A method for producing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx

Abstract

The application provides a method for producing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer PHBHHx, the production of PHBHHx is realized by heterologous expression of PHA synthase PhaC and enoyl-CoA hydratase PhaJ, and the endogenous enoyl-CoA hydratase FadB of halophilic bacteria is inactivated, and / or the composition of the related carbon source is adjusted to control the 3HHx molar ratio in PHBHHx, so as to regulate the material performance of PHBHHx.

Description

Technical Field

[0001] This invention relates to the fields of microbial metabolic engineering, fermentation engineering, and synthetic biology. Specifically, it relates to a method for producing a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid, PHBHHx. Background Technology

[0002] Polyhydroxyalkanoate (PHA) is an environmentally friendly biopolyester that can be produced by fermentation using various bacteria, and it is one of the most promising alternatives to traditional petroleum-based polyesters. PHA also has broad application prospects in healthcare, biodegradable materials, packaging coatings, and animal feed. To date, there are over 160 monomer structures that make up PHA, giving PHA materials significant performance differences and better meeting the needs of various applications. Poly(3-hydroxybutyrate-3-hydroxyhexanoate) copolyester is... shake -3-hydroxyhexanote), abbreviated as P(3HB- shake -3HHx) or PHBHHx is a PHA polymerized from short-chain (C4) and medium-to-long-chain (C6) monomers. It is also one of the PHAs with relatively complete large-scale production and industrial application, and has a very strong commercial prospect.

[0003] Traditional fermentation technologies suffer from drawbacks such as complex sterilization processes, high consumption of fresh water, and susceptibility to contamination by other microorganisms, severely hindering the rapid development of modern industrial biotechnology. To address these issues, Next Generation Industrial Biotechnology (NGIB), developed based on the extremophile *Bacillus thuringiensis*, enables open, continuous fermentation without consuming large amounts of energy. Its simpler engineering process significantly improves the robustness of the fermentation process. (Halophilic bacteria) Halomonas bluephagenesis It possesses broad environmental adaptability and tolerance to salt and alkali, making it one of the important chassis strains in the NGIB process. Wild type H. bluephagenesis It can accumulate over 80% PHA content under sterile conditions using glucose as the sole carbon source. Furthermore, the research group of Chen Guoqiang at Tsinghua University, through knocking out the endogenous PHA polymerase gene in this strain... phase td ), while heterologously expressing Aeromonas hydrophila 4AK4 phaCJ The operons (PhaC, PHA polymerase; PhaJ, enoyl-CoA hydratase) enabled the production of PHBHHx using hexanoic acid as a carbon source on a plasmid expression system. However, this strain exhibited drawbacks in PHBHHx production, including poor cell growth, low carbon source conversion rate, and the need for additional expensive antibiotics.

[0004] Therefore, in the face of increasing market demand, developing a more economical, sustainable and efficient method for producing PHBHHx based on halophilic bacteria chassis and NGIB process will play a very important role in further reducing production costs and improving overall profitability. Summary of the Invention

[0005] To efficiently produce PHBHHx using recombinant halophilic bacteria, this patent combines multiple metabolic engineering strategies to achieve a systematic iterative transformation of halophilic bacteria from being able to synthesize PHBHHx to efficiently synthesizing it, and further to synthesizing PHBHHx without relying on antibiotics, as well as producing PHBHHx with adjustable 3HHx monomer ratios. By optimizing gene expression intensity and improving carbon source conversion rate, production costs are further reduced by eliminating the use of antibiotics. Customized production of PHBHHx containing different 3HHx monomer ratios is possible by adjusting the carbon source ratio; furthermore, diverse materials can meet more application needs. The realization of the above engineering strategies has significant practical implications for improving the product competitiveness and large-scale production efficiency of PHBHHx.

[0006] In a first aspect, the present invention provides a recombinant halophilic bacterium.

[0007] Preferably, the recombinant halophilic bacteria express exogenous... phase Genes and / or phase Genes, and / or, inactivation of key proteins in the β-oxidation cycle pathway in the recombinant halophilic bacteria.

[0008] Preferably, the recombinant halophilic bacteria also express other polymerases capable of polymerizing 3-hydroxybutyric acid and 3-hydroxyhexanoic acid.

[0009] Preferably, the exogenous source phase Genes and / or phase Genes originate from Aeromonas caviae FA440 and / or Aeromonas hydriphila 4AK4.

[0010] In one specific embodiment of the present invention, the exogenous source... phase Genes and phase Genes originate from Aeromonas caviae FA440.

[0011] In one specific embodiment of the present invention, the exogenous source... phase Genes and phase Genes originate from Aeromonas hydriphila 4AK4.

[0012] In one specific embodiment of the present invention, the exogenous source... phase Genes originate from Aeromonas hydraphila 4AK4, external source phase Genes originate from Aeromonas caviae FA440.

[0013] In one specific embodiment of the present invention, the exogenous source... phase Genes originate from Aeromonas guinea pigs FA440, exogenous phase Genes originate from Aeromonas hydriphila 4AK4.

[0014] Preferably, the inactivation includes:

[0015] A) Knock out all or part of the gene encoding a key protein in the β-oxidation cycle pathway;

[0016] B) Mutating certain bases in a gene that encodes a key protein in the β-oxidation cycle pathway can prevent the gene from expressing the protein normally, or reduce or eliminate the activity of the expressed protein.

[0017] In one specific embodiment of the present invention, the key protein of the β-oxidation cycle pathway includes enoyl-CoA hydratase. The enoyl-CoA hydratase is FadB protein, and the amino acid sequence of the FadB protein is shown in SEQ ID NO: 38.

[0018] The nucleotide sequence encoding the FadB protein is shown in SEQ ID NO: 27.

[0019] Preferably, the recombinant halophilic bacteria include, but are not limited to, those mentioned above. Halomonas bluephagenesis , Halomonas Campania , Halomonas aydingkolgenesis .

[0020] In one specific embodiment of the present invention, the recombinant halophilic bacteria is... Halomonas blue phagenesis TD01, CGMCC No. 4353 Halomonas campaniensis LS21, CGMCC No. 6593 Halomonas aydingkolgenesis M1, CGMCC No. 19880 Halomonas bluephagenesis TDH4AB, CGMCC No. 22795.

[0021] Among them, the Halomonas bluephagenesis TDH4AB is Halomonas blue phagenesis The TD01 strain was selected through mutagenesis and is a strain that can tolerate low salt.

[0022] Depending on the specific implementation requirements, the recombinant bacteria may also be those with endogenous PHA synthase (SEQ ID NO: 23) knocked out. Halomonas bluephagenesis TD01; or, the endogenous PHA synthase (SEQ ID NO: 24) was knocked out. Halomonas campaniensis LS21.

[0023] Preferably, the recombinant halophilic bacteria can be obtained using any of the preparation methods in the prior art.

[0024] In a second aspect, the present invention provides a method for preparing recombinant halophilic bacteria.

[0025] Preferably, the preparation method includes introducing one or more of the following groups into halophilic bacteria:

[0026] 1) phase Genes and / or phase Gene;

[0027] 2) The gene encoding sgRNA, upstream and downstream homologous arms, and / or Cas9 protein; preferably, the sgRNA targets... fadB The gene, the upstream and downstream homologous arms mentioned are derived from fadB Gene.

[0028] Preferably, the one described in 1) phase Genes and / or phase Genes originate from Aeromonas caviae FA440 or / and Aeromonas hydriphila 4AK4.

[0029] In one specific embodiment of the present invention, the... phase Genes originate from Aeromonas guinea pigs FA440, whose nucleotide sequence contains SEQ ID NO: 1, or has more than 90% homology with SEQ ID NO: 1, preferably has the nucleotide sequence shown in SEQ ID NO: 1.

[0030] In one specific embodiment of the present invention, the... phase Genes originate from Aeromonas hydraphila 4AK4, whose nucleotide sequence contains SEQ ID NO: 2, or has more than 90% homology with SEQ ID NO: 2, preferably whose nucleotide sequence is as shown in SEQ ID NO: 2.

[0031] In one specific embodiment of the present invention, the... phase Genes originate from Aeromonas guinea pigsFA440, whose nucleotide sequence contains SEQ ID NO:3, or has more than 90% homology with SEQ ID NO:3, preferably has the nucleotide sequence shown in SEQ ID NO:3.

[0032] In one specific embodiment of the present invention, the... phase Genes originate from Aeromonas hydraphila 4AK4, whose nucleotide sequence contains SEQ ID NO:4, or has more than 90% homology with SEQ ID NO:4, preferably whose nucleotide sequence is as shown in SEQ ID NO:4.

[0033] Preferably, the aforementioned phase Genes and / or phase Genes are regulated by inducible promoters and / or constitutive promoters.

[0034] Preferably, the nucleotide sequence of the sgRNA described in 2) comprises the nucleotide sequence shown in SEQ ID NO: 37.

[0035] Preferably, the preparation method includes using a carrier to... phase Genes and / or phase Genes were introduced into halophilic bacteria.

[0036] Preferably, the vector contains a promoter (e.g., an inducible promoter and / or a constitutive promoter), a ribosome binding site (RBS), and / or a terminator (T).

[0037] Preferably, the inducible promoter includes, but is not limited to, P lux promoters and / or P lac Promoter.

[0038] The P mentioned lux The promoter is an AHL (homoserine lactone) type inducible promoter.

[0039] Preferably, the AHL induction concentration is 0-2 mM, and more preferably any value in the range of 0.0005-0.001 mM, 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, 2 mM.

[0040] The P mentioned lac The promoter is an IPTG (isopropyl-β-D-thiogalactoside) type inducible promoter.

[0041] Preferably, the IPTG induction concentration is 0-5 g / L, preferably any value in the range of 0.02-2 g / L, such as 0, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 1.5, 2 g / L.

[0042] In one specific embodiment of the present invention, the P lux The promoter has a nucleotide sequence that contains SEQ ID NO: 5, or has more than 90% homology with SEQ ID NO: 5, preferably as shown in SEQ ID NO: 5.

[0043] In one specific embodiment of the present invention, the P lac The promoter has a nucleotide sequence that contains SEQ ID NO: 6, or has more than 90% homology with SEQ ID NO: 6, preferably as shown in SEQ ID NO: 6.

[0044] Preferably, the constitutive promoters include, but are not limited to, wild-type P. porin Or its mutants.

[0045] More preferably, the P porin Mutants include, but are not limited to, mutant P porin58 Mutant P porin42 Mutant P porin68 Mutant P porin278 Mutant P porin194 Mutant P porin221 Mutant P porin203 .

[0046] In one specific embodiment of the present invention, the wild-type P porin The nucleotide sequence is shown in SEQ ID NO: 7.

[0047] In one specific embodiment of the present invention, the mutant P porin58 Mutant P porin42 Mutant P porin68 Mutant P porin278 Mutant P porin194 Mutant P porin221 Mutant P porin203 The nucleotide sequence contains SEQ ID NO: 8-14, or has more than 90% homology with SEQ ID NO: 8-14, preferably the nucleotide sequence shown in SEQ ID NO: 8-14.

[0048] Preferably, the sequence of the ribosome binding site includes SEQ ID NO: 19 (ribosome binding site 1) or 20 (ribosome binding site 2), or has more than 90% homology with SEQ ID NO: 19 or 20, and preferably the sequence of the ribosome binding site is as shown in SEQ ID NO: 19 or 20.

[0049] Preferably, the sequence of the terminator includes SEQ ID NO: 21 (terminator 1) or 22 (terminator 2), or has more than 90% homology with SEQ ID NO: 21 or 22, and preferably the sequence of the terminator is as shown in SEQ ID NO: 21 or 22.

[0050] Preferably, the imported phase Genes and / or phase Genes are expressed on plasmids and / or integrated into the genome for expression.

[0051] In one specific embodiment of the present invention, the imported phase Genes and / or phase Genes are expressed on plasmids.

[0052] In one specific embodiment of the present invention, the imported phase Genes and / or phase The gene is integrated into the genome of a halophilic bacterium and expressed.

[0053] Preferably, the promoter is an inductive promoter. Preferably, the vector may or may not contain a terminator.

[0054] In one specific embodiment of the present invention, the carrier phase Gene, phase The order of gene, promoter, ribosome binding site, and terminator is: inducible promoter, ribosome binding site 1, phase Gene, terminator 1, inducible promoter, ribosome binding site 2 , phaJ Gene, terminator 2.

[0055] In one specific embodiment of the present invention, the carrier does not contain a terminator, and the carrier contains... phase Gene, phase The order of gene, promoter, and ribosome binding site is: inducible promoter, ribosome binding site 1. phase Gene, inducible promoter, ribosome binding site 2 , phaJ Gene.

[0056] Depending on the specific implementation requirements, phase Genes and phaseThe order of the gene, ribosome binding site 1 and ribosome binding site 2, terminator 1 and terminator 2 in the vector can be interchanged, as long as normal expression is possible.

[0057] Preferably, inductive promoters include, but are not limited to, P lux promoters and / or P lac Promoter.

[0058] Preferably, the promoter is a constitutive promoter. Preferably, the carrier may or may not contain a terminator.

[0059] In one specific embodiment of the present invention, the carrier phase Gene, phase The order of gene, promoter, ribosome binding site, and terminator is: constitutive promoter, ribosome binding site 1, phase Gene, terminator 1, constitutive promoter, ribosome binding site 2 , phaJ Gene, terminator 2.

[0060] In one specific embodiment of the present invention, the carrier does not contain a terminator, and the carrier contains... phase Gene, phase The order of gene, promoter, and ribosome binding site is: constitutive promoter, ribosome binding site 1. phase Gene, constitutive promoter, ribosome binding site 2 , phaJ Gene.

[0061] Depending on the specific implementation requirements, phase Genes and phase The order of the gene, ribosome binding site 1 and ribosome binding site 2, terminator 1 and terminator 2 in the vector can be interchanged, as long as normal expression is possible.

[0062] Preferably, the constitutive promoters include, but are not limited to, wild-type P. porin and / or its mutants.

[0063] Preferably, the vector contains a nucleotide sequence as shown in SEQ ID NO: 32, or contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 32.

[0064] Preferably, the import is to... phase Genes and / or phase Genes are introduced into any one, two, three, or four of the G3 (SEQ ID NO: 15), G4 (SEQ ID NO: 16), G7 (SEQ ID NO: 17), and / or G51 (SEQ ID NO: 18) sites in the genome of halophilic bacteria.

[0065] Preferably, the imported phase Genes and / or phase The gene is a single copy or multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more copies).

[0066] Depending on the specific implementation requirements, the single or multiple copies can be achieved by inserting the target gene (as described in this application) into the target gene. phase Genes and / or phase The target gene is introduced into one or more sites in the genome. For example, introducing the target gene into any one of the G3 (SEQ ID NO: 15), G4 (SEQ ID NO: 16), G7 (SEQ ID NO: 17) and / or G51 (SEQ ID NO: 18) sites in the genome achieves a single copy; introducing the target gene into any two of the G3 (SEQ ID NO: 15), G4 (SEQ ID NO: 16), G7 (SEQ ID NO: 17) and / or G51 (SEQ ID NO: 18) sites in the genome achieves a double copy; introducing the target gene into any three of the G3 (SEQ ID NO: 15), G4 (SEQ ID NO: 16), G7 (SEQ ID NO: 17) and / or G51 (SEQ ID NO: 18) sites in the genome achieves a triple copy; introducing the target gene into the G3 (SEQ ID NO: 15), G4 (SEQ ID NO: 16), G7 (SEQ ID NO: 17) and G51 (SEQ ID NO: 18) sites in the genome achieves a quadruple copy.

[0067] Preferably, the multiple copies can also be integrated multiple times at a single site in the genome to achieve multiple copies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more copies).

[0068] Preferably, the multiple copies can be combined in a way that integrates multiple times at a single site in the genome and integrates separately at multiple sites to achieve multiple copies (e.g., 3, 4, 5, 6, 7, 8, 9 or more copies).

[0069] In one specific embodiment of the present invention, the import is to... phase Genes and / or phase The gene is inserted into any one of the G3, G4, G7 or G51 sites in the genome of a halophilic bacterium.

[0070] In one specific embodiment of the present invention, the import is to... phase Genes and / or phaseGenes are introduced into any one of the following sites in the genome of a halophilic bacterium: G3 and G4, G3 and G7, G3 and G51, G4 and G7, G4 and G51, or G7 and G51.

[0071] In one specific embodiment of the present invention, the import is to... phase Genes and / or phase Genes are introduced into any one of the following sites in the genome of a halophilic bacterium: G3, G4 and G7, G3, G4 and G51, G3, G7 and G51, or G4, G7 and G51.

[0072] In one specific embodiment of the present invention, the import is to... phase Genes and / or phase Genes were introduced into the G3, G4, G7, and G51 sites of the halophilic bacteria genome.

[0073] Preferably, the vector contains a nucleotide sequence as shown in any of SEQ ID NO: 33-36, or contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in any of SEQ ID NO: 33-36.

[0074] In one specific embodiment of the present invention, the preparation method includes:

[0075] 1) Constructing inducible or constitutive promoters for expression phase and phase Gene expression plasmids;

[0076] Preferably, the inducible promoter includes, but is not limited to, P lux Promoter (SEQ ID NO: 5) and / or P lac Promoter (SEQ ID NO: 6);

[0077] Preferably, the constitutive promoters include, but are not limited to, wild-type P. porin (SEQ ID NO: 7) or a mutant thereof; more 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 porin278 (SEQ ID NO: 11), mutant P porin194 (SEQ ID NO: 12), mutant P porin221 (SEQ ID NO: 13), mutant P porin203 (SEQ ID NO: 14);

[0078] The aforementioned phase and phase Genes originate from Aeromonas caviae FA440 and / or Aeromonas hydraphila 4AK4;

[0079] 2) The result obtained in 1) phase and phase Gene expression plasmids were conjugated and transformed into halophilic bacteria; preferably, the halophilic bacteria include, but are not limited to, those expressed in the form of gene expression plasmids. Halomonas bluephagenesis , Halomonas campaniensis , Halomonas aydingkolgenesis Further optimized Halomonas bluephagenesis TD01, CGMCC No. 4353 Halomonas campaniensis LS21, CGMCC No. 6593 Halomonas aydingkolgenesis M1, CGMCC No. 19880 Halomonas bluephagenesis TDH4AB, CGMCC No. 22795.

[0080] Among them, the Halomonas bluephagenesis TDH4AB is Halomonas bluephagegenesis The TD01 strain was selected through mutagenesis and is a strain that can tolerate low salt.

[0081] Depending on the specific implementation requirements, the recombinant bacteria may also be those with endogenous PHA synthase (SEQ ID NO: 23) knocked out. Halomonas bluephagenesis TD01; or, the endogenous PHA synthase (SEQ ID NO: 24) was knocked out. Halomonas campaniensis LS21.

[0082] In one specific embodiment of the present invention, the preparation method includes:

[0083] 1) Knockout of halophilic bacteria fadB Genes, preferably, are knocked out using the CRISPR / Cas9 genome editing method. fadB Gene; preferably, the CRISPR / Cas9 genome editing method includes the use of sgRNA, preferably the nucleotide sequence of the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 37.

[0084] 2) Constructing inducible or constitutive promoters for expression phaC and phaJ Gene expression plasmid; preferably, the inducible promoter includes, but is not limited to, P lux Promoter (SEQ ID NO: 5) and / or P lacPromoter (SEQ ID NO: 6);

[0085] Preferably, the constitutive promoters include, but are not limited to, wild-type P. porin (SEQ ID NO: 7) or a mutant thereof; more 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 porin278 (SEQ ID NO: 11), mutant P porin194 (SEQ ID NO: 12), mutant P porin221 (SEQ ID NO: 13), mutant P porin203 (SEQ ID NO: 14).

[0086] The aforementioned phaC and phaJ Genes originate from Aeromonas caviae FA440 and / or Aeromonas hydrophila 4AK4.

[0087] 3) The result obtained in 2) phaC and phaJ Gene expression plasmid conjugation transformation to 1) obtained fadB Among halophilic bacteria whose genes have been knocked out; preferably, the halophilic bacteria include, but are not limited to, those with gene knockout. Halomonas bluephagenesis , Halomonas campaniensis , Halomonas aydingkolgenesis Further optimized Halomonas bluephagegenesis TD01, CGMCC No. 4353 Halomonas campaniensis LS21, CGMCC No. 6593 Halomonas aydingkolgenesis M1, CGMCC No. 19880 Halomonas bluephagenesis TDH4AB, CGMCC No. 22795; wherein, the aforementioned Halomonas bluephagenesis TDH4AB is Halomonas bluephagegenesis The TD01 strain was selected through mutagenesis and is a strain that can tolerate low salt.

[0088] Depending on the specific implementation requirements, the recombinant bacteria may also be those with endogenous PHA synthase (SEQ ID NO: 23) knocked out. Halomonas bluephagenesis TD01; or, the endogenous PHA synthase (SEQ ID NO: 24) was knocked out. Halomonas campaniensis LS21.

[0089] In one specific embodiment of the present invention, the preparation method includes:

[0090] 1) Constructing inducible or constitutive promoters for expression phaC and phaJ Gene expression plasmid; preferably, the inducible promoter includes, but is not limited to, P lux Promoter (SEQ ID NO: 5) and / or P lac Promoter (SEQ ID NO: 6);

[0091] Preferably, the constitutive promoters include, but are not limited to, wild-type P. porin (SEQ ID NO: 7) or a mutant thereof; more 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 porin278 (SEQ ID NO: 11), mutant P porin194 (SEQ ID NO: 12), mutant P porin221 (SEQ ID NO: 13), mutant P porin203 (SEQ ID NO: 14);

[0092] The aforementioned phaC and phaJ Genes originate from Aeromonas caviae FA440 and / or Aeromonas hydrophila 4AK4;

[0093] 2) The result obtained in 1) phaC and phaJ Gene expression plasmids were conjugated and transformed into halophilic bacteria; preferably, the halophilic bacteria include, but are not limited to, those expressed in the form of gene expression plasmids. Halomonas bluephagenesis , Halomonas campaniensis , Halomonas aydingkolgenesis Further optimized Halomonas bluephagenesis TD01, CGMCC No. 4353 Halomonas campaniensis LS21, CGMCC No. 6593 Halomonas aydingkolgenesis M1, CGMCC No. 19880 Halomonas bluephagenesis TDH4AB, CGMCC No. 22795; wherein, the aforementioned Halomonas bluephagegenesis TDH4AB is Halomonas bluephagenesisThe TD01 strain was selected through mutagenesis and is a strain that can tolerate low salt.

[0094] Depending on the specific implementation requirements, the recombinant bacteria may also be those with endogenous PHA synthase (SEQ ID NO: 23) knocked out. Halomonas bluephagenesis TD01; or, the endogenous PHA synthase (SEQ ID NO: 24) was knocked out. Halomonas campaniensis LS21.

[0095] Depending on the specific implementation requirements, the recombinant bacteria may also be those with knockout bacteria. fadB Halophilic bacteria with preferred genes, knocked out fadB Genes can be knocked out using any method in existing technologies, with a preference for CRISPR / Cas9 genome editing. fadB Gene; preferably, the CRISPR / Cas9 genome editing method includes the use of sgRNA, preferably the nucleotide sequence of the sgRNA comprises the nucleotide sequence shown in SEQ ID NO: 37.

[0096] Preferably, the conjugation is transformed into the genome of the halophilic bacteria, and more preferably into one, two, three or four sites of the G3, G4, G7 and / or G51 sites of the halophilic bacteria genome;

[0097] Preferably, the conjugation is transformed into the genome of the halophilic bacteria. phaC and phaJ Genes can be single or multiple copies.

[0098] A third aspect of the present invention provides a recombinant halophilic bacterium obtained by the preparation method described in the second aspect above.

[0099] In a fourth aspect, the present invention provides a carrier.

[0100] Preferably, the carrier comprises:

[0101] 1) phaC Genes and / or phaJ Gene;

[0102] 2) The gene encoding sgRNA, upstream and downstream homologous arms, and / or Cas9 protein; preferably, the sgRNA targets... fadB The gene, the upstream and downstream homologous arms mentioned are derived from fadB Gene;

[0103] Preferably, the one described in 1) phaC Genes and / or phaJ Genes originate from Aeromonas caviae FA440 or / and Aeromonas hydrophila 4AK4.

[0104] Preferably, the aforementioned phaC Genes and / or phaJ Genes are regulated by inducible promoters and / or constitutive promoters.

[0105] Preferably, the inducible promoter includes, but is not limited to, P lux and / or P lac ;

[0106] Preferably, the constitutive promoters include, but are not limited to, P porin or its mutants;

[0107] More preferably, the P porin Mutants include mutant P porin58 Mutant P porin42 Mutant P porin68 Mutant P porin278 Mutant P porin194 Mutant P porin221 Mutant P porin203 .

[0108] Preferably, the vector further comprises a ribosome binding site and / or a terminator; more preferably, the ribosome binding site and / or terminator can be any ribosome binding site and / or terminator sequence in the prior art.

[0109] Preferably, the promoter is an inductive promoter.

[0110] Preferably, the carrier may or may not contain a terminator.

[0111] In one specific embodiment of the present invention, the carrier phaC Gene, phaJ The order of gene, promoter, ribosome binding site, and terminator is: inducible promoter, ribosome binding site 1, phaC Gene, terminator 1, inducible promoter, ribosome binding site 2 、phaJ Gene, terminator 2.

[0112] In one specific embodiment of the present invention, the carrier does not contain a terminator, and the carrier contains... phaC Gene, phaJ The order of gene, promoter, and ribosome binding site is: inducible promoter, ribosome binding site 1, phaC Gene, inducible promoter, ribosome binding site 2 、phaJ Gene.

[0113] Depending on the specific implementation requirements, phaC Genes and phaJ The order of the gene, ribosome binding site 1 and ribosome binding site 2, terminator 1 and terminator 2 in the vector can be interchanged, as long as normal expression is possible.

[0114] Preferably, the promoter is a compositional promoter.

[0115] Preferably, the carrier may or may not contain a terminator.

[0116] In one specific embodiment of the present invention, the carrier phaC Gene, phaJ The order of gene, promoter, ribosome binding site, and terminator is: constitutive promoter, ribosome binding site 1, phaC Gene, terminator 1, constitutive promoter, ribosome binding site 2 、phaJ Gene, terminator 2.

[0117] In one specific embodiment of the present invention, the carrier does not contain a terminator, and the carrier contains... phaC Gene, phaJ The order of gene, promoter, and ribosome binding site is: constitutive promoter, ribosome binding site 1, phaC Gene, constitutive promoter, ribosome binding site 2 、phaJ Gene.

[0118] Depending on the specific implementation requirements, phaC Genes and phaJ The order of the gene, ribosome binding site 1 and ribosome binding site 2, terminator 1 and terminator 2 in the vector can be interchanged, as long as normal expression is possible.

[0119] Preferably, the vector contains a nucleotide sequence as shown in SEQ ID NO: 32, or contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 32.

[0120] Preferably, the vector contains a nucleotide sequence as shown in any of SEQ ID NO: 33-36, or contains a nucleotide sequence that is at least 90% identical to any of the nucleotide sequences shown in SEQ ID NO: 33-36.

[0121] Preferably, the nucleotide sequence of the sgRNA described in 2) comprises the nucleotide sequence shown in SEQ ID NO: 37.

[0122] Preferably, the coding genes for the sgRNA, upstream and downstream homologous arms and / or Cas9 protein described in 2) can be located at any position in the vector, as long as homologous recombination can be completed normally.

[0123] In a fifth aspect, the present invention provides an expression system comprising the carrier described in the fourth aspect above.

[0124] Preferably, the ribosome binding site in the expression system includes ribosome binding site 1 (SEQ ID NO: 19).

[0125] Preferably, the ribosome binding site in the expression system includes ribosome binding site 2 (SEQ ID NO: 20).

[0126] Preferably, the terminator in the expression system includes terminator 1 (SEQ ID NO: 21).

[0127] Preferably, the terminator in the expression system includes terminator 2 (SEQ ID NO: 22).

[0128] Preferably, the expression system can be an inductive expression system or a constitutive expression system.

[0129] Preferably, the expression system is an inducible expression system, and more preferably, the inducible expression system uses an inducible promoter.

[0130] Preferably, the inducible expression system may or may not contain a terminator.

[0131] In one specific embodiment of the present invention, the inducible expression system comprises the following elements in the following order: an inducible promoter, a ribosome binding site 1, and... phaC Gene, terminator 1, inducible promoter, ribosome binding site 2 、phaJ Gene, terminator 2.

[0132] In one specific embodiment of the present invention, the inducible expression system does not include a terminator, and the elements included in the inducible expression system and their order are: inducible promoter, ribosome binding site 1, ... phaC Gene, inducible promoter, ribosome binding site 2 phaJ Gene.

[0133] Depending on the specific implementation requirements, phaC Genes and phaJ The order of gene, ribosome binding site 1 and ribosome binding site 2, terminator 1 and terminator 2 in the inducible expression system can be interchanged, as long as normal expression is possible.

[0134] Preferably, the inducible promoter includes, but is not limited to, P lux and / or P lac .

[0135] Preferably, the expression system is a constitutive expression system, and more preferably, the constitutive expression system uses a constitutive promoter.

[0136] Preferably, the constitutive expression system may or may not include a terminator.

[0137] In one specific embodiment of the present invention, the constitutive expression system comprises the following elements in the following order: constitutive promoter, ribosome binding site 1, ... phaC Gene, terminator 1, constitutive promoter, ribosome binding site 2 、phaJ Gene, terminator 2.

[0138] In one specific embodiment of the present invention, the constitutive expression system does not include a terminator, and the elements included in the constitutive expression system and their order are: constitutive promoter, ribosome binding site 1, ... phase Genes, constitutive promoters, ribosome binding sites 2 phase Gene.

[0139] Depending on the specific implementation requirements, phase Genes and phase The order of gene, ribosome binding site 1 and ribosome binding site 2, terminator 1 and terminator 2 can be interchanged in a constitutive expression system.

[0140] Preferably, the constitutive promoters include, but are not limited to, wild-type P. porin and / or its mutants.

[0141] Preferably, the constitutive expression system comprises a nucleotide sequence as shown in SEQ ID NO: 32, or comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 32.

[0142] Preferably, the constitutive expression system contains a nucleotide sequence as shown in any of SEQ ID NO: 33-36, or contains a nucleotide sequence that is at least 90% identical to any of the nucleotide sequences shown in SEQ ID NO: 33-36.

[0143] In a sixth aspect, the present invention provides a cell comprising the above-described vector and / or the above-described expression system.

[0144] A seventh aspect of the invention provides the use of the above-described vector, expression system, and / or cells in the production of PHA (especially PHBHHx).

[0145] In an eighth aspect, the present invention provides a fermentation method comprising fermenting and culturing the above-described recombinant halophilic bacteria, and / or the recombinant halophilic bacteria obtained by the above-described preparation method.

[0146] Preferably, the fermentation method does not require sterilization.

[0147] Preferably, the fermentation products include, but are not limited to, PHBHHx.

[0148] Preferably, the fermentation medium is a conventional medium or the composition of the medium is appropriately adjusted to suit the survival of microorganisms and the production of products.

[0149] Preferably, the fermentation conditions can be adjusted appropriately according to the specific recombinant bacteria.

[0150] Preferably, the fermentation equipment can be a shake flask, a small-scale fermenter, a pilot-scale fermenter, or a large-scale industrial fermenter.

[0151] Preferably, the carbon source in the fermentation process includes, but is not limited to, hexanoic acid, hexanoate, and / or glucose.

[0152] More preferably, the hexanoate includes, but is not limited to, sodium hexanoate and / or potassium hexanoate.

[0153] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid.

[0154] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid and hexanoate (preferably sodium hexanoate).

[0155] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoate (preferably sodium hexanoate).

[0156] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid and glucose.

[0157] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoate (preferably sodium hexanoate) and glucose.

[0158] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid, hexanoate (preferably sodium hexanoate), and glucose.

[0159] A ninth aspect of the present invention provides a method for preparing recombinant bacteria, the method comprising introducing into halophilic bacteria. phase and phase Gene expression plasmids.

[0160] Preferably, the preparation method further includes knocking out halophilic bacteria. fadB Gene.

[0161] Preferred, for phase and phase Gene expression plasmids, fadB The limitations regarding gene knockout and halophilic bacteria are the same as those in the second aspect of this invention.

[0162] In a tenth aspect, the present invention provides a method for preparing recombinant bacteria, the method comprising knocking out halophilic bacteria. fadB Gene.

[0163] Preferably, the preparation method further includes introducing halophilic bacteria. phase and phase Gene expression plasmids.

[0164] Preferred, for phase and phase Gene expression plasmids, fadB The limitations regarding gene knockout and halophilic bacteria are the same as those in the second aspect of this invention.

[0165] In an eleventh aspect, the present invention provides a method for producing PHBHHx.

[0166] Preferably, the method includes fermenting and culturing the recombinant halophilic bacteria described above, and / or the recombinant halophilic bacteria obtained by the preparation method described above.

[0167] Preferably, the carbon source in the fermentation process includes, but is not limited to, hexanoic acid, hexanoate, and / or glucose.

[0168] More preferably, the hexanoate includes, but is not limited to, sodium hexanoate and / or potassium hexanoate.

[0169] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid.

[0170] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid and hexanoate (preferably sodium hexanoate).

[0171] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoate (preferably sodium hexanoate).

[0172] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid and glucose.

[0173] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoate (preferably sodium hexanoate and glucose).

[0174] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid, hexanoate (preferably sodium hexanoate and glucose).

[0175] In a twelfth aspect of the present invention, a method is provided for increasing the molar ratio of 3HHx monomer in the production of PHBHHx by halophilic bacteria.

[0176] Preferably, the method includes:

[0177] 1) Fermentation culture of the above-mentioned recombinant halophilic bacteria, and / or, the recombinant halophilic bacteria obtained by the above preparation method; and / or,

[0178] 2) Adjust the carbon source during the fermentation process.

[0179] Preferably, the carbon source described in 2) includes, but is not limited to, hexanoic acid hexanoate, and / or, glucose.

[0180] More preferably, the hexanoate includes, but is not limited to, sodium hexanoate and / or potassium hexanoate.

[0181] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid.

[0182] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid and sodium hexanoate.

[0183] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid and glucose.

[0184] In one specific embodiment of the present invention, the carbon source in the fermentation process is sodium hexanoate and glucose.

[0185] In one specific embodiment of the present invention, the carbon source in the fermentation process is hexanoic acid, sodium hexanoate, and glucose.

[0186] Preferably, the molar ratio of 3HHx in PHBHHx can be adjusted by adjusting the ratio of glucose and sodium hexanoate in the added carbon source.

[0187] Preferably, the adjustable range of the molar ratio of 3HHx in PHBHHx is 0-40 mol%. For example, the molar ratio of 3HHx in PHBHHx can be adjusted to 0, 5, 8, 8.21, 9, 10, 10.01, 13, 13.94, 14, 15, 20, 25, 30, 35, 38, 38.17, 39, and 40 mol%.

[0188] The terms “comprising” or “including” in this invention are open-ended descriptions, containing the specified components or steps described, as well as other specified components or steps that do not materially 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 this invention.

[0189] The term "and / or" in this invention encompasses all combinations of the items connected by the term, and should be regarded as each combination having been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As 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".

[0190] The English-Chinese translation of this application is shown in Table 1:

[0191] Table 1. English-Chinese translation of this application

[0192]

[0193] The above only summarizes some aspects of the present invention and is not, and should not be considered as limiting the present invention in any way.

[0194] All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. Those skilled in the art will recognize that certain modifications may be made to this invention without departing from its spirit or scope. The following embodiments further illustrate the invention in detail and should not be construed as limiting the scope of the invention or the specific methods described herein. Attached Figure Description

[0195] Figure 1 Schematic diagram of the PHBHHx synthesis pathway. fadL Genes encoding membrane transport proteins; fadD It is the gene encoding acyl-CoA synthase; fadE It is the gene encoding acyl-CoA dehydrogenase; phase It is the gene encoding enoyl-CoA hydratase; fadB It is the gene encoding enoyl-CoA hydratase; fadA It is the gene encoding ketoacyl-CoA thiolytic enzyme; phase It is the gene encoding β-ketothiolase; phaB It is the gene encoding NADPH / NADH-dependent acetyl reductase; phase This is the gene encoding PHA hydratase;

[0196] Figure 2Intensities of fluorescent proteins from halophilic bacteria TDC-pDI-dfp at different AHL concentrations;

[0197] Figure 3 Intensities of fluorescent proteins from halophilic bacteria TDC-pDI-dfp at different IPTG concentrations;

[0198] Figure 4 : Halophilic bacteria TDC-pDI-CJ FA440 Production of PHBHHx under different combinations of inducer concentrations;

[0199] Figure 5 Unit point integrated functional module for the recombinant halophilic bacteria to produce PHBHHx;

[0200] Figure 6 : Multi-copy integration functional module for recombinant halophilic bacteria to produce PHBHHx;

[0201] Figure 7 The recombinant halophilic bacterium TDC-G34 produces PHBHHx through fermentation using sodium hexanoate as a carbon source.

[0202] Figure 8 Recombinant halophilic bacteria TDC-G34 produces PHA using glucose as a carbon source;

[0203] Figure 9 The recombinant halophilic bacterium TDC-G34 produces PHBHHx using a mixed carbon source. Detailed Implementation

[0204] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0205] The present invention will be described in detail below by way of examples.

[0206] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0207] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0208] Escherichia coli was grown in LB medium containing 10 g / L sodium chloride, 10 g / L peptone, and 5 g / L yeast extract.

[0209] Unless otherwise specified, halophilic bacteria are cultured on LB60 medium. The composition of LB60 medium is the same as that of LB, except that the sodium chloride concentration is adjusted to 60 g / L.

[0210] The growth temperature for both Escherichia coli and halophilic bacteria is 37°C and 200 rpm.

[0211] The halophilic bacteria gene editing technology used in this patent is CRISPR / Cas9 technology, which includes endogenous DNA knockout and heterologous DNA integration technology. See Qin et al. CRISPR / Cas9 editing genome of extremophile. Halomonas spp. . Metabolic Engineering. 47 (2018) 219-229.

[0212] Shake-flask fermentation of PHBHHx medium:

[0213] 60LB fermentation medium: 60g / L sodium chloride, 5g / L yeast extract, 10g / L tryptone, 0.1-50g / L related carbon source combination.

[0214] Basic culture medium: 0.1-50 g / L of relevant carbon source combination, 55-70 g / L of sodium chloride, 1-10 g / L of yeast extract, 3-6 g / L of urea, 1.5-5.2 g / L of potassium dihydrogen phosphate, 0.2-0.4 g / L of magnesium sulfate, 8.5-10 g / L of disodium hydrogen phosphate, 7-15 ml / L of component III, and 1-5 ml / L of component IV.

[0215] Component III: 5 g / L ferric ammonium citrate, 2 g / L calcium chloride dihydrate, 41.7 ml concentrated hydrochloric acid (12 mol / L), and water to a final volume of 1000 ml.

[0216] 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, and 30 mg / L sodium molybdate dihydrate.

[0217] All of the above culture media can be prepared using standard preparation methods.

[0218] Methods for determining cell dry weight:

[0219] Weigh the 50mL empty centrifuge tube; collect a certain volume of bacterial culture after being cultured in a shake flask or fermenter using the centrifuge tube, centrifuge at 10000×g for 10min, discard the supernatant and collect the bacterial cells; resuspend the bacterial cells in an appropriate amount of deionized water, centrifuge at 10000×g for 10min, discard the supernatant and collect the bacterial cells again; freeze the obtained bacterial cell precipitate at -80℃ for more than 3 hours, then place it in a vacuum freeze dryer until constant weight; weigh the total weight of the centrifuge tube and the dried bacterial cells inside; calculate the dry weight of the cells using the differential method.

[0220] PHBHHx detection method:

[0221] Take 30-40 mg of dried bacterial cells or about 15 mg of standard (P3HB or methyl 3-hydroxyhexanoate) and place it in an esterification tube. Add 2 mL of esterification solution (chromatographically pure methanol solution with 3% (v / v) concentrated sulfuric acid and 0.5 g / L benzoic acid) and 2 mL of chloroform, and seal the tube. React at 100 °C for 4 h, then cool to room temperature. Add 1 mL of deionized water to each tube, shake to mix, and let stand until the liquid completely separates into layers. Take an appropriate amount of the lower chloroform sample for GC analysis. The GC analysis program is as follows: raise the column temperature from room temperature to 80 °C and hold for 90 s, raise the temperature at a rate of 0.5 °C / s to 140 °C and hold for 0 s, raise the temperature at a rate of 0.7 °C / s to 240 °C and hold for 120 s, then cool to room temperature to end the analysis. Use the internal standard normalization method to quantify PHA based on the peak area value and calculate the molar ratio of PHA, 3HB, and 3HHx to the dry weight of the cells.

[0222] The embodiments described in this patent are a detailed description of the construction method and application of recombinant halophilic bacteria for producing PHBHHx. They are illustrative rather than limiting. Several embodiments can be listed according to the defined scope. Therefore, changes and modifications that do not depart from the overall concept of this invention should be within the protection scope of this invention.

[0223] Example 1: Constructing a system containing inductive... phaC-phaJ Recombinant halophilic bacteria expressing plasmid modules produce PHBHHx.

[0224] Construct a good dual-induction type phaC-phaJ After heterologous expression of the plasmid module, it was conjugated and transformed into halophilic bacteria to obtain recombinant halophilic bacteria containing the functional module. PHBHHx was then produced by fermentation in shake flasks with different concentrations of relevant carbon sources and inducers.

[0225] The specific implementation process is as follows:

[0226] (1) Induced type phaC-phaJ Construction of expression plasmids

[0227] strain Aeromonas caviae FA440 or / and AeromonashydriphilaUsing the 4AK4 genome as a template, specific primers were designed for PCR amplification. phase Gene elements and phase Gene elements. Direct synthesis of AHL-inducible promoter elements (including the AHL promoter and regulatory module) and IPTG-inducible promoter elements (including the IPTG promoter and regulatory module). Direct synthesis of ribosome binding sites RBS1 and RBS2. Direct synthesis of terminators Terminator1 and Terminator2. Using Gibson Assembly technology, the expression module "P" is... lux -RBS1- phase -T1-P lac -RBS2- phase The "-T2" gene was integrated into the multiple restriction enzyme sites of the low-copy plasmid pSEVA321. After confirmation by colony PCR and gene sequencing, the successfully constructed plasmid was named pDI-CJ.

[0228] Specifically, phase and phase All from strains Aeromonas caviae The plasmid pDI-CJ of FA440 is named: pDI-CJ FA440 ;

[0229] Specifically, phase and phase All from strains Aeromonashydriphila The plasmid pDI-CJ of 4AK4 is named: pDI-CJ 4AK4 ;

[0230] (2) Construction of recombinant halophilic bacteria containing plasmid pDI-CJ

[0231] The constructed plasmid pDI-CJ was first transformed into... E.coli In S17-1, the plasmid pDI-CJ was then transformed into different species of halophilic bacteria through conjugation transformation to obtain different recombinant halophilic bacterial strains.

[0232] Specifically, the plasmid pDI-CJ 4AK4 Transformation into halophilic bacteria H. bluephagenesis The recombinant halophilic bacteria obtained from TD01 was named TD-pDI-CJ. 4AK4 ;

[0233] Specifically, the plasmid pDI-CJ 4AK4 Transformation into PHA synthase-deficient halophilic bacteria H. blue phagenesis The recombinant halophilic bacteria obtained from TD01 was named TDC-pDI-CJ. 4AK4 ;

[0234] Specifically, the plasmid pDI-CJ4AK4 Transformation into halophilic bacteria H.campaniensi The recombinant halophilic bacteria obtained from LS21 was named LS-pDI-CJ. 4AK4 ;

[0235] Specifically, the plasmid pDI-CJ 4AK4 Transformation into PHA synthase-deficient halophilic bacteria H.campaniensi The recombinant halophilic bacteria obtained from LS21 was named LSC-pDI-CJ. 4AK4 ;

[0236] Specifically, the plasmid pDI-CJ 4AK4 Transformation into halophilic bacteria Halomonasaydingkolgenesis The recombinant halophilic bacteria obtained from M1 was named M1-pDI-CJ. 4AK4 ;

[0237] Specifically, the plasmid pDI-CJ FA440 Transformation into halophilic bacteria H. bluephagenesis The recombinant halophilic bacteria obtained from TD01 was named TD-pDI-CJ. FA440 .

[0238] Specifically, the plasmid pDI-CJ FA440 Transformation into PHA synthase-deficient halophilic bacteria H. blue phagenesis The recombinant halophilic bacteria obtained from TD01 was named TDC-pDI-CJ. FA440 ;

[0239] Specifically, the plasmid pDI-CJ FA440 Transformation into halophilic bacteria H.campaniensi The recombinant halophilic bacteria obtained from LS21 was named LS-pDI-CJ. FA440 ;

[0240] Specifically, the plasmid pDI-CJ FA440 Transformation into PHA synthase-deficient halophilic bacteria H. campaniensis The recombinant halophilic bacteria obtained from LS21 was named LSC-pDI-CJ. FA440 ;

[0241] Specifically, the plasmid pDI-CJ FA440 Transformation into halophilic bacteria Halomonasaydingkolgenesis The recombinant halophilic bacteria obtained from M1 was named M1-pDI-CJ. FA440 .

[0242] (3) Shake-flask fermentation experiment

[0243] The recombinant halophilic bacteria strains were inoculated into 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) and cultured for 10-12 h. Then, they were transferred to a new 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture for shake-flask fermentation.

[0244] 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium, and a shake-flask experiment was conducted. The concentration of chloramphenicol was 25 μg / mL, and the concentration of AHL was 100 × 10⁻⁶. -4 The concentrations were mM, IPTG 200 mg / L, sodium hexanoate or hexanoic acid 5 g / L, and the shaker temperature was 37℃ with a rotation speed of 200 rpm. After 48 h of culture, cell dry weight and PHBHHx content were measured. Each experiment was performed in triplicate, and the average results were taken. The results are shown in Table 2.

[0245] Table 2 includes induced types. phaC-phaJ Recombinant halophilic bacteria producing PHBHHx from expression plasmid modules

[0246]

[0247] The results showed that all 10 recombinant halophilic strains could produce PHBHHx using hexanoic acid or sodium hexanoate as the relevant carbon source, and sodium hexanoate was a better carbon source than hexanoic acid, which fully proves the introduction of... phaC-phaJ The effectiveness and broad-spectrum nature of gene-based production of PHBHHx.

[0248] Example 2: Constructing a compositional type phaC-phaJ Recombinant halophilic bacteria expressing plasmid modules produce PHBHHx.

[0249] In Example 1, the use of an inducible promoter necessitates the addition of an expensive inducer during the fermentation process to produce PHBHHx, increasing production costs. To eliminate the need for an inducer, this example utilizes a constitutive promoter instead of the inducible promoter, thus avoiding dependence on expensive inducers. Furthermore, to optimize the constitutive promoter, this patent further constructs a dual-inducible expression system. Moreover, by establishing a rational relationship between the strength of the inducible promoter and the strength of the constitutive promoter through the dual-inducible expression system, a mathematical foundation is laid for optimizing the constitutive promoter.

[0250] The specific implementation process is as follows:

[0251] (1) Construction of dual-induction characterization plasmid

[0252] Using plasmid pDI-CJ from Example 1 FA440Based on this, green fluorescent protein GFP (SEQ ID NO: 25) and red fluorescent protein RFP (SEQ ID NO: 26) were used to replace PHA synthase PhaC and enoyl-CoA hydratase PhaJ, respectively, to construct a double-inducible expression intensity characterization plasmid, named: pDI-dfp.

[0253] Specifically, the plasmid pDI-dfp is transmitted through... E.coli S17-1 conjugation to PHA synthase-deficient halophilic bacteria H. bluephagenesis The recombinant halophilic bacteria obtained from TD01 was named: TDC-pDI-dfp.

[0254] The halophilic bacteria TDC-pDI-dfp were cultured in a 2 mL deep-well plate system, and different concentrations of AHL and IPTG inducers were added. The expression intensity of fluorescent proteins at the corresponding concentrations was determined by flow cytometry.

[0255] Specifically, the preferred AHL concentration gradients are: 0 mM, 0.1 × 10⁻⁶ ... -4 mM, 0.5×10 -4 mM, 1×10 -4 mM, 5×10 -4 mM, 10×10 -4 mM, 50×10 -4 mM, 100×10 -4 mM;

[0256] Specifically, the preferred IPTG concentration gradients are: 0 mg / L, 0.5 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 100 mg / L, 200 mg / L, and 2000 mg / L;

[0257] The test results are shown below. Figure 2-3 .

[0258] (2) Induced type phase and phase Establishment of the expression intensity matrix and shake-flask fermentation experiment:

[0259] The recombinant halophilic strain TDC-pDI-CJ from Example 1 was used. FA440 Different concentrations of AHL and IPTG inducer combinations were added during shake-flask fermentation. The specific effects of these combinations were determined by analyzing cell dry weight and PHBHHx content. phase and phase The effect of expression intensity on PHBHHx production.

[0260] Recombinant halophilic bacteria TDC-pDI-CJ FA440Inoculate into 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) and culture for 10-12 h. Then, transfer to a new 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) at a volume ratio of 1% and continue culturing for 8-12 h to obtain the seed culture for shake-flask fermentation.

[0261] 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium for a shake-flask experiment. Chloramphenicol was used at a concentration of 25 μg / mL. Different concentrations of AHL and IPTG were added. The shaker temperature was 37 °C, and the rotation speed was 200 rpm. 20 g / L glucose and 5 g / L sodium hexanoate were used as the mixed carbon source for this fermentation.

[0262] Specifically, three AHL concentration gradients were selected: 1×10 -4 mM (L), 10×10 -4 Mm(M), 100×10 -4 mM(H);

[0263] Specifically, three IPTG concentration gradients were selected: 20 mg / L (L), 100 mg / L (M), and 200 mg / L (H).

[0264] Specifically, the number of AHL and IPTG concentration combinations is 3. 3 = 9;

[0265] After 48 hours of culture, cell dry weight and PHBHHx content were measured. Each experiment was conducted in triplicate, and the results were averaged. See below for detailed results. Figure 4 The results showed that different expression intensities phase and phase It has a significant impact on the production of PHBHHx by recombinant strains. Specifically, it affects low and medium expression levels. phase Genes, and low, medium, and high expression levels phase The combination of genes showed good results in all indicators (cell dry weight, PHA content, and 3HHx ratio).

[0266] (3) Constitutive type phaC-phaJ Construction of expression plasmid module

[0267] References Stimulus response-based fine-tuning of polyhydroxyalkanoatepathway in Halomonas The porin gene was characterized in (Ye, et al. Metabolic Engineering, 2020.). porinThe strength of the mutant promoter library. Based on the preferred inducible promoter strength in this embodiment, further, the constitutive P expression strength corresponding to the preferred expression strength is... porin Promoter.

[0268] Specifically, the pDI-CJ constructed in Example 1 FA440 Based on the promoter, P is used respectively porin58 and P porin68 The promoters replace their AHL-inducible and IPTG-inducible promoters to construct a well-defined constitutive promoter. phaC-phaJ The expression plasmid is named: pDC-CJ FA440 .

[0269] (4) Shake-flask fermentation experiment

[0270] pass E.coli S17-1 combined experiments with plasmid pDC-CJ FA440 It is transferred to some halophilic bacteria.

[0271] Specifically, the plasmid pDC-CJ FA440 Transformation into halophilic bacteria H. bluephagenesis The recombinant halophilic bacteria obtained from TD01 was named TD-pDC-CJ. FA440 .

[0272] Specifically, the plasmid pDC-CJ FA440 Transformation into PHA synthase-deficient halophilic bacteria H. bluephagenesis The recombinant halophilic bacteria obtained in TD was named TDC-pDC-CJ. FA440 .

[0273] Specifically, the plasmid pDI-CJ FA440 Transformation into halophilic bacteria H. campaniensis The recombinant halophilic bacteria obtained from LS21 was named LS-pDC-CJ. FA440 .

[0274] Specifically, the plasmid pDC-CJ FA440 Transformation into PHA synthase-deficient halophilic bacteria H. campaniensis The recombinant halophilic bacteria obtained from LS21 was named LSC-pDC-CJ. FA440 .

[0275] The recombinant halophilic bacteria strain was inoculated into 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) and cultured for 10-12 h. Then, it was transferred to a new 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture for shake-flask fermentation.

[0276] 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium, and a shake-flask experiment was conducted. The concentration of chloramphenicol was 25 μg / mL, the concentration of sodium hexanoate was 7.5 g / L, the shaker temperature was 37℃, and the shaking speed was 200 rpm. The results are shown in Table 3.

[0277] Table 3 includes compositional types phaC-phaJ Recombinant halophilic bacteria producing PHBHHx from expression plasmid modules

[0278]

[0279] The results showed that constitutive promoter expression phase and phase Genes can play a role in different halophilic bacterial strains.

[0280] Preferably, by constructing a compositional phaC-phaJ Expression plasmid module. Reduces the use of expensive inducers and optimizes gene expression intensity.

[0281] Example 3: Constructing recombinant halophilic bacteria with inactivated key genes of the β-oxidation cycle pathway to produce PHBHHx.

[0282] according to Figure 1 It is known that inactivating key genes in the β-oxidation cycle pathway can further improve the efficiency of substrate conversion to 3HHx monomers, thereby increasing the molar ratio of 3HHx in PHBHHx. In this embodiment, the endogenous enoyl-CoA hydratase gene in the halophilic bacteria β-oxidation cycle pathway was knocked out. fadB (SEQ ID NO: 27, 39-48) to increase the molar ratio of 3HHx in PHBHHx.

[0283] The specific steps are as follows:

[0284] (1) Knockout of halophilic bacteria fadB Gene

[0285] halophilic bacteria H. bluephagenesis TD01 genome annotation information shows that this strain has 11 potential genes. fadB Gene. The target gene was knocked out using the CRISPR / Cas9 genome editing method. Specifically, the plasmid containing sgRNA and the recombination template was constructed as follows: 1000bp homologous arms upstream and downstream, the sgRNA expression module, and other DNA fragments were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the GibsonAssembly method. The plasmid arrangement order was: sgRNA expression module - upstream homologous arm - downstream homologous arm.

[0286] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transmitted via E. coli. E.coli S17-1 conjugates into the corresponding halophilic bacteria.

[0287] Primers were designed using colony PCR to screen for gene knockout. fadB Mutant strains were identified and confirmed by gene sequencing. Colony PCR was performed as a routine procedure. Furthermore, strains that had successfully undergone genome editing were identified by streaking them multiple times in liquid medium onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, respectively, to facilitate the next round of genome editing.

[0288] Finally, through colony PCR and gene sequencing, it was confirmed that the corresponding halophilic bacteria genome contained... fadB The gene has been knocked out.

[0289] Specifically, halophilic bacteria H. bluephagenesis In the TD01 genome fadB The recombinant halophilic bacteria obtained by gene knockout was named TDB.

[0290] Specifically, PHA synthase-deficient halophilic bacteria H. bluephagenesis In the TD01 genome fadB The recombinant halophilic bacteria obtained by gene knockout is named TDCBn, where n represents fadB Gene number.

[0291] (2) Heterogeneous phaC-phaJ Construction of recombinant halophilic bacteria using expression plasmid modules

[0292] pass E.coli S17-1 Combined with experiments, the plasmid pDC-CJ from Example 2 was used... FA440 Transformed into halophilic bacteria.

[0293] Specifically, the plasmid pDC-CJ FA440 The recombinant halophilic bacterium obtained by transformation into halophilic bacteria TDCB was named TDCBn-pDC-CJ. FA440 where n represents fadB Gene number.

[0294] (3) Shake flask verification of the ability of recombinant halophilic bacteria to produce PHBHHx

[0295] The recombinant halophilic strains were inoculated into 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) and cultured for 10-12 h. Then, they were transferred to a new 20 mL of LB60 medium (containing 25 μg / mL chloramphenicol) at a volume ratio of 1% and cultured for another 8-12 h to obtain the fermentation seed culture.

[0296] 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium for a shake-flask experiment. The concentration of chloramphenicol was 25 μg / mL, the sodium hexanoate concentration was 7.5 g / L, the shaker temperature was 37℃, and the shaking speed was 200 rpm. After 48 h of culture, the cell dry weight and PHBHHx content were measured. Each experiment was performed in triplicate, and the mean value was taken. The results are shown in Table 4.

[0297] Table 4 Inactivation fadB Recombinant halophilic bacteria produce PHBHHx

[0298]

[0299] By comparing the halophilic bacteria TD-pDC-CJ in Example 2 FA440 The shake-flask fermentation data shows that fadB1 It is a halophilic bacterium H. bluephagenesis A key gene in the β-oxidation cycle pathway of its derived strains. And through inactivation... fadB1 It can increase the molar ratio of 3HHx monomer in PHBHHx.

[0300] Example 4: Single-copy expression at different sites in the genome phaC-phaJ The functional module uses recombinant halophilic bacteria to produce PHBHHx.

[0301] The use of plasmid expression modules during fermentation requires the addition of large amounts of antibiotics, further increasing production costs and making post-fermentation broth processing more difficult. In the shake-flask fermentation experiments of Examples 1, 2, and 3 of this patent, exogenous antibiotics were added. Therefore, this patent optimizes expression sites on the halophilic bacteria genome and uses CRISPR / Cas9 gene editing technology to... phaC-phaJ The functional modules are integrated into designated sites on the genome, enabling the recombinant halophilic bacteria to stably produce PHBHHx without the need for antibiotics.

[0302] The specific steps are as follows:

[0303] (1) Selecting suitable expression sites on the genome of halophilic bacteria

[0304] In this embodiment, four functional module integration sites were selected from the halophilic bacteria genome for subsequent experiments.

[0305] Specifically, the preferred halophilic bacteria are: the PHA synthase-deficient halophilic bacteria in Example 1. H. bluephagenesis It was named TDC.

[0306] Specifically, based on the early transcriptome data of this strain, the four preferred genomic integration sites are: G3 (guide RNA sequence see SEQ ID NO: 28), G4 (guide RNA sequence see SEQ ID NO: 29), G7 (guide RNA sequence see SEQ ID NO: 30), and G51 (guide RNA sequence see SEQ ID NO: 31).

[0307] Specifically, preferred phaC-phaJ The functional module is: "P" porin58 -RBS1- phase FA440 -T1-P porin68 -RBS2- phase FA440 The "-T2" sequence is shown in SEQ ID NO: 32.

[0308] (2) phaC-phaJ Construction of functional module integration plasmid

[0309] The CRISPR / Cas9 genome editing method was used to integrate the target DNA sequence. Specifically, the plasmid containing sgRNA and the recombination template was constructed as follows: 1000bp homologous arms were inserted upstream and downstream, along with the sgRNA expression module. phaC-phaJ DNA fragments, including functional modules, were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson Assembly method. The order of arrangement on the plasmid was as follows: sgRNA expression module - upstream homologous arm - " phaC-phaJ "Functional Module" - Downstream Homologous Arm.

[0310] Specifically, the plasmid sequence integrated into the G3 site is shown in SEQ ID NO: 33;

[0311] Specifically, the plasmid sequence integrated into the G4 site is shown in SEQ ID NO: 34;

[0312] Specifically, the plasmid sequence integrated into the G7 site is shown in SEQ ID NO: 35;

[0313] Specifically, the plasmid sequence integrated into the G51 site is shown in SEQ ID NO: 36;

[0314] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transmitted via E. coli. E.coli S17-1 conjugation was transferred into the halophilic bacteria TDC.

[0315] Primers were designed and screened using colony PCR. phaC-phaJFunctional module knock-in mutants were identified and confirmed by gene sequencing. Colony PCR was performed as a routine procedure. Furthermore, strains that had successfully undergone genome editing were identified by streaking them multiple times in liquid medium onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, respectively, to facilitate the next round of genome editing.

[0316] Finally, colony PCR and DNA sequencing confirmed that different sites in the genome of the halophilic bacterium TDC had been knocked in. phaC-phaJ Functional modules.

[0317] Specifically, the G3 site in the genome of the halophilic bacteria TDC was knocked in phaC-phaJ The recombinant halophilic bacteria obtained from the functional module was named TDC-G3.

[0318] Specifically, the G4 site in the genome of the halophilic bacterium TDC was knocked in... phaC-phaJ The recombinant halophilic bacteria obtained from the functional module was named TDC-G4.

[0319] Specifically, the G7 site in the genome of the halophilic bacterium TDC was knocked in... phaC-phaJ The recombinant halophilic bacteria obtained from the functional module was named TDC-G7.

[0320] Specifically, the G51 site in the genome of the halophilic bacterium TDC was knocked in... phaC-phaJ The recombinant halophilic bacteria obtained from the functional module was named TDC-G51.

[0321] (3) Shake flask test to verify the ability of recombinant halophilic bacteria to produce PHBHHx

[0322] The recombinant halophilic strains from this example were inoculated into 20 mL of LB60 medium and cultured for 10-12 h. Afterward, they were transferred at a 1% volume ratio to fresh 20 mL of LB60 medium and cultured for another 8-12 h to obtain the fermentation seed culture. 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium for a shake-flask experiment. The sodium hexanoate concentration was 7.5 g / L, the shaker temperature was 37°C, and the shaking speed was 200 rpm. After 48 h of culture, cell dry weight and PHBHHx content were measured. Each experiment was performed in triplicate, and the average results were taken. The results are shown in [Figure number missing]. Figure 5 The results show that by... phaC-phaJ Integrating functional modules into preferred genomic sites of halophilic bacteria can achieve the goal of stable production of PHBHHx without relying on antibiotics.

[0323] Preferably, the expression efficiency of the integration sites is in the following order: G3>G4>G7>G51.

[0324] Example 5: Expression using multiple copies at different sites in the genome phaC-phaJ The functional module uses recombinant halophilic bacteria to produce PHBHHx.

[0325] In Example 4, by phaC-phaJ The functional modules are integrated into the preferred genomic sites of halophilic bacteria, achieving stable production of PHBHHx without relying on antibiotics. Furthermore, this embodiment achieves this by... phaC-phaJ Functional modules are sequentially integrated into designated sites on the genome, improving the genome's... phaC-phaJ The number of copies of the functional modules can further improve the ability of recombinant halophilic bacteria to produce PHBHHx.

[0326] The specific steps are as follows:

[0327] (1) phaC-phaJ Construction of multi-copy recombinant halophilic bacteria for functional modules

[0328] The different sites constructed in Example 4 phaC-phaJ Functional modules were integrated sequentially into halophilic bacteria to obtain genomes containing different phaC-phaJ Recombinant halophilic bacteria with a high copy number of functional modules.

[0329] The CRISPR / Cas9 genome editing method was used to integrate the target DNA sequence. Specifically, the plasmid containing sgRNA and the recombination template was constructed as follows: 1000bp homologous arms were inserted upstream and downstream, along with the sgRNA expression module. phaC-phaJ DNA fragments, including functional modules, were inserted into the original expression plasmid pSEVA241 (containing kanamycin and spectinomycin resistance genes) using the Gibson Assembly method. The plasmid arrangement was as follows: sgRNA expression module - upstream homologous arm - " phaC-phaJ "Functional Module" - Downstream Homologous Arm.

[0330] The pSEVA241 plasmid expressing sgRNA and recombinant template and the pQ08 plasmid expressing Cas9 were transmitted via E. coli. E. coli S17-1 conjugation was transferred into the halophilic bacteria TDC.

[0331] Primers were designed and screened using colony PCR. phaC-phaJ Functional module knock-in mutants were identified and confirmed by gene sequencing. Colony PCR was performed as a routine procedure. Furthermore, strains that had successfully undergone genome editing were identified by streaking them multiple times in liquid medium onto spectinomycin-resistant, chloramphenicol-resistant, and non-resistant plates, respectively, to facilitate the next round of genome editing.

[0332] Finally, colony PCR and DNA sequencing confirmed that a specific site in the recombinant halophilic bacteria genome had been knocked in. phaC-phaJ Functional modules.

[0333] Furthermore, by repeating the CRISPR / Cas9 genome editing method, the amount of recombinant halophilic bacteria in the genome was sequentially increased. phaC-phaJ Number of copies of the functional module.

[0334] Specifically, the G3 site in the genome of the halophilic bacteria TDC was knocked in phaC-phaJ The recombinant halophilic bacterium obtained from the functional module was named TDC-G3. This recombinant halophilic bacterium has one copy of the genome. phaC-phaJ Functional modules.

[0335] Specifically, the G3 and G4 sites in the genome of the halophilic bacterium TDC were knocked in. phaC-phaJ The recombinant halophilic bacterium obtained from the functional module was named TDC-G34. This recombinant halophilic bacterium has two copies of its genome. phaC-phaJ Functional modules.

[0336] Specifically, the G3, G4, and G7 sites in the genome of the halophilic bacterium TDC were knocked in. phaC-phaJ The recombinant halophilic bacterium obtained from the functional module was named TDC-G34-7. This recombinant halophilic bacterium has 3 copies of its genome. phaC-phaJ Functional modules.

[0337] Specifically, the G3, G4, G7, and G51 sites in the genome of the halophilic bacterium TDC were knocked in. phaC-phaJ The recombinant halophilic bacterium obtained from the functional module was named TDC-G34-7-51. This recombinant halophilic bacterium has 4 copies of its genome. phaC-phaJ Functional modules.

[0338] (2) Shake flask verification of the ability of recombinant halophilic bacteria to produce PHBHHx

[0339] The recombinant halophilic strains in this example were inoculated into 20 mL of LB60 medium and cultured for 10-12 h. Then, they were transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the fermentation seed culture.

[0340] 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium for a shake-flask experiment. The sodium hexanoate concentration was 7.5 g / L, the shaker temperature was 37℃, and the shaking speed was 200 rpm. After 48 h of culture, the cell dry weight and PHBHHx content were measured. Each experiment was performed in triplicate, and the average results were taken. The results are shown in the figure. Figure 6 The results showed that by increasing phaC-phaJIntegrating functional modules into the halophilic bacteria genome increases the strain's ability to produce PHBHHx. Specifically, the 2-copy group showed higher cell dry weight and PHA content than other groups, while the 4-copy group had a higher proportion of 3HHx.

[0341] Example 6: Production of PHBHHx using recombinant halophilic bacteria under low-salt conditions.

[0342] Halophilic bacteria require a high concentration of sodium chloride (60 g / L NaCl) to maintain osmotic pressure during fermentation, but this leads to a very complex high-salt wastewater treatment process after fermentation, directly increasing the overall production cost. In this patent, by using halophilic bacteria that can tolerate low salt levels as the substrate strain to produce PHBHHx, production costs can be directly reduced.

[0343] The specific steps are as follows:

[0344] (1) Construction of recombinant low-salt halophilic bacteria

[0345] The composition in Example 2 phaC-phaJ Expression plasmid (pDC-CJ) FA440 )pass E. coli S17-1 conjugation was transferred into low-salt-tolerant halophilic bacteria to obtain recombinant low-salt-tolerant halophilic bacteria.

[0346] Specifically, the preferred low-salt-tolerant halophilic bacteria are: Halomonas bluephagenesis TDH4AB.

[0347] Specifically, the plasmid pDC-CJ FA440 Transformation into halophilic bacteria Halomonas bluephagenesis The recombinant halophilic bacteria obtained from TDH4AB was named TDH4AB-pDC-CJ. FA440 .

[0348] (2) Shake flask verification of the ability of recombinant low-salt halophilic bacteria to produce PHBHHx

[0349] The recombinant halophilic bacterium TDH4AB-pDC-CJ in this embodiment FA440 Inoculate into 20 mL of LB medium and culture for 10-12 h. Then, transfer to a new 20 mL of LB medium at a volume ratio of 1% and continue culturing for 8-12 h to obtain the fermentation seed culture.

[0350] 2.5 mL of the fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of LB fermentation medium (NaCl concentration: 10 g / L) for a shake-flask experiment. The concentration of chloramphenicol was 25 μg / mL, the sodium hexanoate concentration was 7.5 g / L, the shaker temperature was 37℃, and the shaking speed was 200 rpm. After 48 h of culture, the cell dry weight and PHBHHx content were measured. Each experiment was performed in triplicate, and the results were averaged. The results are shown in Table 5.

[0351] Table 5 Production of PHBHHx by Recombinant Low-Salt Halophilic Bacteria

[0352]

[0353] The results showed that PHBHHx could also be produced using relevant carbon sources in recombinant low-salt halophilic bacteria.

[0354] Preferably, low-salt-tolerant halophilic bacteria are used. Halomonas bluephagenesis TDH4AB, as a chassis strain, can reduce the amount of NaCl used and simplify the fermentation wastewater treatment process.

[0355] Example 7: Production of PHBHHx by recombinant halophilic bacteria using sodium hexanoate as the sole carbon source in a fermenter

[0356] Laboratory-scale fermenter experiments serve both as validation of shake-flask techniques and as the foundation for large-scale fermentation production. Using the recombinant halophilic bacterium TDC-G34 constructed in Example 5 as the fermentation strain, PHBHHx was produced in a 7L fermenter with sodium hexanoate as the sole carbon source.

[0357] The specific steps are as follows:

[0358] Recombinant halophilic bacteria TDC-G34 was inoculated into 20 ml of LB60 medium and cultured for 12-16 h. Then, it was transferred to new LB60 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 the 7 L bioreactor (NBSBioflo3000).

[0359] Prepare 2.7L of substrate culture medium. The concentrations of each component in the substrate are as follows: sodium chloride (135g), potassium chloride (15g), yeast extract (30g), urea (9g), disodium citrate (7.8g), anhydrous magnesium sulfate (0.6g), potassium dihydrogen phosphate (15.6g), component III (30mL), and component IV (3mL).

[0360] During fermentation, oxygen concentration is adjusted by stirring and aeration; the pH of the culture medium is set to 8.5 and automatically adjusted by NaOH; the temperature is set to 37 ℃ and automatically controlled by the instrument.

[0361] Sodium hexanoate was added after 8 hours of fermentation. The concentration of sodium hexanoate was monitored online by HPLC, and the concentration in the tank was controlled to not exceed 7.5 g / L. Experimental results are shown below. Figure 7 .

[0362] The results showed that after 48 hours of fermentation, the molar ratio of 3HHx monomer in PHBHHx produced by recombinant halophilic bacteria was close to 40 mol.

[0363] Preferably, recombinant halophilic bacteria can produce PHBHHx with a high 3HHx molar ratio using sodium hexanoate as the sole carbon source.

[0364] Example 8: Production of PHA by recombinant halophilic bacteria using glucose as the sole carbon source in a fermenter

[0365] Using the recombinant halophilic bacterium TDC-G34 constructed in Example 5 as the fermentation strain, PHA was produced in a 7L fermenter with glucose as the sole carbon source.

[0366] The specific steps are as follows:

[0367] Recombinant halophilic bacteria TDC-G34 was inoculated into 20 ml of LB60 medium and cultured for 12-16 h. Then, it was transferred to new LB60 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 the 7 L bioreactor (NBSBioflo3000).

[0368] Prepare 2.7L of substrate culture medium. The concentrations of each component in the substrate are as follows: glucose (60g), sodium chloride (135g), potassium chloride (15g), yeast extract (30g), urea (9g), disodium citrate (7.8g), anhydrous magnesium sulfate (0.6g), potassium dihydrogen phosphate (15.6g), component III (30mL), and component IV (3mL).

[0369] The feed I culture medium contains glucose (200g), yeast extract (8g) and urea (32g).

[0370] The feed II medium contains glucose (200g), yeast extract (4g) and urea (28g).

[0371] During fermentation, oxygen concentration is adjusted by stirring and aeration; the pH of the culture medium is set to 8.5 and automatically adjusted by NaOH; the temperature is set to 37℃ and automatically controlled by the instrument.

[0372] After 8 hours of fermentation, feed I and feed II were added sequentially. The residual glucose content in the fermenter was monitored in real-time using a glucometer, and its concentration was controlled between 5-10 g / L. Experimental results are shown below. Figure 8 .

[0373] The results showed that the recombinant halophilic bacterium TDC-G34, using glucose as the sole carbon source, produced PHAs of P3HB after 48 hours of fermentation, and could not produce PHBHHx.

[0374] Example 9: Production of PHBHHx by recombinant halophilic bacteria using a mixed carbon source in a fermenter

[0375] Using the recombinant halophilic bacterium TDC-G34 constructed in Example 5 as the fermentation strain, PHBHHx was produced in a 7L fermenter with glucose and sodium hexanoate as a mixed carbon source.

[0376] The specific steps are as follows:

[0377] Recombinant halophilic bacteria TDC-G34 was inoculated into 20 ml of LB60 medium and cultured for 12-16 h. Then, it was transferred to new LB60 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 the 7L bioreactor (NBSBioflo3000).

[0378] Prepare 2.7L of substrate culture medium. The concentrations of each component in the substrate are as follows: glucose (60g), sodium chloride (135g), potassium chloride (15g), yeast extract (30g), urea (9g), disodium citrate (7.8g), anhydrous magnesium sulfate (0.6g), potassium dihydrogen phosphate (15.6g), component III (30mL), and component IV (3mL).

[0379] The feed I culture medium contains glucose (200g), yeast extract (8g) and urea (32g).

[0380] The feed II medium contains glucose (200g), yeast extract (4g) and urea (28g).

[0381] During fermentation, oxygen concentration is adjusted by stirring and aeration; the pH of the culture medium is set to 8.5 and automatically adjusted by NaOH; the temperature is set to 37℃ and automatically controlled by the instrument.

[0382] After 8 hours of fermentation, feed I and feed II were added sequentially. The residual glucose content in the fermenter was monitored in real time using a blood glucose meter, and its concentration was controlled between 5-10 g / L.

[0383] Simultaneously, sodium hexanoate was added after 8 hours of fermentation. The concentration of sodium hexanoate was monitored online by HPLC, and the concentration in the tank was controlled to not exceed 7.5 g / L. Experimental results are shown below. Figure 9 .

[0384] The results showed that after 48 hours of fermentation, the molar ratio of 3HHx in PHBHHx was approximately 12 mol.

[0385] Preferably, in conjunction with Examples 7 and 8, the molar ratio of 3HHx in PHBHHx can be controlled by adjusting the ratio of sodium hexanoate to glucose.

[0386] Example 10: Production of PHBHHx by recombinant halophilic bacteria using a mixed carbon source in shake flasks

[0387] Using the recombinant halophilic bacterium TDC-G34 constructed in Example 5 as the fermentation strain, PHBHHx was produced in a shake flask with glucose and sodium hexanoate as a mixed carbon source.

[0388] The specific steps are as follows:

[0389] Recombinant halophilic bacteria TDC-G34 was inoculated into 20 mL of LB60 medium and cultured for 10-12 h. Then, it was transferred to a new 20 mL of LB60 medium at a volume ratio of 1% and cultured for another 8-12 h to obtain the seed culture for shake-flask fermentation.

[0390] 2.5 mL of the shake-flask fermentation seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of 60 LB fermentation medium and cultured. The final concentrations of sodium hexanoate and glucose are shown in Table 6. The shaker temperature was 37℃ and the rotation speed was 200 rpm. After 48 h of culture, the cell dry weight and PHBHHx content were measured. Each experiment was performed in triplicate, and the results were averaged. The results of PHBHHx production by strain TDC-G34 under mixed carbon source conditions are shown in Table 6. Further verification showed that the molar ratio of 3HHx in PHBHHx could be controlled by adjusting the ratio of sodium hexanoate and glucose. The adjustable range of the molar ratio of 3HHx in PHBHHx was 0-38.17 mol.

[0391] Table 6. Production of PHBHHx by strain TDC-G34 under mixed carbon source conditions

[0392]

[0393] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of 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 protection scope of the present invention.

Claims

1. A recombinant halophilic bacterium, characterized in that, The recombinant halophilic bacteria expressed exogenous phaC Genes and phaJ Genes, as described phaC Genes and phaJ Genes originate from Aeromonas caviae ; The recombinant halophilic bacteria are inactivated by enol-CoA hydratase; the inactivation includes knocking out the gene encoding enol-CoA hydratase. The enoyl-CoA hydratase mentioned above is the FadB protein; The amino acid sequence of the FadB protein is shown in SEQ ID NO: 38; The recombinant halophilic bacteria are Halomonas bluephagenesis .

2. The recombinant halophilic bacteria according to claim 1, characterized in that, The aforementioned phaC The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. The recombinant halophilic bacteria according to claim 1, characterized in that, The aforementioned phaJ The nucleotide sequence of the gene is shown in SEQ ID NO:

3.

4. The recombinant halophilic bacteria according to claim 1, characterized in that, The nucleotide sequence encoding the FadB protein is shown in SEQ ID NO:

27.

5. The recombinant halophilic bacteria according to claim 1, characterized in that, The nucleotide sequence of the sgRNA used to knock out the gene encoding enzymatic coenzyme A hydratase is shown in SEQ ID NO:

37.

6. A method for preparing the recombinant halophilic bacteria according to any one of claims 1-5, characterized in that, The preparation method includes introducing the following 1) and 2) into halophilic bacteria: 1) phaC Genes and phaJ Gene; 2) The encoding genes of sgRNA and Cas9 protein, wherein the encoding genes of sgRNA and Cas9 protein are used to knock out the gene encoding enoyl-CoA hydratase.

7. The preparation method according to claim 6, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

37.

8. The preparation method according to claim 6, characterized in that, The aforementioned phaC Genes and / or phaJ Genes are regulated by inducible promoters and / or constitutive promoters.

9. The preparation method according to claim 6, characterized in that, The phaC Genes and / or phaJ Genes are expressed on plasmids and / or integrated into the genome for expression.

10. The preparation method according to claim 9, characterized in that, The integration into the genome refers to integration into the G3, G4, G7 and / or G51 sites in the halophilic bacteria genome; The nucleotide sequence of the G3 site is shown in SEQ ID NO: 15; The nucleotide sequence of the G4 site is shown in SEQ ID NO: 16; The nucleotide sequence of the G7 site is shown in SEQ ID NO: 17; The nucleotide sequence of the G51 site is shown in SEQ ID NO:

18.

11. The preparation method according to claim 6, characterized in that, phaC Genes and / or phaJ Genes in halophilic bacteria can be single or multiple copies.

12. A method for producing PHBHHx, characterized in that, The method includes fermenting and culturing the recombinant halophilic bacteria according to any one of claims 1-5, or fermenting and culturing the recombinant halophilic bacteria obtained by the preparation method according to any one of claims 6-11.

13. The method according to claim 12, characterized in that, The carbon source in the fermentation process includes hexanoic acid, hexanoate, and / or glucose.

14. The method according to claim 13, characterized in that, The hexanoates include sodium hexanoate and / or potassium hexanoate.

15. A method for increasing the molar ratio of 3HHx monomer in the production of PHBHHx by halophilic bacteria, characterized in that, The method includes fermenting and culturing the recombinant halophilic bacteria according to any one of claims 1-5, or fermenting and culturing the recombinant halophilic bacteria obtained by the preparation method according to any one of claims 6-11.

Images