Method for producing 5-carbon compound or polymer thereof

Abstract

The present invention provides a recombinant strain for producing a 5-carbon compound or a polymer thereof and a use thereof. A strain which originally cannot produce a 5-carbon compound is modified, and a 5-carbon compound synthetic gene is added, so as to construct a recombinant strain capable of producing a 5-carbon compound or a polymer containing a 5-carbon compound monomer, so that production of high-added-value compounds such as 5-aminovaleric acid, 5-hydroxyvaleric acid and 5-hydroxyvaleric acid-related PHA is achieved.

Description

A method for producing a 5-carbon compound or a polymer thereof Technical Field

[0001] The present invention relates to the fields of microbial synthetic biology and fermentation engineering, and more particularly to a method for producing 5-carbon compounds and 5-hydroxyvaleric acid-related PHA. Background Art

[0002] Five-carbon compounds include the high-value-added but expensive 5-aminovaleric acid (5-AVA) and 5-hydroxyvaleric acid (5HV). 5-AVA is a key monomer in the synthesis of novel bio-based nylon 5, which exhibits the highest remanent polarizability and is an ideal material for many applications, including ferroelectric polymers, such as sensors and dielectric energy storage media. Furthermore, 5-AVA can serve as an additive to enhance the mechanical integrity of mixed peroxide films. It also has broad application prospects in the medical, mechanical, food, and chemical industries. In addition to its inherent functions, 5-AVA is a precursor to many five-carbon compounds, serving as an intermediate hub for the construction of high-value-added five-carbon platform compounds. This opens up significant industrial value and broad development potential for downstream derivative applications. 5-AVA can be used to synthesize five-carbon compounds such as 5-hydroxyvaleric acid, glutaric acid, δ-valerolactone, and 1,5-pentanediol. 5-Hydroxyvaleric acid can be used as a drug synthesis intermediate, serving as a carbon skeleton or increasing the biocompatibility of drugs during the synthesis process. Polyhydroxyalkanoates (PHAs) containing 5-Hydroxyvaleric acid monomers have excellent material properties and biodegradability.

[0003] Polyhydroxyalkanoate (PHA) is a class of environmentally friendly biopolyesters that can be produced by fermentation with a variety of bacteria. It is one of the most promising alternatives to traditional petroleum-based products. 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, which not only give PHA materials significant performance differences but also better meet different application scenarios. Poly (3-hydroxybutyrate-co-5-hydroxyvalerate), or P(3HB-co-5HV), is a PHA polymerized from four- and five-carbon monomers. It can provide more flexible material properties to adapt to a wider range of applications. It is one of the newer and higher-performance PHAs with strong commercial prospects. The biosynthetic pathway has been finely optimized in modules to convert L-lysine into 5-aminovaleric acid, and on this basis, the production of 5-hydroxyvaleric acid and P(5HV-co-3HB) is achieved in sequence.

[0004] Traditional fermentation technologies are complex to sterilize, consume large amounts of freshwater resources, and are prone to bacterial contamination, severely hindering the development of modern industrial biotechnology. However, a salt- and alkali-tolerant halophile isolated by Chen Guoqiang's research group at Tsinghua University from Ayding Lake in Xinjiang has been discovered. Wild-type H. bluephagenesis can accumulate over 80% of P3HB using glucose as the sole carbon source under sterile conditions. It is considered a key platform for Next Generation Industrial Biotechnology (NGIB).

[0005] Therefore, in the face of increasing market demand, the development of more economical, sustainable and efficient methods for producing five-carbon compounds and 5-hydroxyvaleric acid-related PHAs based on the halophilic chassis and NGIB process will play an important role in further reducing production costs and improving sustainable development.

[0006] Summary of the Invention

[0007] This application modifies Halomonas, a bacterium that originally does not produce compounds such as 5-aminovaleric acid and 5-hydroxyvaleric acid, to produce 5-aminovaleric acid, 5-hydroxyvaleric acid, and 5-hydroxyvaleric acid-related PHAs. This has important implications for the industrial production of high-value-added 5-carbon compounds and high-performance PHAs.

[0008] A first aspect of the present invention provides a recombinant bacterium, wherein the recombinant bacterium is Halomonas.

[0009] Preferably, the genome of the recombinant bacteria contains a synthesis gene for a 5-carbon compound, and the synthesis gene for the 5-carbon compound includes one or more of an L-lysine monooxygenase encoding gene (davB), a 5-aminovaleramidase encoding gene (davA) or an alcohol dehydrogenase encoding gene (yqhD).

[0010] Preferably, the 5-carbon compound is a 5-carbon alkyl substituted by an amino group, a hydroxyl group and / or a carboxyl group, or the 5-carbon compound is a 5-carbon ring substituted by an amino group, a hydroxyl group and / or a carboxyl group, or the 1-position or 5-position is substituted by an amino group or a carboxyl group to form a ring.

[0011] Preferably, the 5-carbon compound includes but is not limited to 5-aminovaleric acid, 5-hydroxyvaleric acid, glutaric acid, δ-valerolactam or 1,5-pentanediol.

[0012] Preferably, the gene encoding glutaric semialdehyde dehydrogenase (gabD) and / or the gene encoding 4-aminobutyrate aminotransferase (gabT) in the recombinant bacteria is knocked down or knocked out.

[0013] More preferably, the gene encoding glutaric semialdehyde dehydrogenase is gabD2 or gabD3.

[0014] More preferably, the 4-aminobutyrate aminotransferase encoding gene is gabT1 or gabT2.

[0015] Preferably, the recombinant bacteria lacks the gene encoding glutaric semialdehyde dehydrogenase (gabD) and / or the gene encoding 4-aminobutyrate aminotransferase (gabT).

[0016] Preferably, the recombinant bacteria lacks the gene encoding glutaric semialdehyde dehydrogenase (gabD), and further preferably, the recombinant bacteria lacks gabD2 and / or gabD3.

[0017] Preferably, the recombinant bacteria lacks the 4-aminobutyrate aminotransferase encoding gene (gabT), and further preferably, the recombinant bacteria lacks gabT1 and / or gabT2.

[0018] Preferably, the recombinant bacteria lacks, knocks down or knocks out one or more of gabD2, gabD3, gabT1 or gabT2.

[0019] Preferably, the amino acid sequence corresponding to GabD2 is shown in SEQ ID NO: 6, and the amino acid sequence corresponding to GabD3 is shown in SEQ ID NO: 7.

[0020] Preferably, the amino acid sequence corresponding to GabT1 is shown in SEQ ID NO: 8, and the amino acid sequence corresponding to GabT2 is shown in SEQ ID NO: 9.

[0021] Preferably, the L-lysine monooxygenase encoding gene is derived from Pseudomonas, preferably from Pseudomonas putida.

[0022] Preferably, the 5-aminovaleramidase encoding gene is derived from Pseudomonas, preferably from Pseudomonas putida.

[0023] Preferably, the alcohol dehydrogenase encoding gene is derived from Halomonas or Escherichia (eg, Escherichia coli).

[0024] Preferably, the amino acid sequence of the L-lysine monooxygenase includes SEQ ID NO: 1, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 1.

[0025] Preferably, the amino acid sequence of the 5-aminovaleramidase includes SEQ ID NO: 2, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 2.

[0026] Preferably, the amino acid sequence of the alcohol dehydrogenase includes SEQ ID NO: 3, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 3.

[0027] The recombinant bacteria can produce 5-carbon compounds. Preferably, the recombinant bacteria can produce 5-carbon compounds using glucose, L-lysine, or 5-aminovaleric acid as substrates.

[0028] In a specific embodiment of the present invention, the recombinant bacteria can produce 5-aminovaleric acid using glucose and / or L-lysine as substrates.

[0029] In a specific embodiment of the present invention, the recombinant bacteria can produce 5-hydroxyvaleric acid using glucose and / or L-lysine as substrates.

[0030] In a specific embodiment of the present invention, the recombinant bacteria can produce 5-hydroxyvaleric acid using 5-aminovaleric acid as a substrate.

[0031] Preferably, the recombinant bacteria comprises an L-lysine to 5-aminovaleric acid pathway;

[0032] The recombinant bacteria include:

[0033] A) gabD2 and gabD3 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene; or

[0034] B) gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene; or

[0035] C) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis genes include L-lysine monooxygenase encoding gene and 5-aminovaleramidase encoding gene.

[0036] Preferably, the recombinant bacteria comprises a 5-aminovaleric acid to 5-hydroxyvaleric acid pathway, and the recombinant bacteria comprises:

[0037] a) gabD2 and gabD3 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene includes an alcohol dehydrogenase encoding gene; or

[0038] b) gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene includes an alcohol dehydrogenase encoding gene; or

[0039] c) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene includes an alcohol dehydrogenase encoding gene.

[0040] Preferably, the recombinant bacteria comprises an L-lysine to 5-hydroxyvalerate pathway;

[0041] The recombinant bacteria include:

[0042] A) gabD2 and gabD3 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene; or

[0043] B) gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene; or

[0044] C) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis genes include L-lysine monooxygenase encoding gene, 5-aminovaleramidase encoding gene and alcohol dehydrogenase encoding gene.

[0045] Preferably, the number of copies of the synthetic gene inserted into the genome is one or more.

[0046] Preferably, the synthetic gene can be integrated into the chromosomal genome or contained in one or more recombinant plasmid vectors and released in the recombinant bacteria.

[0047] The synthetic gene is overexpressed in the recombinant bacteria.

[0048] In a specific embodiment of the present invention, the synthetic gene is expressed at different locations on the chromosome.

[0049] Preferably, the synthetic gene is expressed by a promoter, which may be constitutive and / or inducible.

[0050] In a specific embodiment of the present invention, the constitutive promoter can be a constitutive porin gene porin promoter or a mutant thereof. The constitutive promoter can be a low strength promoter, a medium strength promoter or a high strength promoter. For example, porin 、P porin29 、P porin88 、P porin221 、P porin194 、P porin251 、P porin278 、P porin68 、P porin42 、P porin58 、P porin226 、P porin183 、P porin140 or P porin141 .

[0051] In a specific embodiment of the present invention, the inducible promoter may be an IPTG (isopropyl-β-D-thiogalactopyranoside) inducible T7 promoter or an AHL (homoserine lactone) inducible promoter.

[0052] Preferably, the inducible promoter includes but is not limited to P tac Promoter, P lux Promoter and / or P lac Promoter.

[0053] Preferably, the inducer includes but is not limited to IPTG or AHL.

[0054] Preferably, the AHL induction concentration is 0-2 mM, for example 0, 5×10 -6 , 10 -5 , 5×10 -5 , 10 -4 , 5×10 -4 , 10 -3 , 0.005, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2 mM, preferably 5 × 10 -6 -10 -3 Any value in mM.

[0055] Preferably, the IPTG induction concentration is 0-5 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, preferably any value between 0.02-2 g / L.

[0056] Preferably, the synthetic gene is expressed under the control of one or more promoters, and can also be combined into an operon and expressed under the control of one promoter.

[0057] Preferably, each gene in the synthetic gene can be on one plasmid or several genes can be combined on one or more plasmids.

[0058] The genus Halomonas includes Halomonas bluephagenesis, Halomonas campaniensis or Halomonas aydingkolgenesis and any derivatives thereof, especially those genetically engineered and physicochemically mutagenized.

[0059] Preferably, the synthetic gene is introduced into either or both of the G4 and G7 sites in the genome of the recombinant bacterium.

[0060] Preferably, each synthetic gene is a single copy or a double copy.

[0061] Depending on the specific implementation, the single or multiple copies can be achieved by introducing the synthetic gene into one or more sites in the genome. For example, introducing the synthetic gene into either the G4 or G7 sites in the genome achieves a single copy; introducing the synthetic gene into both the G4 and G7 sites in the genome achieves a double copy.

[0062] Preferably, the upstream sequence of the G4 site includes SEQ ID NO: 14, and the downstream sequence includes SEQ ID NO: 15.

[0063] Preferably, the upstream sequence of the G7 site includes SEQ ID NO: 16, and the downstream sequence includes SEQ ID NO: 17.

[0064] In a second aspect, the present invention provides a recombinant bacterium, wherein the recombinant bacterium is of the genus Halomonas.

[0065] Preferably, the genome of the recombinant bacterium includes a synthetic gene for a polymer comprising a 5-carbon compound monomer, and the synthetic gene for the polymer comprising a 5-carbon compound monomer includes a PHA synthase encoding gene (phaC) and / or a 5-hydroxyvalerate coenzyme A transferase encoding gene (abfT).

[0066] In a specific embodiment of the present invention, the genome of the recombinant bacterium contains a synthesis gene for a 5-carbon compound and a synthesis gene for a polymer.

[0067] The recombinant bacteria can produce a polymer comprising a 5-carbon compound monomer. Preferably, the recombinant bacteria can produce a polymer comprising a 5-carbon compound monomer using glucose, L-lysine, or 5-aminovaleric acid as a substrate.

[0068] Preferably, the recombinant bacteria comprises a pathway for producing PHA comprising a 5-hydroxyvaleric acid monomer from a substrate comprising L-lysine, and the recombinant bacteria comprises the recombinant bacteria described in the first aspect further introduced with a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene.

[0069] The recombinant bacteria include any of the following groups of recombinant bacteria into which a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene are further introduced:

[0070] A) gabD2 and gabD3 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene; or

[0071] B) gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene; or

[0072] C) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis genes include L-lysine monooxygenase encoding gene and 5-aminovaleramidase encoding gene.

[0073] The recombinant bacteria include any of the following groups of recombinant bacteria into which a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene are further introduced:

[0074] A) gabD2 and gabD3 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene; or

[0075] B) gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene; or

[0076] C) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis genes include L-lysine monooxygenase encoding gene, 5-aminovaleramidase encoding gene and alcohol dehydrogenase encoding gene.

[0077] Preferably, the recombinant bacteria comprises a PHA pathway comprising a substrate comprising 5-aminovaleric acid to produce a 5-hydroxyvaleric acid monomer, and the recombinant bacteria comprises the recombinant bacteria described in the first aspect further introduced with a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene.

[0078] Preferably, the recombinant bacteria include any one of the following groups of recombinant bacteria into which a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene are further introduced:

[0079] a) gabD2 and gabD3 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene includes an alcohol dehydrogenase encoding gene; or

[0080] b) gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene includes an alcohol dehydrogenase encoding gene; or

[0081] c) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked out or knocked down, and the 5-carbon compound synthesis gene includes an alcohol dehydrogenase encoding gene.

[0082] Preferably, the PHA synthase encoding gene is derived from Aeromonas or Ralstonia; more preferably, it is derived from Aeromonas caviae FA440 or Ralstonia eutropha.

[0083] Preferably, the 5-hydroxyvalerate-CoA transferase encoding gene is derived from Escherichia, preferably from Escherichia coli.

[0084] Preferably, the amino acid sequence of the PHA synthase includes SEQ ID NO: 5, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 5.

[0085] Preferably, the amino acid sequence of the 5-hydroxyvalerate CoA transferase includes SEQ ID NO: 4, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 4.

[0086] Preferably, the 5-carbon compound is a 5-carbon alkyl substituted by an amino group, a hydroxyl group and / or a carboxyl group, or the 5-carbon compound is a 5-carbon ring substituted by an amino group, a hydroxyl group and / or a carboxyl group, or the 1-position or 5-position is substituted by an amino group or a carboxyl group to form a ring.

[0087] Preferably, the 5-carbon compound includes but is not limited to 5-aminovaleric acid, 5-hydroxyvaleric acid, glutaric acid, δ-valerolactam or 1,5-pentanediol.

[0088] Preferably, the polymer is a 5-hydroxyvaleric acid homopolymer, or a copolymer of 5-hydroxyvaleric acid and other monomers.

[0089] Preferably, the copolymer includes a random copolymer, an alternating copolymer, a block copolymer or a graft copolymer.

[0090] Preferably, the other monomers include but are not limited to one or more of 3-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid or 3-hydroxydodecanoic acid.

[0091] Preferably, the number of copies of the synthetic gene inserted into the genome is one or more.

[0092] Preferably, the synthetic gene can be integrated into the chromosomal genome or contained in one or more recombinant plasmid vectors and released in the recombinant bacteria.

[0093] The synthetic gene is overexpressed in the recombinant bacteria.

[0094] In a specific embodiment of the present invention, the synthetic gene is expressed at different locations on the chromosome.

[0095] Preferably, the synthetic gene is expressed by a promoter, which may be constitutive and / or inducible.

[0096] In a specific embodiment of the present invention, the constitutive promoter can be a constitutive porin gene porin promoter or a mutant thereof. The constitutive promoter can be a low strength promoter, a medium strength promoter or a high strength promoter. For example, porin 、P porin29 、P porin88 、P porin221 、Pporin194 、P porin251 、P porin278 、P porin68 、P porin42 、P porin58 、P porin226 、P porin183 、P porin140 or P porin141 .

[0097] In a specific embodiment of the present invention, the inducible promoter may be an IPTG (isopropyl-β-D-thiogalactopyranoside) inducible T7 promoter or an AHL (homoserine lactone) inducible promoter.

[0098] Preferably, the inducible promoter includes but is not limited to P tac Promoter, P lux Promoter and / or P lac promoter.

[0099] Preferably, the inducer includes but is not limited to IPTG or AHL.

[0100] Preferably, the AHL induction concentration is 0-2 mM, for example 0, 5×10 -6 , 10 -5 , 5×10 -5 , 10 -4 , 5×10 -4 , 10 - 3 , 0.005, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2 mM, preferably 5 × 10 -6 -10 -3 mM, preferably any value between 0.0005-0.001 mM.

[0101] Preferably, the IPTG induction concentration is 0-5 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, preferably any value between 0.02-2 g / L.

[0102] Preferably, the synthetic gene is expressed under the control of one or more promoters, and can also be combined into an operon and expressed under the control of one promoter.

[0103] Preferably, each gene in the synthetic gene can be on one plasmid or several genes can be combined on one or more plasmids.

[0104] The genus Halomonas includes Halomonas bluephagenesis, Halomonas campaniensis or Halomonas aydingkolgenesis and any derivatives thereof, especially those genetically engineered and physicochemically mutagenized.

[0105] Preferably, each synthetic gene is a single copy, a double copy, or three or more copies.

[0106] In a third aspect of the present invention, an expression vector is provided, comprising a gene for synthesizing a 5-carbon compound, wherein the gene for synthesizing a 5-carbon compound comprises at least one or more of an L-lysine monooxygenase encoding gene (davB), a 5-aminovaleramidase encoding gene (davA), or an alcohol dehydrogenase encoding gene (yqhD).

[0107] Preferably, the amino acid sequence of the L-lysine monooxygenase includes SEQ ID NO: 1, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 1.

[0108] Preferably, the amino acid sequence of the 5-aminovaleramidase includes SEQ ID NO: 2, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 2.

[0109] Preferably, the amino acid sequence of the alcohol dehydrogenase includes SEQ ID NO: 3, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 3.

[0110] Preferably, the expression vector comprises a promoter, and the promoter may be constitutive and / or inducible.

[0111] In a specific embodiment of the present invention, the constitutive promoter can be a constitutive porin gene porin promoter or a mutant thereof. The constitutive promoter can be a low strength promoter, a medium strength promoter or a high strength promoter. For example, porin 、P porin29 、P porin88 、P porin221 、P porin194 、P porin251 、P porin278 、P porin68 、P porin42 、P porin58 、P porin226 、P porin183 、P porin140 or P porin141 .

[0112] In a specific embodiment of the present invention, the inducible promoter may be an IPTG (isopropyl-β-D-thiogalactopyranoside) inducible T7 promoter or an AHL (homoserine lactone) inducible promoter.

[0113] Preferably, the inducer includes but is not limited to IPTG or AHL.

[0114] Preferably, the inducible promoter includes but is not limited to P tac Promoter, P lux Promoter and / or P lac promoter.

[0115] Preferably, the expression vector is capable of replication, transcription, and translation in the host cell. Therefore, it also contains other conventional expression elements, such as ribosome binding sites, terminators, enzyme cleavage sites, etc.

[0116] Preferably, the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, preferably a prokaryotic expression vector.

[0117] Preferably, the expression vector is a plasmid.

[0118] In a fourth aspect of the present invention, an expression vector is provided, wherein the expression vector comprises a synthetic gene for a polymer comprising a 5-carbon compound monomer, wherein the synthetic gene comprises a PHA synthase encoding gene (phaC) and / or a 5-hydroxyvalerate coenzyme A transferase encoding gene (abfT).

[0119] Preferably, the amino acid sequence of the PHA synthase includes SEQ ID NO: 5, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 5.

[0120] Preferably, the amino acid sequence of the 5-hydroxyvalerate CoA transferase includes SEQ ID NO: 4, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 4.

[0121] Preferably, the expression vector further comprises one or more of an L-lysine monooxygenase encoding gene (davB), a 5-aminovaleramidase encoding gene (davA) or an alcohol dehydrogenase encoding gene (yqhD).

[0122] Preferably, the amino acid sequence of the L-lysine monooxygenase includes SEQ ID NO: 1, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 1.

[0123] Preferably, the amino acid sequence of the 5-aminovaleramidase includes SEQ ID NO: 2, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 2.

[0124] Preferably, the amino acid sequence of the alcohol dehydrogenase includes SEQ ID NO: 3, or includes an amino acid sequence having more than 80% homology with SEQ ID NO: 3.

[0125] Preferably, the expression vector comprises a promoter, and the promoter may be constitutive and / or inducible.

[0126] In a specific embodiment of the present invention, the constitutive promoter can be a constitutive porin gene porin promoter or a mutant thereof. The constitutive promoter can be a low strength promoter, a medium strength promoter or a high strength promoter. For example, porin 、P porin29 、P porin88 、P porin221 、P porin194 、P porin251 、P porin278 、P porin68 、P porin42 、P porin58 、P porin226 、P porin183 、P porin140 or P porin141 .

[0127] In a specific embodiment of the present invention, the inducible promoter may be an IPTG (isopropyl-β-D-thiogalactopyranoside) inducible T7 promoter or an AHL (homoserine lactone) inducible promoter.

[0128] Preferably, the inducer includes but is not limited to IPTG or AHL.

[0129] Preferably, the inducible promoter includes but is not limited to P tac Promoter, P lux Promoter and / or P lac Promoter.

[0130] Preferably, the expression vector is capable of replication, transcription, and translation in the host cell. Therefore, it also contains other conventional expression elements, such as ribosome binding sites, terminators, enzyme cleavage sites, etc.

[0131] Preferably, the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, preferably a prokaryotic expression vector.

[0132] Preferably, the expression vector is a plasmid.

[0133] The fifth aspect of the present invention provides a recombinant bacterium comprising the above expression vector.

[0134] In a sixth aspect, the present invention provides a method for preparing the above-mentioned recombinant bacteria, which comprises introducing a synthetic gene for a 5-carbon compound and / or a synthetic gene for a polymer comprising a 5-carbon compound monomer into the recombinant bacteria.

[0135] Preferably, the preparation method comprises introducing the above-mentioned expression vector into recombinant bacteria.

[0136] Preferably, the synthetic gene can be integrated into the chromosomal genome or contained in one or more recombinant plasmid vectors and released in the recombinant bacteria.

[0137] The introduction can be performed by inserting the synthetic gene into a non-functional region of the recombinant bacteria.

[0138] The construction method includes overexpression of the synthetic gene in a recombinant bacterium.

[0139] Preferably, the synthetic gene is introduced into the recombinant bacteria using a plasmid, so that the synthetic gene is overexpressed in the recombinant bacteria.

[0140] Preferably, the plasmid contains a synthetic gene for a 5-carbon compound and / or a synthetic gene for a polymer containing a 5-carbon compound monomer.

[0141] Preferably, the preparation method is preferably a gene editing technology, such as CRISPR-Cas9 or LoxP-Cre system. In a specific embodiment of the present invention, the gene is introduced into Halomonas by conjugation transformation with Escherichia coli.

[0142] Preferably, the introduction is to insert a synthetic gene for a 5-carbon compound and / or a synthetic gene for a polymer comprising a 5-carbon compound monomer into the genome of Halomonas.

[0143] In a seventh aspect, the present invention provides a method for producing a 5-carbon compound or a polymer containing a 5-carbon compound monomer, wherein the method comprises culturing (preferably fermenting) the above-mentioned recombinant bacteria.

[0144] The culture medium used in the culture may be liquid, solid or semi-solid.

[0145] The culture medium can be a natural culture medium, a synthetic culture medium and / or a semi-synthetic culture medium. Preferably, the culture medium can adopt a culture medium conventional in the prior art, or other substances containing nutrients. The conventional culture medium comprises a mineral salt culture medium (MMG), a Luria-Bertani culture medium (LB), etc.

[0146] Preferably, the culture medium contains a carbon source and / or a nitrogen source, etc. The nitrogen source includes an inorganic nitrogen source and / or an organic nitrogen source. Further preferably, the culture medium further contains vitamins and / or growth factors.

[0147] Preferably, the carbon source includes but is not limited to glucose or sugar, fat or protein that can be decomposed into glucose; or, L-lysine or protein that can be decomposed into L-lysine; or, 5-aminovaleric acid or 5-hydroxyvaleric acid.

[0148] Preferably, the carbon source is glucose and / or L-lysine.

[0149] Preferably, the inorganic nitrogen source includes but is not limited to ammonium sulfate, nitrate, ammonia water or urea.

[0150] The organic nitrogen source includes but is not limited to soybean cake powder, peanut cake powder, cottonseed cake powder, corn steep liquor, peptone, yeast extract or fish meal.

[0151] Preferably, the culture medium contains inorganic salts, including but not limited to those that can provide Na + , K + , Ca 2+ Mg 2+ 、Cl － PO4 3- 、SO4 2- 、HCO3 － Inorganic salts with other ingredients.

[0152] Preferably, the culture medium comprises urea and / or yeast extract.

[0153] Preferably, the urea concentration in the culture medium is 0.5 g / L-2 g / L, such as 0.5 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.5 g / L, 1.6 g / L or 2 g / L.

[0154] Preferably, the concentration of yeast extract in the culture medium is 0.5 g / L-2 g / L, such as 0.5 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.5 g / L, 1.6 g / L or 2 g / L.

[0155] Preferably, the salt concentration in the culture medium is 10 g / L-50 g / L, such as 10 g / L, 20 g / L, 30 g / L, 40 g / L or 50 g / L.

[0156] Preferably, the carbon source concentration is 5 g / L-50 g / L, such as 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L or 50 g / L.

[0157] Preferably, the culture method is open fermentation.

[0158] Preferably, the culture method is a whole-cell catalytic method.

[0159] Preferably, the whole-cell catalysis method includes adding L-lysine, Triton-X100 and / or ethanol to improve the permeability of the cell membrane.

[0160] Preferably, the fermentation and culture 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.

[0161] In an eighth aspect, the present invention provides a method for producing 5-aminovaleric acid, wherein the method comprises fermenting and culturing the recombinant bacteria.

[0162] In a specific embodiment of the present invention, the recombinant bacteria comprises a 5-aminovaleric acid synthesis gene, and the 5-aminovaleric acid synthesis gene comprises an L-lysine monooxygenase encoding gene and / or a 5-aminovaleramidase encoding gene.

[0163] Preferably, the recombinant bacteria lacks, knocks out or knocks down one or more of gabD2, gabD3, gabT1 or gabT2.

[0164] Preferably, the fermentation method is open fermentation.

[0165] Preferably, the fermentation method is a whole-cell catalytic method.

[0166] Preferably, the whole-cell catalytic method is to add different concentrations of L-lysine and Triton-X100 and / or ethanol at different time points to improve the permeability of the cell membrane.

[0167] Preferably, the carbon source in the fermentation process includes but is not limited to glucose or sugar, fat or protein that can be decomposed into glucose; or L-lysine or protein that can be decomposed into L-lysine.

[0168] Preferably, the fermentation product further comprises PHA.

[0169] Preferably, the fermentation medium is a conventional medium or the composition of the medium is appropriately adjusted to suit the survival of the microorganisms and the production of products.

[0170] Preferably, 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.

[0171] A ninth aspect of the present invention provides a method for producing 5-hydroxyvaleric acid, wherein the method comprises fermenting and culturing the recombinant bacteria.

[0172] Preferably, the fermentation medium contains glucose or sugar that can be decomposed into glucose, fat or protein; L-lysine or protein that can be decomposed into L-lysine, or 5-aminovaleric acid.

[0173] In a specific embodiment of the present invention, the recombinant bacteria contains a 5-hydroxyvaleric acid synthesis gene, and the synthesis gene contains one or a combination of two or more of the L-lysine monooxygenase encoding gene, the 5-aminovaleramidase encoding gene or the alcohol dehydrogenase encoding gene.

[0174] Preferably, the recombinant bacteria lacks, knocks out or knocks down one or more of gabD2, gabD3, gabT1 or gabT2.

[0175] Preferably, the fermentation is carried out under open conditions.

[0176] Preferably, the fermentation product further comprises PHA.

[0177] Preferably, the production method is a whole cell catalysis method

[0178] Preferably, the fermentation medium contains Triton X-100 at different concentrations.

[0179] In a tenth aspect, the present invention provides a method for producing 5-hydroxyvaleric acid-related PHA, wherein the method comprises fermenting and culturing the recombinant bacteria.

[0180] Preferably, the fermentation medium contains glucose or sugar, fat or protein that can be decomposed into glucose; or L-lysine or protein that can be decomposed into L-lysine, or 5-aminovaleric acid or 5-hydroxyvaleric acid.

[0181] Preferably, the recombinant bacteria comprises a synthetic gene, wherein the synthetic gene comprises a 5-hydroxyvalerate CoA transferase encoding gene and / or a PHA synthase encoding gene.

[0182] In a specific embodiment of the present invention, the synthetic gene further comprises an alcohol dehydrogenase encoding gene.

[0183] In a specific embodiment of the present invention, the synthetic gene further comprises a gene encoding L-lysine monooxygenase, a gene encoding 5-aminovaleramidase and a gene encoding alcohol dehydrogenase.

[0184] Preferably, the recombinant bacteria lacks, knocks out or knocks down one or more of gabD2, gabD3, gabT1 or gabT2.

[0185] Preferably, the fermentation product further comprises PHA.

[0186] Preferably, the synthetic gene further comprises a β-ketothiolase encoding gene and / or a NADPH / NADH-dependent acetoacetyl reductase encoding gene.

[0187] Preferably, the fermentation medium contains Triton-X100 at different concentrations.

[0188] In an eleventh aspect, the present invention provides the use of the aforementioned recombinant bacteria, expression vector, preparation method, or a 5-carbon compound or a polymer comprising a 5-carbon compound monomer obtained by the aforementioned production method in the preparation of a biodegradable new biomaterial or drug. Preferably, the use is in the development of medical devices, medical microspheres, surgical sutures, patches, disposable packaging materials, or textile fibers.

[0189] The "polymer" described in the present invention includes 5-hydroxyvaleric acid-related PHA, and the PHA is a homopolymer PHA and / or a copolymer PHA. Preferably, the PHA is selected from 3-hydroxybutyric acid (3HB) homopolymer PHB. 3-hydroxybutyric acid (3HB) and 5-hydroxyvaleric acid (5HV) binary copolymer P3HB5HV. 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV) and 4-hydroxybutyric acid (4HB), or 3-hydroxyvaleric acid (3HV), or 3-hydroxyhexanoic acid (3HHx) or 3-hydroxypropionic acid (3HP) are abbreviated as P(3HB-co-5HV-co-4HB), P(3HB-co-5HV-co-3HV), P(3HB-co-5HV-co-3HHx), P(3HB-co-5HV-co-3HP). The tetrapolymer includes PHA composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB) and 3-hydroxyvaleric acid (3HV), or a tetramer composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV) 4-hydroxybutyric acid (4HB) and 3-hydroxyhexanoic acid (3HHx), or a polymer composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV) 4-hydroxybutyric acid (4HB), 3-hydroxypropionic acid (3HP), or a tetramer composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 3-hydroxyvaleric acid (3HV), 3-hydroxyhexanoic acid (3HHx), or a tetramer composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 3-hydroxypentanoic acid The polymers composed of 3-hydroxybutyric acid (3HV) and 3-hydroxypropionic acid (3HP), or the tetramers composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 3-hydroxyhexanoic acid (3HHx), and 3-hydroxypropionic acid (3HP), are abbreviated as P(3HB-co-5HV-co-4HB-co-3HV), P(3HB-co-5HV-co-4HB-co-3HHx), P(3HB-co-5HV-co-4HB-co-3HP), P(3HB-co-5HV-co-3HV-co-HHx), P(3HB-co-5HV-co-3HV-co-3HP), and P(3HB-co-5HV-co-3HHx-co-3HP), respectively.Pentapolymers such as PHA composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxyvaleric acid (3HV) and 3-hydroxyhexanoic acid (3HHx), or PHA composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxyvaleric acid (3HV), 3-hydroxypropionic acid (3HP), 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxyvaleric acid (3HV), 3-hydroxypropionic acid (3HP), 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxyvaleric acid (3HV), 3-hydroxypropionic acid (3HP), 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxypropionic acid (3HP), 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 3-hydroxyhexanoic acid (3HHx ... PHAs composed of 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxyvaleric acid (3HV), 3-hydroxyhexanoic acid (3HHx), and 3-hydroxypropionic acid (3HP) are abbreviated as P(3HB-co-5HV-co-4HB-co-3HV-co-3HHx), P(3HB-co-5HV-co-4HB-co-3HV-co-3HP), and P(3HB-co-5HV-co-3HV-co-3HHx-3HP), respectively. PHAs composed of hexapolymers such as 3-hydroxybutyric acid (3HB), 5-hydroxyvaleric acid (5HV), 4-hydroxybutyric acid (4HB), 3-hydroxyvaleric acid (3HV), 3-hydroxyhexanoic acid (3HHx), and 3-hydroxypropionic acid (3HP) are abbreviated as (3HB-co-5HV-co-4HB-co-3HV-co-3HHx-3HP). The above PHAs do not represent all types of PHAs, and other new monomers are also included.

[0190] The "synthetic genes" described herein can be introduced into cells in various ways, and can be either genes already present in the cells or genes not already present. Of course, their specific sequences can be adjusted based on actual needs. For example, if the genes are derived from other species or from Gram-negative or Gram-positive bacteria, they can be codon-optimized to be more suitable for Halomonas. For example, davBA, after codon optimization, is more suitable for Halomonas bluephagenesis.

[0191] The terms "include" or "comprising" of the present invention are open-ended descriptions, containing the specified components or steps described, as well as other specified components or steps that will not substantially affect them; when used to describe a protein or nucleic acid sequence, 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.

[0192] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed 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."

[0193] The "overexpression" mentioned in the present invention refers to up-regulating the expression of a gene to a level higher than the original natural expression level or causing a gene that was not expressed to be expressed.

[0194] The abbreviation and full name of this application are as follows:

[0195] DavB: L-lysine monooxygenase;

[0196] DavA: 5-aminopentanamidase;

[0197] GabD: glutarate semialdehyde dehydrogenase;

[0198] GabT: 4-aminobutyrate aminotransferase;

[0199] AbfT: 5-hydroxyvalerate coenzyme A transferase;

[0200] PhaA: β-ketothiolase encoding gene;

[0201] PhaB: NADPH / NADH-dependent acetoacetyl reductase encoding gene;

[0202] PhaC: PHA synthase;

[0203] YqhD: alcohol dehydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0204] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0205] Figure 1: The technical route of the present application, a recombinant Halomonas strain, a schematic diagram of producing 5-carbon compounds or their polymers using L-lysine and glucose as substrates, wherein the pathway for producing 3-hydroxybutyric acid is an endogenous pathway of Halomonas.

[0206] Figure 2: Schematic diagram of the inducible plasmid for 5-aminovaleric acid production in module 1, pSEVA321-P J23110 -luxR-P lux -davBA.

[0207] Figure 3: Metabolic pathway modification of 5-aminovaleric acid.

[0208] Figure 4: Secondary mass spectrometry results of 5-hydroxyvaleric acid (a) standard mass-to-charge ratio (b) sample mass-to-charge ratio and secondary mass spectrometry results of 5-aminovaleric acid (c) standard mass-to-charge ratio (d) sample mass-to-charge ratio.

[0209] Figure 5: Results of fermentation experiments for the scale-up of 5-aminovaleric acid production by Halomonas bluephagenes YF117. DETAILED DESCRIPTION

[0210] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments 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 shall fall within the scope of protection of the present invention.

[0211] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0212] Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial sources.

[0213] The culture medium formula used in the examples is:

[0214] 1) Escherichia coli was grown in LB medium containing 5 g / L yeast extract, 10 g / L sodium chloride, and 10 g / L peptone.

[0215] Unless otherwise specified, halophilic bacteria were cultured in 60 LB or 50 MM medium containing 5 g / L yeast extract, 60 g / L sodium chloride, and 10 g / L peptone.

[0216] The culture conditions for E. coli and halophilic bacteria were 37°C and 200 rpm.

[0217] 2) Shake flask fermentation of 5-aminovaleric acid, 5-hydroxyvaleric acid and 5-hydroxyvaleric acid-related PHA medium:

[0218] 60LB fermentation medium: 60g / L sodium chloride, 5g / L yeast extract, 10g / L tryptone, 0.1-50g / L relevant carbon source combination.

[0219] MM medium: 0.1-50 g / L of the relevant carbon source combination, 10-50 g / L sodium chloride, 1-10 g / L yeast extract, 3-6 g / L urea, 1.5-5.2 g / L potassium dihydrogen phosphate, 0.2-1 g / L magnesium sulfate, 8.5-10 g / L sodium dihydrogen phosphate, 7-15 ml / L of component III, and 1-5 ml / L of component IV. 50 MM medium refers to MM medium with a salt content of 50 g / L.

[0220] Component III: 5g / L ammonium ferric citrate, 2g / L calcium chloride dihydrate, 41.7ml concentrated hydrochloric acid (12mol / L), add water to make up to 1000ml.

[0221] 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.

[0222] The above culture media can be prepared using standard preparation methods.

[0223] 3) The halophile gene editing technology used in this application is CRISPR / Cas9 or CRISPR / AID technology, including 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.

[0224] 4) Cell dry weight determination method:

[0225] Use a centrifuge tube to collect a certain volume of bacterial liquid after culture in a shake flask or fermenter, centrifuge at 12000 rpm for 30 minutes, discard the supernatant and collect the bacteria; resuspend the bacteria in an appropriate amount of deionized water and centrifuge at 12000 rpm for 30 minutes, discard the supernatant and collect the bacteria again; place the obtained bacterial pellet in a -80℃ low-temperature refrigerator and freeze it for more than 1 hour, then place it in a vacuum freeze dryer to constant weight; weigh the total weight of the centrifuge tube and the dried bacteria in the tube; calculate the cell dry weight by the difference method.

[0226] 5) 5-Hydroxyvaleric acid-related PHA detection method:

[0227] 30-40 mg of dried bacterial cells or approximately 10-30 mg of a standard sample (poly 3-hydroxybutyrate or 5-hydroxyvalerate) 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 in chromatographic-grade methanol) and 2 mL of chloroform were added. The tube was sealed with a lid. 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 layer sample was aspirated for GC analysis. The GC analysis procedure was as follows: the column temperature was raised from room temperature to 80°C, then held for 90 seconds, then raised at a rate of 0.5°C / s to 140°C, then held for 0 seconds, then raised at a rate of 0.7°C / s to 240°C, then 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 molar ratio of PHA, 3HB, and 5HV to the dry cell weight was calculated.

[0228] 6) HPLC detection method for 5-aminovaleric acid and 5-hydroxyvaleric acid

[0229] (1) 5-aminovaleric acid was detected by derivatization of amino acids and their derivatives with diethyl ethoxymethylenemalonate (DEEMM) and analysis by HPLC (HPLC-20A, Japan). The analytical column used was a C18 column (Agilent, USA). The analytical column was characterized by a 4.6 × 250 mm diameter and a 5 μm particle size.

[0230] The experimental procedure was as follows: Mobile phases A and B were prepared. Mobile phase A consisted of pure acetonitrile, filtered through a 0.22 μm nylon membrane; mobile phase B consisted of 25 mM sodium acetate (pH adjusted to 4.8), filtered through a 0.22 μm cellulose membrane. Both mobile phases A and B were ultrasonically degassed for 30 minutes to ensure that no bubbles were present.

[0231] The specific HPLC procedure is: mobile phase A at 20-25% by volume for 0-2 minutes; 25-60% by volume for 2-20 minutes; and 60-20% by volume for 20-30 minutes. The UV absorbance wavelength is 284 nm, the temperature is 35°C, and the flow rate is 1 mL / min.

[0232] (2) HPLC determination of 5-hydroxyvaleric acid content: The fermentation broth was centrifuged at 12,000 rpm for 10 min, 200 μL of the supernatant was taken, and 1 mL of water was added to dilute the reaction solution 6 times. The diluted liquid was filtered using a 0.22 μm cellulose membrane, and the production of 5-hydroxyvaleric acid was detected by HPLC. The analytical column used was HPX-87H (Agilent, USA). The mobile phase was 5 mM dilute sulfuric acid, the flow rate was 0.5 mL / min, and the column temperature reached 60°C. The standard concentrations of 5-hydroxyvaleric acid were 0.25 g / L, 0.5 g / L, 1 g / L, 2 g / L, and 4 g / L, respectively.

[0233] For the sites of Halomonas in the examples, such as G4 and G7, refer to the article: Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas (Ye JW et al, Metabolic Engineering, 2020).

[0234] The wild mushroom species used in the examples are from:

[0235] Halomonas bluephagenesis TD01: Patent application publication number CN102120973A; the public can obtain this bacterium from Tsinghua University.

[0236] Halomonas campaniensis LS21 strain: Patent application publication number CN102925382A and the article "Jiang X, Yao Z, Chen G Q. Controlling cell volume for efficient PHB production by Halomonas[J]. Metabolic Engineering, 2017, 44:30-37" are available from Tsinghua University.

[0237] Halomonas aydingkolgenesis M1: described in patent application publication number CN111593006A; the public can obtain this bacterium from Tsinghua University.

[0238] The present application uses a recombinant Halomonas strain to produce 5-aminovaleric acid, 5-hydroxyvaleric acid, and 5-hydroxyvaleric acid-related PHAs such as P(5HV-co-3HB) using L-lysine and glucose as substrates. The specific technical route is shown in Figure 1. The present application achieves the production of 5-aminovaleric acid by heterologously expressing L-lysine monooxygenase (encoding gene davB) and 5-aminovaleramidase (encoding gene davA), further forming 5-hydroxyvaleric acid through the action of 4-aminobutyrate transaminase (encoding gene davT / gabT) and semialdehyde dehydrogenase (encoding gene yqhD / yahK), forming 5-hydroxyvaleric acid under the catalysis of 5-hydroxyvalerate coenzyme A transferase (encoding gene abfT), and further forming 5-hydroxyvaleric acid-related PHA under the action of PHA synthase (encoding gene phaC).

[0239] The embodiments described in this application are detailed descriptions of the construction methods and applications of recombinant halophilic bacteria for producing 5-aminovaleric acid, 5-hydroxyvaleric acid, and 5-hydroxyvaleric acid-related PHAs. They are illustrative rather than restrictive, and several embodiments can be listed within the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

[0240] Example 1: Construction and Optimization of a Plasmid System for the Production of 5-Aminovaleric Acid (Module 1: L-Lysine-5-Aminovaleric Acid)

[0241] The strain H. bluephagenesis Y86 is based on the wild-type Halomonas bluephagenesis TD01 CGMCC.No.4353, in which gabD2 and gabD3 are knocked out (Ye JW et al, Metabolic Engineering, 2018). The use of this strain can reduce the production of the byproduct glutaric acid. The amino acid sequence corresponding to GabD2 is shown in SEQ ID NO: 6, and the amino acid sequence corresponding to GabD3 is shown in SEQ ID NO: 7. The specific knockout scheme can be found in the literature (Jianwen Ye, et al. "Engineering of Halomonas bluephagenesis for Low Cost Production of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from Glucose." Metabolic Engineering). Construction of the inducible plasmid pSEVA321-P lux -davBA. This plasmid uses AHL as an inducer to induce the expression of davBA for the production of 5-aminovaleric acid using L-lysine as a substrate, as shown in Figure 2. This plasmid is named PYF17. The plasmid was then transformed into the halophilic bacterium H. bluephagenesis Y86. The amino acid sequence corresponding to DavB is shown in SEQ ID NO: 1, and the amino acid sequence corresponding to DavA is shown in SEQ ID NO: 2.

[0242] First, the induction level was optimized to obtain the optimal concentration of AHL for producing 5-aminovaleric acid, as shown in Table 1. The substrate L-lysine was 5 g / L. The optimal addition concentration of AHL was 5×10 -4 M.

[0243] Table 1 Induction of pSEVA321-P by different AHL concentrations lux -davBA 5-aminovaleric acid production

[0244] Secondly, the culture medium conditions were optimized. 50MM was used as a control for shake flask fermentation. During the shake flask experiment, the concentrations of yeast extract and urea were optimized and combined for culture to further test the effect on 5-aminovaleric acid production. The shake flask experiment conditions were to initially add 5×10 AHL. -4M, 5 g / L of the substrate L-lysine, in 20 mL of culture medium at 37°C, 200 rpm, and pH 8.5-9.0 for 48 hours. Through orthogonal optimization of these two variables, the optimal combination concentrations were both 5 g / L. Excerpted data are shown in Table 2. The culture medium with the highest yield was renamed 50MM-YU.

[0245] Table 2 Effect of optimization of different culture medium components on 5-aminovaleric acid production

[0246] In addition, to increase 5-aminovaleric acid production, endogenous gabT1 and gabT2 genes need to be knocked out to prevent 5-AVA degradation (Figure 3). The amino acid sequence corresponding to GabT1 is shown in SEQ ID NO: 8, and the amino acid sequence corresponding to GabT2 is shown in SEQ ID NO: 9. The sgRNA sequences for knocking out gabT1 are sgRNA1-aacaaaaatatgtcgctaatgg (SEQ ID NO: 10) and sgRNA2-caattcgctgaccgtgctg (SEQ ID NO: 11). The sgRNA sequences for knocking out gabT2 are sgRNA3-aaagcagaagtatgtcgcag (SEQ ID NO: 12) and sgRNA4-aaccgctttatcgacttcgc (SEQ ID NO: 13). After knockout, 5-AVA production was further improved, as shown in Table 3. Strain Y86 and strain Y86ΔgabT1ΔgabT2 were transformed into PYF17, named YF90 and YF91, respectively. At this time, 5 g / L L-lysine was initially added, and AHL was 5 × 10 -5 M.

[0247] Table 3 Effects of gabT1 and gabT2 knockout on 5-aminovaleric acid production

[0248] Example 2: Construction and Optimization of a Genome-Integrated 5-Aminovaleric Acid-Producing Strain (Module 1: L-Lysine—5-Aminovaleric Acid)

[0249] The engineered strains Halomonas bluephagenesis YF116 and YF117 were further constructed. Among them, YF116 had a copy of the davBA gene inserted at the G4 site on the genome (P porin183-davBA), while YF117 inserts two copies of davBA gene at the G4 and G7 sites of the genome, achieving stable expression of davBA module and efficient accumulation of 5-aminovaleric acid. porin183 -davBA, inserted into P at G7 site porin42 -davBA. The upstream sequence of the G4 site is shown in SEQ ID NO: 14, and the downstream sequence is shown in SEQ ID NO: 15. The upstream sequence of the G7 site is shown in SEQ ID NO: 16, and the downstream sequence is shown in SEQ ID NO: 17.

[0250] The specific method for constructing a recombinant bacterium producing 5-aminovaleric acid is as follows: the davBA gene cluster is inserted into the genome, and a gene integration plasmid pSEVA341-P containing the target gene and the corresponding promoter and RBS element is constructed. porinxx Finally, davBA was verified by PCR and sequencing and integrated into the genome of Halomonas bluephagenesis Y86 to construct recombinant strains YF116 and YF117.

[0251] The recombinant bacteria were inoculated into 20mL of 60LB medium, and after culturing for 12h, they were transferred to a new 20mL of 60LB medium at a volume ratio of 1% and continued to be cultured for 10h. After culturing for 12h, the bacterial liquid was used as the seed bacterial liquid. 1mL of the seed bacterial liquid was inoculated into a 100mL conical flask containing 19mL of 50MM-YU medium for a shake flask experiment. The shaker temperature was 37°C, the speed was 200rpm, and the fermentation time was 48h. After the fermentation was completed, the supernatant was centrifuged and analyzed for the content of 5-aminovaleric acid by high-performance liquid chromatography. As shown in Figure 4, it was confirmed that 5-aminovaleric acid was successfully produced.

[0252] The expression of 5-aminovaleric acid was further tested after inserting different copies and different strengths of the davBA gene, and the results are shown in Table 4. 20 g / L of L-lysine was initially added, and the shake flask experiment was completed in 20 mL of 50 MM-YU culture medium at 37 ° C, 200 rpm, pH 8.5-9.0 for 48 hours. Three replicates were set for each experiment. The intracellular supernatant was added to the cells and the cells were broken by a high-pressure homogenizer. The broken samples and supernatant were derivatized with diethyl ethoxymethylmalonate (DEEMM) and then detected by HPLC. When the double-copy davBA strain YF117 was cultured for 24 hours, 20 g / L L-lysine and 0.2% Triton-X100 were added to produce 5-aminovaleric acid. As shown in Table 4, the highest yield was 7.83 g / L. Halomonas bluephagenesis YF117 was tested in a fermentation tank experiment and the production of 5-aminovaleric acid was increased to 23.46 g / L, as shown in Figure 5.

[0253] Table 4 Effects of different copy numbers of davBA gene cluster inserted into the gene cluster on 5-aminovaleric acid production

[0254] Example 3 Construction of recombinant halophilic bacteria Halomonas campaniensis LS21 CGMCC No.6593 producing 5-aminovaleric acid

[0255] First, according to the article Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose (Ye JW et al, Metabolic Engineering, 2018) and the method described above, gabD2, gabD3, gabT1, and gabT2 were knocked out in Halomonas campaniensis LS21 to obtain the strain LSΔgabD2ΔgabD3ΔgabT1ΔgabT2. The plasmid PYF17 (pSEVA321-P J23110 -luxR-P lux The davBA) plasmid was ligated into LSΔgabD2ΔgabD3ΔgabT1ΔgabT2 to achieve the production of 5-aminovaleric acid.

[0256] The specific operations are as follows:

[0257] The chloramphenicol-resistant plasmid PYF17 was transformed into LSΔgabD2ΔgabD3ΔgabT1ΔgabT2 using the conjugation transformation method, and the cells were cultured on plates containing chloramphenicol for one day until single colonies grew.

[0258] The LSΔgabD2ΔgabD3ΔgabT1ΔgabT2 strains conjugated with plasmid PYF17 were cultured in 60LB medium at 37°C for 12 h as the primary seed solution. The solution was then transferred to a new 60LB medium at a 1% transfer rate and cultured for 12 h as the secondary seed solution. The solution was then transferred to a shake flask containing 50MM-YU medium at a 5% transfer rate. The lysine addition rate was 5 g / L. The AHL induction intensity was 5X10 -4 M, pH value of about 9, cultured at 37°C for 48 hours, 200 rpm. Liquid chromatography analysis showed that the recombinant bacteria produced 2.24 g / L of 5-aminovaleric acid after 48 hours of shake flask fermentation, with a cell dry weight of 7.31 g / L.

[0259] The results showed that the optimized 5-aminovaleric acid biosynthesis gene cluster could be expressed in Halomonas campaniensis LS21 and successfully synthesize 5-aminovaleric acid.

[0260] Example 4 Construction of recombinant bacteria Halomonas aydingkolgenesis M1 CGMCC No. 19880 producing 5-aminovaleric acid

[0261] First, according to the method described in the article: Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose (Ye JW et al, Metabolic Engineering, 2018) and Example 1, gabD2, gabD3, ΔgabT1, and ΔgabT2 in Halomonas aydingkolgenesis M1 were knocked out to obtain strain M1ΔgabD2gabD3ΔgabT1ΔgabT2. The plasmid pYF17 (pSEVA321-P J23110 -luxR-P lux The davBA) plasmid was ligated into M1ΔgabD2gabD3ΔgabT1ΔgabT2 to achieve the production of 5-aminovaleric acid.

[0262] The specific operations are as follows:

[0263] The chloramphenicol-resistant plasmid PYF17 was transformed into M1ΔgabD2gabD3ΔgabT1ΔgabT2 using the conjugation transformation method, and the cells were cultured on plates containing chloramphenicol for one day until single colonies grew.

[0264] The M1ΔgabD2gabD3ΔgabT1ΔgabT2 strains conjugated with plasmid PYF17 were cultured in 60LB medium at 37°C for 12 h as the primary seed solution, which was then transferred to a new 60LB medium at a transfer rate of 1% and cultured for 12 h as the secondary seed solution, which was then transferred to a shake flask containing 50MM-YU medium at a transfer rate of 5%. The amount of lysine added was 5 g / L. The induction intensity of AHL was 5X10 -4 M, pH value of about 9, cultured at 37°C for 48 hours, 200 rpm. Liquid chromatography analysis showed that the recombinant bacteria produced 3.43 g / L of 5-aminovaleric acid after 48 hours of shake flask fermentation, with a cell dry weight of 7.83 g / L.

[0265] The results showed that the optimized 5-aminovaleric acid biosynthesis gene cluster could be expressed in Halomonas aydingkolgenesis M1 and successfully synthesize 5-aminovaleric acid.

[0266] Example 5: Construction of 5-hydroxyvaleric acid expression module (Module 2: 5-aminovaleric acid - 5-hydroxyvaleric acid)

[0267] Construction of pSEVA321-P porinxx -yqhD, P here porinxx It consists of a range of expression intensities from low to high, including P porin251 , P porin278 , P porin42 , P porin88 , P porin58 , P porin29 , P porin183 The promoters were used to overexpress the plasmid into the strain H. bluephagenesis Y86. The corresponding strains were YF80, YF81, YF82, YF83, YF103, YF104 and YF106, and the production of 5-hydroxyvaleric acid was detected, as shown in Table 5 (5 g / L of 5-aminovaleric acid was added to each group). The amino acid sequence corresponding to YqhD is shown in SEQ ID NO: 3. The shake flask experiment was completed in 20 mL of 50MM culture medium at 37 ° C, 200 rpm, pH 8.5-9.0 for 48 h. Three replicates were set for each group of experiments. Strain YF82 was used, i.e., promoter P porin42 , a maximum of 2.26 g / L of 5-hydroxyvaleric acid can be produced with 5 g / L of 5-aminovaleric acid.

[0268] Table 5 Yield of 5-hydroxyvaleric acid produced by 5-aminovaleric acid (Module 2)

[0269] Example 6 Construction of 5-Hydroxyvaleric Acid-Related PHA Expression System (Module 3: 5-Hydroxyvaleric Acid-Related PHA)

[0270] 5-Hydroxyvaleric acid can also be used as a precursor of PHA. (Yan X, Liu X, Yu LP, et al. Biosynthesis of diverse α, ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis [J]. Metabolic Engineering, 2022, 72: 275-288.) It can be polymerized into PHA through coenzyme A ligase AbfT and PHA synthase PhaC, such as P(3HB-co-5HV). In this embodiment, this process was achieved in Halomonas by expressing abfT and phaC genes at different intensities. The amino acid sequence corresponding to AbfT is shown in SEQ ID NO: 4, and the amino acid sequence corresponding to PhaC is shown in SEQ ID NO: 5.

[0271] The specific implementation process is as follows:

[0272] (1) Construction of the constitutive phaC-abfT expression plasmid module

[0273] Two porin promoters with different strengths were selected to express the constitutive expression plasmid pSEVA341-P porin251 -phaC-abfT and pSEVA341-P porin278 -phaC-abfT, named as: PYF143 and PYF144 respectively.

[0274] The production of 5-hydroxyvalerate-related PHA was achieved by adding 5-hydroxyvalerate.

[0275] Plasmids PYF143 and PYF144 were transformed into the halophilic bacterium H. bluephagenes TD01 via an E. coli S17-1 conjugation assay and designated TD-PYF143 and TD-PYF144. Recombinant halophiles obtained by transforming plasmids PYF143 and PYF144 into the halophilic bacterium H. campaniensis LS21 were designated LS-PYF143 and LS-PYF144. Recombinant halophiles obtained by transforming plasmids PYF143 and PYF144 into the PHA synthase-deficient halophilic bacterium Halomonas aydingkolgenesis M1 were designated M1-PYF143 and M2-PYF144. Shake flask experiments were performed in 20 mL of 50 mm Hg culture medium at 37°C, 200 rpm, and a pH of 8.5-9.0 for 48 hours, with three replicates per experiment. The addition amount of 5-hydroxyvaleric acid was 5 g / L. The results are shown in Table 6.

[0276] Table 6 Production of P(5HV-co-3HB) by recombinant halophiles containing a constitutive phaC-abfT expression plasmid module

[0277] Example 7: Production of 5-hydroxyvaleric acid using L-lysine as a substrate (Module 1+2: L-lysine - 5-hydroxyvaleric acid)

[0278] Preferred recombinant bacteria for producing 5-hydroxyvaleric acid:

[0279] The plasmid used in YF82 in Example 5 was introduced into the strain Halomonas bluephagenesis YF117, resulting in the strain designated YF117-2. The expression bacteria were inoculated into 20 mL of 60 LB medium and, after 12 hours of culture, transferred to a fresh 20 mL of 60 LB medium at a 1% volume ratio and cultured for another 10 hours. After 10 hours of culture, the seed culture was obtained, and 1 mL of the seed culture was inoculated into 19 mL of 50 MM culture medium for a shake flask experiment. The shaker was maintained at 37°C and 200 rpm for 48 or 60 hours of fermentation (initially adding L-lysine for 48 hours, followed by 24 hours of addition of 20 g / L L-lysine and Triton-X100, and continued for another 48 hours, for a total of 60 hours). After fermentation, the supernatant was centrifuged and analyzed for 5-hydroxyvaleric acid by high-performance liquid chromatography.

[0280] The recombinant Halomonas bluephagenesis YF117-2 constructed to produce 5-hydroxyvaleric acid used L-lysine as a substrate and was treated with 0.2% Triton-X100 to increase cell membrane permeability. During the shake flask experiment, the maximum production of 5-hydroxyvaleric acid from L-lysine was 6.12 g / L, as shown in Table 7.

[0281] Table 7 The content of 5-hydroxyvaleric acid produced by engineered strain YF117-2 using L-lysine as substrate

[0282] Example 8 Production of 5-hydroxyvaleric acid-related PHA using 5-aminovaleric acid as a substrate (Module 2+3: 5-aminovaleric acid-related PHA)

[0283] Combining Examples 5 and 6, the constitutive plasmid system PYF278 (pSEVA341-P Porin278 -phaC-yqhD-abfT) and PYF251(pSEVA341-P Porin251 -phaC-yqhD-abfT), and the plasmid was transformed into halophilic bacteria H.bluephagenesis Y86 and named TD-PYF278 and TD-PYF251 respectively. After culturing for 12 hours, it was transferred to a new 20mL 60LB culture medium at a volume ratio of 1%, and continued to be cultured for 10 hours as a shake flask fermentation seed liquid. 1mL of the fermentation seed liquid was inoculated into 19mL50MM for a shake flask experiment. The concentration of 5-aminovaleric acid was 5g / L, the shaker temperature was 37°C, and the speed was 200rpm. Shake flask fermentation was carried out using 50MM culture medium, and the PHA content and the molar ratio of 5HV are shown in Table 8.

[0284] Table 8 Production of 5-hydroxyvaleric acid-related PHA using 5-aminovaleric acid as substrate (Module 2 + Module 3)

[0285] Example 9 Synthesis of 5-Hydroxyvaleric Acid-Related PHA Using L-Lysine (Module 1+2+3: L-Lysine-Related PHA)

[0286] Based on the results obtained in Example 8, plasmid pYF278 was transferred into H. bluephagenesis YF117 to obtain the recombinant strain Halomonas bluephagenesis YF117-3, which produces 5-hydroxyvaleric acid-related PHAs using L-lysine as a substrate, at an addition level of 20 g / L. After treatment with 0.2% Triton-X100 to increase cell membrane permeability, shake flask fermentation was performed using 50 mm flasks. During the shake flask experiment, YF117-3 achieved a maximum PHA production of 35%, with a molar ratio of 5-hydroxyvaleric acid of 5.52%, as shown in Table 9.

[0287] Table 9 Synthesis of P(5HV-co-3HB) using L-lysine as substrate (module 1+2+3)

[0288] 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.

[0289] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A recombinant bacterium for producing a 5-carbon compound, characterized in that: The recombinant bacteria is of the genus Halomonas, and the genome of the recombinant bacteria contains a synthesis gene for a 5-carbon compound, wherein the synthesis gene for the 5-carbon compound contains one or more of an L-lysine monooxygenase encoding gene, a 5-aminopentanamidase encoding gene, or an alcohol dehydrogenase encoding gene.

2. The recombinant bacterium according to claim 1, characterized in that The 5-carbon compound is a 5-carbon alkyl substituted by an amino group, a hydroxyl group and / or a carboxyl group, or the 5-carbon compound is a 5-carbon ring substituted by an amino group, a hydroxyl group and / or a carboxyl group, or the 1st position or the 5th position is substituted by an amino group or a carboxyl group to form a ring.

3. The recombinant bacterium according to claim 2, characterized in that The 5-carbon compound includes 5-aminovaleric acid, 5-hydroxyvaleric acid, glutaric acid, delta-valerolactam or 1,5-pentanediol.

4. The recombinant bacterium according to claim 1, characterized in that The synthetic gene is integrated into the chromosome genome or contained in one or more recombinant plasmid vectors and is free in the recombinant bacteria.

5. The recombinant bacterium according to claim 1, characterized in that The glutaric semialdehyde dehydrogenase encoding gene and / or the 4-aminobutyrate aminotransferase encoding gene in the recombinant bacteria are deleted or knocked down or knocked out; Preferably, the glutaric semialdehyde dehydrogenase encoding gene is gabD2 and / or gabD3; Preferably, the 4-aminobutyrate aminotransferase encoding gene is gabT1 and / or gabT2.

6. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria comprises a pathway from L-lysine to 5-aminovaleric acid; The recombinant bacteria include: A) gabD2 and gabD3 are deleted, knocked down or knocked out, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene; or, B) gabT1 and gabT2 are deleted, knocked down or knocked out, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene; or, C) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked down or knocked out, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene and a 5-aminovaleramidase encoding gene.

7. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria contain a 5-aminovaleric acid to 5-hydroxyvaleric acid pathway, and the recombinant bacteria include: a) gabD2 and gabD3 are deleted, knocked down or knocked out, and the synthesis gene of the 5-carbon compound comprises an alcohol dehydrogenase encoding gene; or, b) gabT1 and gabT2 are deleted, knocked down or knocked out, and the synthesis gene of the 5-carbon compound comprises an alcohol dehydrogenase encoding gene; or, c) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked down or knocked out, and the synthesis gene of the 5-carbon compound comprises an alcohol dehydrogenase encoding gene.

8. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria comprises a pathway from L-lysine to 5-hydroxyvaleric acid; The recombinant bacteria include: A) gabD2 and gabD3 are deleted, knocked down or knocked out, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene; or, B) gabT1 and gabT2 are deleted, knocked down or knocked out, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene; or, C) gabD2, gabD3, gabT1 and gabT2 are deleted, knocked down or knocked out, and the 5-carbon compound synthesis gene comprises an L-lysine monooxygenase encoding gene, a 5-aminovaleramidase encoding gene and an alcohol dehydrogenase encoding gene.

9. The recombinant bacterium according to claim 1, characterized in that The synthetic gene is expressed under the control of a promoter, and the promoter is constitutive and / or inducible.

10. The recombinant bacterium according to claim 9, characterized in that The constitutive promoter is a porin promoter, preferably including P porin or a mutant thereof, the P porin Mutants include P porin29 , P porin88 , P porin221 , P porin194 , P porin251 , P porin278 , P porin68 , P porin42 , P porin58 , P porin226 , P porin183 , P porin140 or P porin141 ; The inducible promoter includes P tac Promoter, P lux Promoter or P lac Promoter.

11. A recombinant bacterium for producing a polymer comprising a 5-carbon compound monomer, characterized in that: The recombinant bacteria is of the genus Halomonas, and the genome of the recombinant bacteria includes a PHA synthase encoding gene and / or a 5-hydroxyvaleric acid coenzyme A transferase encoding gene.

12. The recombinant bacterium according to claim 11, characterized in that The recombinant bacteria include a PHA pathway containing a substrate containing L-lysine to produce a 5-hydroxyvaleric acid monomer, and the recombinant bacteria include the recombinant bacteria described in any one of claims 1-6 or 8-10 further introduced with a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene.

13. The recombinant bacterium according to claim 11, characterized in that The recombinant bacteria include a PHA pathway for producing 5-hydroxyvaleric acid monomers from a substrate containing 5-aminovaleric acid, and the recombinant bacteria include the recombinant bacteria described in any one of claims 1-5, 7 or 9-10, further introduced with a PHA synthase encoding gene and a 5-hydroxyvaleric acid coenzyme A transferase encoding gene.

14. The recombinant bacterium according to any one of claims 1 to 13, characterized in that The genus Halomonas includes Halomonas bluephagenesis, Halomonas campaniensis or Halomonas aydingkolgenesis and any derivatives thereof.

15. A method for producing a 5-carbon compound or a polymer containing a 5-carbon compound monomer, characterized in that The method comprises culturing the recombinant bacteria according to any one of claims 1 to 14.

16. Use of a 5-carbon compound or a polymer containing a 5-carbon compound monomer obtained by the recombinant bacterium according to any one of claims 1 to 14 or the method according to claim 15 in preparing a degradable new biological material or medicine.

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