or introducing the gene encoding enoyl-CoA hydratase into Halomonas.

7. The method according to claim 6, characterized in that The enoyl-CoA hydratase is derived from Aeromonas, preferably Aeromonas caviae, or the amino acid sequence of the enoyl-CoA hydratase comprises SEQ ID NO: 1 or comprises an amino acid sequence with at least 80% homology to SEQ ID NO:

1.

8. The method according to claim 1, characterized in that: The Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function, the Halomonas does not express endogenous PHA hydratase, and the Halomonas expresses PHA hydratase derived from Halomonas or Aeromonas, and expresses enoyl-CoA hydratase derived from Aeromonas.

9. The method according to claim 8, characterized in that The Halomonas bacteria express PHA hydratase derived from Halomonas bluephagenesis, Aeromonas caviae or Aeromonas hydriphila, and express enoyl-CoA hydratase derived from Aeromonas caviae and / or Aeromonas hydriphila.

10. The method according to claim 1, characterized in that The Halomonas is a bacterium in which the 4HB-CoA transferase encoding gene is integrated into the genome, the phaP1 gene is knocked out or knocked down, the lpxL gene is knocked out or knocked down, the lpxM gene is knocked out or knocked down, and the P expressing phaCAB is Mmp1 The promoter was replaced by P porin promoter, and then overexpresses the additional phaCAB associated with the essential gene ompW.

11. The method according to claim 1, characterized in that: The polyhydroxyalkanoate includes one or more of PHV, P3HB3HV, P3HB4HB3HV or P3HB3HV3HHx.

12. The method according to any one of claims 1 to 11, characterized in that: The Halomonas include Halomonas bluephagenesis or its derivatives, Halomonas campaniensis or its derivatives or Halomonas aydingkolgenesis or its derivatives.

13. The method according to claim 1, characterized in that The concentration of one or more of the valeric acid, valerate, heptanoic acid or heptanoate in the culture medium is 0.0001-10 g / L.

14. The method according to claim 13, characterized in that The concentration of one or more of the valeric acid, valerate, heptanoic acid or heptanoate in the culture medium is 1-7 g / L.

15. The method according to claim 1, characterized in that The substrate also includes one or more of glucose, gluconate, gluconate ester, sucrose, fructose, organic acid or organic acid salt.

16. The method according to claim 15, characterized in that The organic acid is selected from one or more of acetic acid, propionic acid, butyric acid, caproic acid or medium-chain fatty acids.

17. The method according to claim 16, characterized in that The medium-chain fatty acid is selected from lauric acid, palm oil or oleic acid.

18. The method according to claim 1 or 15, characterized in that: The substrate is contained in a culture medium. When the culture medium is a liquid culture medium, the concentration of the substrate is 0.0001-100 g / L.

19. The method according to claim 18, characterized in that The substrate concentration is 1-50 g / L.

20. The method according to claim 15, characterized in that The substrate comprises: Component A: one or more of valeric acid, valerate, heptanoic acid or heptanoate; Component B: glucose; Among them, the mass ratio of component A to component B is (0.0001-7): (0-30).

21. The method according to any one of claims 1 or 12, characterized in that: The salt is potassium salt, calcium salt, sodium salt, magnesium salt, aluminum salt, zinc salt or iron salt.

22. The method according to claim 1 or 20, characterized in that The valerate is potassium valerate, calcium valerate, sodium valerate, magnesium valerate, aluminum valerate, zinc valerate or iron valerate.

23. The method according to claim 1 or 20, characterized in that The enanthate salt is potassium enanthate, calcium enanthate, sodium enanthate, magnesium enanthate, aluminum enanthate, zinc enanthate or iron enanthate.

24. The method according to claim 1, characterized in that The molar ratio of 3-hydroxyvaleric acid monomer in the polyhydroxyalkanoate is 0.001-98%.

25. A polyhydroxyalkanoate obtained by the method according to any one of claims 1 to 24.

26. Use of the method according to any one of claims 1 to 24 or the polyhydroxyalkanoate according to claim 25 in the preparation of a biodegradable material.

27. The use according to claim 26, characterized in that The applications described include medical implant materials, degradable packaging materials, food-grade coating materials and animal feed.

28. A recombinant halobacterium for producing polyhydroxyalkanoate, characterized in that: The recombinant Halomonas does not express fatty acid oxidation complex subunit α or the expressed fatty acid oxidation complex subunit α has no function or has weakened function; the fatty acid oxidation complex subunit α is FadB; The polyhydroxyalkanoate is a homopolymer or copolymer composed of monomers, and the monomers include 3-hydroxyvaleric acid.

29. A recombinant Halomonas for producing polyhydroxyalkanoate, characterized in that: The recombinant Halomonas overexpresses enoyl-CoA hydratase; The polyhydroxyalkanoate is a homopolymer or copolymer composed of monomers, wherein the monomers include 3-hydroxypentyl acid.

30. A fermentation method, characterized in that: The fermentation method comprises fermenting and culturing the recombinant salt mononas described in claim 28 or 29.

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