Construction method of engineering bacteria for synthesizing polyvalerolactone (PVL)

Abstract

The invention discloses a recombinant bacterium for producing polyvalerolactone (PVL). According to the recombinant bacteria disclosed by the invention, endogenous beta-ketothiolase (PhaA), acetoacetyl coenzyme A reductase (PhaB), polyhydroxyalkanoate PHA polymerase (PhaC) and alkenyl acyl coenzyme A hydratase (FadB) are knocked out or knocked down, and exogenous polyhydroxyalkanoate polymerase (PhaCin) and 4-hydroxybutyryl coenzyme A transferase (AbfT) are introduced into a genome. Biosynthesis of the polyvalerolactone in the halomonas is realized, and the molecular weight and the mechanical property of the obtained polyvalerolactone are obviously superior to those of the polyvalerolactone obtained by chemical synthesis.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biology. More specifically, the present invention constructs a non-natural metabolic pathway for synthesizing polyvalerolactone from delta-valerolactone in, for example, Halomonas bluephagenesis, and realizes the biosynthesis of polyvalerolactone in Halomonas bluephagenesis. Background Art

[0002] Halomonas bluephagenesis is a halophilic microorganism that is resistant to high temperatures and high pH. It has the ability to naturally synthesize polyhydroxyalkanoic acid (poly 3-hydroxybutyric acid) (PHB) and is an excellent strain for the next generation of industrial biotechnology. Its open continuous fermentation has irreplaceable advantages such as energy saving and cost reduction, and has unique fermentation advantages and market competitiveness. At present, the genome annotation and gene editing methods for halophilic strains are relatively mature, so it has the potential to develop rich products.

[0003] Polyvalerolactone (PVL) is a polyhydroxyalkanoic acid homopolymer. Due to its biodegradability, biocompatibility and permeability, PVL and PVL-based copolymers have been reported to be used in drug delivery carriers, cell culture matrices, food and drug packaging and coatings, etc., and have great application potential in food, medicine and industrial applications.

[0004] First, H. bluephagenesis naturally synthesizes PHB depending on endogenous β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoic acid polymerase (PhaC), but does not have the ability to synthesize PVL. Secondly, due to its excellent ability to naturally synthesize PHB, it has a strong ability to polymerize 3-hydroxybutyric acid (3HB) monomers. Therefore, the polyhydroxyalkanoates (PHAs) synthesized in H. bluephagenesis currently almost all contain a high proportion of 3HB monomers.

[0005] Due to the strict specificity and catalytic activity of the enzyme to the catalytic substrate, screening for the coenzyme A ligase (CoA ligase) that can specifically recognize the 5-hydroxybutyrate monomer and PhaC that polymerizes 5-hydroxyvaleryl-CoA in H. bluephagenesis is the first step to achieve the biosynthesis of PVL in H. bluephagenesis.

[0006] Since H. bluephagenesis naturally has a strong ability to synthesize PHB, and the known PhaC can recognize and polymerize 3-hydroxybutyryl-CoA, the pathway for the bacteria to further synthesize PHB by glycolysis of glucose as a raw material needs to be inhibited.

[0007] In addition, even if the pathway for synthesizing PHB is blocked, there are still other sources of 3-hydroxybutyryl-CoA (3HB-CoA). In order to further increase the proportion of 5HV monomers in the polymer, the genes related to the 3HB-CoA pathway from other sources in bacteria except glycolysis were knocked out.

[0008] Currently, PVL is mainly synthesized through chemical reactions based on ring-opening polymerization. Ring-opening polymerization uses δ-valerolactone as the raw material, uses a cationic catalyst, and reacts in an organic solvent at high temperature to produce polyvalerolactone. There are also studies reporting that the thermophilic esterase of Archaeoglobus fulgidus can catalyze the ring-opening polymerization of δ-valerolactone in toluene at 70°C, and the reaction takes 72 hours to obtain polyvalerolactone. Recently, a study was published in Escherichia coli to achieve the production of PVL from α-ketoglutarate by constructing a non-natural metabolic pathway.

[0009] PVL obtained by chemical reaction of ring-opening polymerization has poor ductility (ε B <30%), low molecular weight (<5KDa), and contamination by metal catalysts and organic solvents, and its mechanical properties are still not sufficient for packaging or medicine. The production of PVL using thermophilic esterases in archaea requires purification to obtain active esterases. The process is cumbersome, and the temperature and reaction conditions need to be strictly controlled. The resulting materials are also contaminated with organic reagents and have a very low molecular weight (<2KDa). The polyvalerolactone produced by constructing a metabolic pathway in Escherichia coli has a low yield (<412mg / L) and a low percentage of cell dry weight (20.5%), so it cannot be used in industrial production. At the same time, the PVL material synthesized in Escherichia coli has not been determined by NMR and may contain contamination from other hydroxy fatty acid monomers in the cell, such as 3-hydroxybutyric acid. And its material mechanical properties have not been characterized.

[0010] In summary, the PVL obtained by the existing PVL synthesis methods has the disadvantages of low molecular weight, pollution, and being unsuitable for large-scale industrial production.

[0011] In view of this, there is a need to develop a non-native metabolic pathway for producing PVL in H. bluephagenesis to achieve large-scale biosynthesis of PVL. Summary of the invention

[0012] In order to solve the problems of the prior art, the inventors screened polyhydroxy fatty acid polymerase and 4-hydroxybutyryl-CoA transferase, and transformed host bacteria such as halophilic bacteria that were originally unable to synthesize polyvalerolactone to achieve the biosynthesis of polyvalerolactone, and developed polyvalerolactone-producing bacteria using valerolactone monomer as a substrate, which has far-reaching significance for the industrial production of polyvalerolactone by biological methods.

[0013] Therefore, the present invention provides an engineered bacterium for producing polyvalerolactone (PVL), which expresses or overexpresses exogenous or endogenous polyhydroxy fatty acid polymerase (e.g., PhaC un ) and 5-hydroxybutyrate monomer CoA ligase (e.g. 4-hydroxybutyryl-CoA transferase, AbfT), preferably the engineered bacteria do not have or have knocked out or knocked down endogenous β-ketothiolase (PhaA) gene, acetoacetyl-CoA reductase (PhaB) gene, PHA polymerase (PhaC) gene and enoyl-CoA hydratase (FadB) gene one or more (preferably all). Polyhydroxy fatty acid polymerase (PhaC) can be selected from PhaC un , PhaC re , PhaC 4AK4 , PhaC 61-3 , PhaC 1437 , PhaC ac and PhaC ar , and / or -hydroxybutyrate monomers can be selected from 4-hydroxybutyryl-CoA transferase (AbfT), orfZ, pct540 and alkK.

[0014] In one embodiment, the engineered bacteria cannot or barely produce poly 3-hydroxybutyrate (PHB), for example, in a mineral culture medium with glucose as a carbon source. "Almost unable to produce poly 3-hydroxybutyrate (PHB)" means that the cell dry weight content (wt%) of PHB after fermentation culture is less than 5%, 4%, 3%, 2%, or even 1%.

[0015] In a specific embodiment, the engineered bacteria is selected from halophiles, preferably selected from Halomonas, Pseudomonas, Escherishia, Ralstonia eutropha, Aeromonas, Bacilllus, Alcaligenes latus, Alcaligenes eutropus or a combination thereof, more preferably Halomonas, even more preferably Halomonas bluephagenesis, Halomonasaydingkolgenesis, Halomonas campaniensis or Halomonas lutescens, most preferably Halomonas bluephagenesis TD01 (strain collection number CGMCC No.4353), Halomonasbluephagenesis TD141, Halomonas bluephagenesis TD1.0, Halomonasaydingkolgenesis M1 (Cultivation Number CGMCC No. 19880) or Halomonas campaniensis LS21 (Cultivation Number CGMCC No. 6593).

[0016] In one embodiment, the polyhydroxyalkanoic acid polymerase (PhaC un ) and the CoA ligase for 5-hydroxybutyrate monomer (4-hydroxybutyryl-CoA transferase, AbfT) are independently expressed or overexpressed on the genome of the engineered bacteria or on one or more plasmids.

[0017] In one embodiment, the 4-hydroxybutyryl-CoA transferase encoding gene abfT is derived from Clostridium aminobutyricum, and / or the polyhydroxyalkanoic acid polymerase encoding gene phaC un The uncultured bacteria derived from the red forest swamp or a mutant of the gene, preferably the amino acid sequence of the polyhydroxyalkanoic acid polymerase is as shown in SEQ ID NO: 1, more preferably the phaC unThe nucleotide sequence of abfT is shown in SEQ ID NO:2, and / or preferably the amino acid sequence of 4-hydroxybutyryl-CoA transferase is shown in SEQ ID NO:3, and more preferably the nucleotide sequence of abfT is shown in SEQ ID NO:4.

[0018] In a specific embodiment, the gene encoding polyhydroxyalkanoic acid polymerase (PhaCun) and the gene encoding 4-hydroxybutyryl-CoA transferase (AbfT) are independently expressed under the control of a constitutive or inducible promoter, preferably the inducible promoter is selected from IPTG-inducible Mmp1 promoter, lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof, preferably the constitutive promoter is selected from wild-type Pporin promoter or a mutant thereof, wherein the mutant is selected from porin3, porin 42 、porin 58 、porin 68 、porin 140 、porin 141 、porin 194 、porin 211 、porin 221 、porin 226 、porin 278 , or a combination thereof.

[0019] In one embodiment, the polyhydroxyalkanoic acid polymerase (PhaC un ) and the encoding gene of 4-hydroxybutyryl-CoA transferase (AbfT) are introduced into the engineered bacteria.

[0020] Another aspect of the present invention provides a method for producing polyvalerolactone (PVL), which comprises fermenting and culturing the engineered bacteria according to the present invention.

[0021] In a specific embodiment, the fermentation culture uses a mineral medium, uses valerolactone (δ-valerolactone) as a substrate, and the fermentation is carried out under open conditions, especially when the engineered bacteria is Halomonas.

[0022] Another aspect of the present invention also provides a polyvalerolactone having one or more (preferably all) of the following properties:

[0023] 1) The purity of the polyvalerolactone is greater than 98 mol%, preferably greater than 99 mol%, and more preferably greater than 99.5%. Preferably, the molar percentage of 3HB monomer in the polyvalerolactone is less than 2%, more preferably less than 1.5%, less than 1%;

[0024] 2) the Mw of the polyvalerolactone is greater than 100 kDa, preferably greater than 110 kDa, greater than 120 kDa, greater than 130 kDa, greater than 140 kDa or greater than 150 kDa, and / or less than 500 kDa, less than 400 kDa, less than 300 kDa or less than 200 kDa; the Mn is greater than 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or 100 kDa, and / or less than 200 kDa, less than 150 kDa;

[0025] 3) PDI (Mw / Mn) is about 2;

[0026] 4) The elongation at break is greater than 100%, preferably greater than 200%, greater than 300%, greater than 400%, greater than 500%, greater than 600%, and / or less than 1000%.

[0027] Preferably, the above purity, Mw, Mn, PDI (Mw / Mn) and elongation at break are each measured by the method described in the specification of the present application, especially in Example 6. In particular, the molecular weight is measured by gel permeation chromatography.

[0028] In a specific embodiment, the polyvalerolactone is produced by the method according to the present invention.

[0029] The present invention is the first to develop polyvalerolactone-producing bacteria (halophilic bacteria) using valerolactone (δ-valerolactone) monomer as a substrate through a synthetic biology method. The yield, molecular weight and / or mechanical properties of the obtained polyvalerolactone are significantly better than those obtained by existing chemical synthesis and biosynthesis methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0031] Figure 1 : PHA polymerase, CoA ligase and plasmid construction map;

[0032] Figure 2 : Dry weight and PHA content of copolymers produced by different combinations of PHA polymerase and CoA ligase;

[0033] Figure 3 : molar percentages of 3HB and 5HV monomers in copolymers produced by different PHA polymerase and CoA ligase combinations;

[0034] Figure 4 :The effect of knocking out endogenous phaCAB on reducing 3HB;

[0035] Figure 5: Knocking out the endogenous fadB gene further increased the cell dry weight and the molar percentage of 5HV;

[0036] Figure 6 :Schematic diagram of the metabolic pathway of PVL biosynthesis;

[0037] Figure 7 : Dry cell weight and PVL production of recombinant halophilic bacterium Halomonas bluephagenesis JL02 in an open fermentation tank in a 7L fermenter; and

[0038] Figure 8 : NMR spectrum data of PVL ((A) carbon spectrum and (B) hydrogen spectrum). DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Unless otherwise indicated, the terms used herein have the general technical meanings understood by those skilled in the art. For definitions and terms in the art, it is particularly recommended that the technician refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0041] In the present invention, the singular articles "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety.

[0042] The words “include” or “comprising” described in the present invention are open-ended descriptions, including all the specified components or steps described, as well as other specified components or steps that will not be substantially affected; 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.

[0043] The "and / or" described in the present invention includes all combinations of 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".

[0044] As used herein, the term "engineered bacteria", also known as genetically engineered bacteria, refers to bacteria into which target genes are introduced to express them and produce the desired protein.

[0045] As used herein, the term "knock out" refers to a genetic engineering technique in which an exogenous mutated gene is used to replace an endogenous normal homologous gene through homologous recombination, thereby inactivating the endogenous gene and expressing the mutant's traits.

[0046] As used herein, the term "knock down" refers to the effect of preventing gene expression by degrading mRNA of a target gene with a homologous sequence. It uses double-stranded small RNA to efficiently and specifically degrade homologous mRNA in cells, thereby blocking the expression of the target gene in vivo and causing the cell to show a phenotype of target gene deletion. It is different from gene knockout, which permanently silences the expression of the target gene, but it achieves the effect of preventing gene expression by degrading mRNA of a target gene with a homologous sequence.

[0047] The technology of gene knockout or knock-down is well known in the art, including but not limited to point mutation, insertion, deletion, frameshift, or missense mutation. Another way to knock out a gene is to use zinc finger nuclease. Zinc finger nuclease (ZFN) is an artificial restriction enzyme produced by fusing a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be modified to target the target DNA sequence, which can enable the zinc finger nuclease to target unique sequences in complex genomes. Other genome customization technologies that can be used to knock out genes include TAL effector nucleases (TALENs). Another technology is the genome editing technology CRISPR / Cas system, which can be used to achieve RNA-guided genome modification.

[0048] Techniques for gene knockout or knockdown may also include the use of gapmers, antisense RNA, siRNA, esiRNA, shRNA, miRNA, or RNA aptamers.

[0049] "Antisense RNA" refers to RNA molecules that are complementary to mRNA, and also includes RNA molecules that are complementary to other RNAs. Since ribosomes cannot translate double-stranded RNA, antisense RNA specifically binds to mRNA, thus inhibiting the translation of the mRNA.

[0050] Another special form of the antisense RNA strategy is gapmer. Gapmer is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers that is long enough to induce RNase H cleavage. The design and synthesis of gapmers are well known to those skilled in the art and can be performed by commercial companies.

[0051] "Small interfering RNA (siRNA)", sometimes called short interfering RNA or silencing RNA, is a type of double-stranded RNA molecule, about 20-25 base pairs in length, which acts through the RNA interference (RNAi) pathway. It interferes with the post-transcriptional degradation of mRNA expressing a specific gene with a complementary nucleotide sequence, thereby preventing translation. Techniques for selecting target sequences for siRNA are well known in the art.

[0052] "Short hairpin RNA" (shRNA) is an RNA sequence consisting of two short inverted repeats that can silence gene expression via RNA interference (RNAi).

[0053] The full English name of "esiRNA" is Endoribonuclease-prepared siRNAs, which is a mixture of siRNAs generated by cutting long double-stranded RNA (dsRNA) by RNase III (a ribonuclease) of Escherichia coli. The length is between 18-25bp and can be used to efficiently knock out the expression level of the target gene.

[0054] As used herein, the term "promoter" generally refers to a deoxyribonucleic acid (DNA) sequence that enables a specific gene to be transcribed. The promoter can be recognized by RNA polymerase and begin to transcribe and synthesize RNA. In ribonucleic acid (RNA) synthesis, the promoter can interact with the transcription factors that regulate gene transcription to control the start time and degree of expression of gene expression (transcription). The promoter includes a core promoter region and a regulatory region, which is located in the regulatory sequence that controls gene expression and upstream of the gene transcription start site (5' direction of the DNA antisense strand), and has no compilation function itself. Promoters can include constitutive promoters and inducible promoters.

[0055] There is no particular limitation on the type of promoter used for the expression or overexpression of the gene of the present invention, and it can be an inducible promoter or a constitutive promoter. For example, the promoter can be a promoter for IPTG-induced expression. In one embodiment, the inducible promoter is selected from lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof. In one embodiment, the constitutive promoter is selected from wild-type Pporin promoter or a mutant thereof, wherein the mutant is selected from porin3, porin 42 、porin 58 、porin 68 、porin 140 、porin 141 、porin 194 、porin 211 、porin 221 、porin 226 、porin 278 , or a combination thereof (for wild-type Pporin and its mutants, see, for example, the literature: Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas blue phagenesis (Shen Rui, Yin Jin, Ye Jianwen et al. ACS Synthetic Biology 7 (2018) 1897–1906).

[0056] Wild-type Pporin promoter (SEQ ID NO: 18):

[0057] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0058] Pporin3: (SEQ ID NO: 19)

[0059] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCATGAtagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0060] Pporin42:(SEQ ID NO:20)

[0061] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCACTAtagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0062] Pporin51:(SEQ ID NO:21)

[0063] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCATTTtagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0064] Pporin58:(SEQ ID NO:22)

[0065] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAATAtagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0066] Pporin68:(SEQ ID NO:23)

[0067] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAGACtagagtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0068] Pporin140:(SEQ ID NO:24)

[0069] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatAAGAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0070] Pporin141:(SEQ ID NO:25)

[0071] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatATACtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0072] Pporin194:(SEQ ID NO:26)

[0073] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatCTAAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0074] Pporin211:(SEQ ID NO:27)

[0075] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatCAGAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0076] Pporin221:(SEQ ID NO:28)

[0077] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatGCTAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0078] Pporin226:(SEQ ID NO:29)

[0079] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatAAAGtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0080] Pporin278:(SEQ ID NO:30)

[0081] ATGCCTCCACACCGCTCGTCACAtcctgttgcgtTCACTGGAATCCCAgtatTAAAtTTGACCTGCGAGCAaGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAgagttactagagaaagaggagaaatactag

[0082] In the present invention, the term "overexpression" refers to the transcription of a gene at a high rate, resulting in the production of a large amount of mRNA. un ) gene and 4-hydroxybutyryl-CoA transferase (AbfT) gene overexpression refers to the overexpression of their DNA or RNA expression levels as compared with the control (negative plasmid control or host strain without these genes) by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300%, or even 4, 5, 6, 7, 8, 9, 10, 20, 30, 50 or 100 times or more of the expression level in the control.

[0083] The type of the engineered bacteria of the present invention is not particularly limited, and can be a prokaryotic cell or a eukaryotic cell, preferably a microbial cell, more preferably selected from Halomonas, Pseudomonas, Escherishia, Ralstonia eutropha, Aeromonas, Bacilllus, Alcaligenes latus, Alcaligenes eutropus or a combination thereof, preferably the microorganism is Halomonas.

[0084] As used herein, the term "Halomonas" refers to a class of halophilic microorganisms that can grow under extreme conditions such as high pH, ​​high salt and high temperature. Since the growth of non-halophilic microorganisms will be inhibited under high salt and alkaline conditions, the bacteria of this genus can resist the contamination of foreign bacteria and are often used to establish open fermentation systems with low energy consumption, low fresh water consumption, low fixed capital investment, low cost and continuous production. The halophilic microorganism Halomonas bluephagenesis TD01 (preservation number is CGMCC No.4353) screened from Xinjiang Aiding Lake disclosed in CN102120973A is a PHA super-producing bacterium. It can grow and accumulate PHA under high salt and high pH conditions, thereby realizing continuous fermentation and continuous and efficient production of PHA under non-sterilized conditions. Halomonas bluephagenesis TD1.0 is a strain obtained on the basis of Halomonas bluephagenesis TD01 (see, for example, Zhao, H. et al. (2017) Novel T7-like expression systems used for Halomonas. Metab Eng 39, 128-140). Halomonas bluephagenesis TD141, see Xu Yan et al., 2022. Biosynthesis of diverse α, ω-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis [J]. Microbial Biotechnology.

[0085] As used herein, the term "plasmid" refers to a DNA molecule that is capable of autonomous replication outside of the chromosome or nuclear region of a cell.

[0086] As used herein, the term "transformation" refers to the process by which cells undergo genetic changes by taking up foreign genetic material. In the transformation process, the transformed DNA fragment is called a transformation factor. The transformation factor is usually a plasmid DNA.

[0087] As used herein, the term "conjugation" refers to a phenomenon of genetic material exchange between two bacteria, which is an important stage of bacterial sexual reproduction. When the conjugation phenomenon occurs, the two cells directly join or join through a channel similar to a bridge, and gene transfer occurs.

[0088] Methods for introducing genes into host bacteria to be modified, such as Halomonas, are well known to those skilled in the art. In one embodiment, the gene is introduced into a host bacteria, such as Halomonas, by transformation or conjugation. For example, polyhydroxyalkanoic acid polymerase (PhaC un ) gene and 4-hydroxybutyryl-CoA transferase (AbfT) gene can be expressed on one or more plasmids or integrated into the genome of the engineered bacteria. The plasmid can be an expression plasmid or a gene editing plasmid, preferably a CRISPR-Cas9 gene editing plasmid. For Halomonas, it is preferred to introduce polyhydroxyalkanoic acid polymerase (PhaC un ) gene and / or 4-hydroxybutyryl-CoA transferase (AbfT) gene.

[0089] In the first aspect of the present invention, coenzyme A ligase and polyhydroxyalkanoic acid synthase from different sources were synthesized, and their sequences were codon optimized for H. bluephagenesis. Different gene combinations were expressed on the same plasmid backbone. The gene combinations of coenzyme A transferase and polyhydroxyalkanoic acid synthase that can polymerize 5-hydroxyvaleric acid monomers were screened, which are 4-hydroxybutyryl-CoA transferase gene abfT from Clostridium aminobutyricum and polyhydroxyalkanoic acid polymerase gene phaC from uncultured bacterial strains in red forest swamps. un .

[0090] The second aspect of the present invention provides an engineered bacterium that can hardly accumulate PHB in a mineral culture medium, wherein the endogenous β-ketothiolase gene (phaA), acetoacetyl-CoA reductase gene (phaB) and polyhydroxyalkanoic acid polymerase gene (phaC) are knocked out.

[0091] In the third aspect of the present invention, the 4-hydroxybutyryl-CoA transferase gene abfT from Clostridium aminobutyricum was introduced into the G4 site of the genome. The polyhydroxyalkanoic acid synthase gene phaC from an uncultured bacteria from the red forest swamp was introduced. un .

[0092] In a fourth aspect of the present invention, enoyl-CoA hydratase (FadB) is further knocked out. When 5-hydroxyvaleric acid is polymerized, almost no 3HB monomer is polymerized.

[0093] In a fifth aspect of the present invention, the biological production of PVL is achieved in a 7-L fermenter.

[0094] In a sixth aspect of the present invention, the molecular weight, mechanical properties and thermodynamic properties of biosynthesized PVL were tested.

[0095] The above merely summarizes some aspects of the present invention and is not and should not be considered to limit the present invention in any aspect.

[0096] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0097] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0098] Unless otherwise specified, the reagents and materials used in the examples can be obtained from commercial sources.

[0099] Table 1: Comparison of some English abbreviations and their full names in Chinese and English

[0100]

[0101] 1. Halomonas bluephagenesis TD01 (CGMCC No.4353):

[0102] The applicant previously screened a Gram-negative halophilic bacterium that is salt-tolerant, alkali-tolerant, and naturally produces PHB. This bacterium can accumulate a high content of polyhydroxyalkanoates by itself and has good prospects for industrial production and application. It is recorded in the article "TanDan, Wu Qiong, Chen, Jin-Chun and Chen GQ. Engineering Halomonas TD01 for LowCost Production of Polyhydroxyalkanoates. Metabolic Engineering 26 (2014) 34-47", which can be obtained by the public from the applicant and can only be used to repeat the experiments of the present invention.

[0103] 2. Culture medium formula:

[0104] Escherichia coli was cultured in LB medium, and Halomonas was cultured in LB60 or MM60 medium.

[0105] LB medium contains: 5 g / L yeast extract (OXID, product catalog number LP0021), 10 g / L peptone (OXID, product catalog number LP0042), 10 g / L NaCl, and the rest is water. The pH value is adjusted to 7.0-7.2 and sterilized by high pressure steam.

[0106] LB60 medium is LB medium containing 60 g / L NaCl, and the other components and preparation conditions are the same as LB medium.

[0107] MM60 medium contains: 60g / L NaCl, 1g / L yeast extract (OXID, catalog number LP0021), 20g-55g glucose (depending on the sugar source intake of the microorganism), and the rest is water. The optimal pH for the halophilic bacteria used in the experiment is around 8-9, and the pH is adjusted with NaOH.

[0108] 3. pSEVA321 plasmid and pQ08, pQ41 plasmid:

[0109] Recorded in the following document: "Qin Qin, Ling Chen, Zhao Yiqing, Yang Tian, ​​Yin Jin, Guo Yingying, Chen GQ. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering 47 (2018) 219-229". The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0110] 4. Unless otherwise specified, the gene editing technology used in the present invention is CRISPR-Cas9 gene editing technology. The specific operation method is recorded in the following document: "Qin Qin, Ling Chen, Zhao Yiqing, Yang Tian, ​​Yin Jin, Guo Yingying, Chen GQ. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metabolic Engineering 47 (2018) 219-229". The public can obtain it from the applicant and can only be used to repeat the experiments of the present invention.

[0111] 5. Genes introduced into the recombinant halophilic bacteria in the examples:

[0112] PhaC un Amino acid sequence (SEQ ID NO: 1)

[0113] MASKDSFGKTGDLWSSMFNWMSGTMTAAAQIQQANMRAFAQSMELATSAYARMWGQPVEQVVPADRRFKDEAWTENMAADLLKQSYLITSQWLMEIADGWQAIDPDLHERTRFWTQQLVDATSPANFAMTNPVVMQEIARTGGMNLIQGAQNLLKDAQSGRLTQVPEDAFEVGKDLAITPGKVVYRNRLIELIQYTPATETVHEIPILVVPPWINKYYVMDMQPENSLFKYLVDAGFTLFTISWKNPDETVLDLEWDDYLDLGTLEALRMVKEIMGVEQVNLVGYCLGGIISQVTLAYLAATGDDAQINSATYFTTHQDFSDAGEISVFISRLDVMFLEWLMKISGGYLDGRNLAATFNMLRANDLLWNYVVHNYLLGQEPASFDLLYWNNDGTRVPGKVHSFLLREFFLDNKLKEPEGIQVKGVGIDLGKITTPTYVVTADRDHIVPWRGAFLVRQLQSGPVRFILSGGGHIAGVISPPTKNRGFWINEEEKDDADAWLAGATKHDGSWWVDWIPWLEERSGRRVKPPTAAGSDEFKPLMDAPGTYVLEK

[0114] phaC un The nucleotide sequence of

[0115]

[0116] Amino acid sequence of AbfT (SEQ ID NO:3)

[0117] MDWKKIYEDRTCTADEAVKSIKSGDRVLFAHCVAEPPVLVEAMVANAAAYKNVTVSHMVTLGKGEYSKPEYKENFTFEGWFTSPSTRGSIAEGHGQFVPVFFHEVPSLIRKDIFHVDVFMVMVSPPDHNGFCCVGVSSDYTMQAIKSAKIVLAEVNDQVPVVYGDTFVHVSEIDKFVETSHPLPEIGLPKIGEVEAAIGKHCASLIEDGSTLQLGIGAIPDAVLSQLKDKKHLGIHSEMISDGVVDLYEAGVIDCSQKSIDKGKMAITFLMGTKRLYDFAANNPKVELKPVDYINHPSVVAQCSKMVCINACLQVDFMGQIVSDSIGTKQFSGVGGQVDFVRGASMSIDGKGKAIIAMPSVAKKKDGSMISKIVPFIDHGAAVTTSRNDADYVVTEYGIAEMKGKSLQDRARALINIAHPDFKDELKAEFEKRFNAAF

[0118] The nucleotide sequence of abfT is shown in SEQ ID NO:4:

[0119]

[0120] 6. Coenzyme A ligase gene and hydroxy fatty acid polymerase gene sequences used for screening in other embodiments (codon optimized for Halomonas TD01)

[0121] phaC re The nucleotide sequence is shown in SEQ ID NO:5:

[0122]

[0123] phaC 4AK4 The nucleotide sequence is shown in SEQ ID NO:6:

[0124]

[0125] phaC 61-3 The nucleotide sequence is shown in SEQ ID NO:7:

[0126]

[0127] phaC 1437 The nucleotide sequence is shown in SEQ ID NO:8:

[0128]

[0129] phaC ac The nucleotide sequence is shown in SEQ ID NO:9:

[0130]

[0131] phaC ar The nucleotide sequence is shown in SEQ ID NO: 10:

[0132]

[0133] The nucleotide sequence of orfZ is shown in SEQ ID NO:11:

[0134]

[0135] The nucleotide sequence of pct540 is shown in SEQ ID NO:12:

[0136]

[0137] The nucleotide sequence of alkK is shown in SEQ ID NO:13:

[0138]

[0139] 7. Endogenous gene sequences knocked out in the examples

[0140] The nucleotide sequence of phaA is shown in SEQ ID NO:14:

[0141]

[0142] The nucleotide sequence of phaB is shown in SEQ ID NO:15:

[0143] atggccaatcaagcccccgtcgcctgggtaactggtggaactggtggaatcggaacgtcaatttgtcattcattggcggatgcggggtatttggtagtcgcggggtatcataatcctgagaaagccaaaacctggctggaaactcagcaagccgccggttatgacaacatcgcgttatcgggtgttgatctttccgaccacaatgcctgcctagaaggtgctcgcgaaatccaagaaaaatatgggcctgtcagcgtgttggttaactgcgctggcatcactcgtgatggcactatgaagaagatgtcctatgagcagtggcatcaagtgatcgataccaaccttaatagcgtcttcaacccctgccgcagtgtaattgaaatgatgctggagcagggttacggtcgcatcatcaatatttcttctatcaacggtcgcaaaggccagtttgggcaggtgaactatgcggctgccaaagcaggtatgcatgggttgaccatgtcgctggcgcaagaaacggcaaccaaaggcattacggtgaataccgtatcgccaggctacattgccaccgatatgatcatgaaaattcccgaacaggtgcgtgaagccattcgtgaaaccattccagtgaagcgttacggtacaccggaggagattggtcgtctggtcacgttcttagcagataaagagtcaggttttattactggcgccaatattgatattaacggcggtcagtttatgggctaa

[0144] The nucleotide sequence of phaC is shown in SEQ ID NO:16:

[0145]

[0146] The nucleotide sequence of fadB is shown in SEQ ID NO:17:

[0147]

[0148] Example 1

[0149] Screening for combinations of coenzyme A ligase and polyhydroxyalkanoic acid polymerase for polymerizing 5-hydroxybutyrate monomers

[0150] Different CoA ligases and PHA polymerases were arranged and combined and constructed on the pSEVA321 plasmid. The promoters of the endogenous wild-type porin gene were selected. The spectinomycin-resistant plasmid containing CoA ligase and PHA polymerase ( Figure 1 ) was introduced into Halomonas bluephagenesis TD01 (CGMCC No. 4353). A shake flask experiment was conducted by adding 5 g / L valerolactone as a precursor to LB60 medium. The fermentation conditions were 37°C, 200 rpm, and 48 hours. The bacteria of each group were collected and the samples were subjected to subsequent esterification reaction and gas chromatography analysis (for example, the detection method is referred to CN117925488 A). The cell dry weight and the contents of 5HV and 3HB in different combinations were tested ( Figure 2 ), the molar percentage of monomers in the copolymers obtained by each combination was calculated ( Figure 3 ), PhaC un The optimal combination of these two enzymes, AbfT, can achieve the synthesis of polyhydroxyalkanoic acid P3HB5HV containing a higher proportion of 5HV.

[0151] Example 2

[0152] Reducing 3HB ratio using endogenous phaCAB knockout strain

[0153] In the mineral culture medium MM60, glucose is usually used as the carbon source, and Halomonas bluephagenesis can accumulate PHB greater than 70% of the cell dry weight. In order to obtain PVL homopolymers, a strain in which the endogenous gene phaCAB of Halomonas bluephagenesis has been knocked out is required to block its endogenous metabolic pathway for synthesizing PHB. Our laboratory has an H. bluephagenesis TD1.0 strain (abbreviated as TD (ΔphaCAB)) in which its endogenous phaCAB has been knocked out (for strain construction, see “Xiao-Ran Jiang, Xu Yan, Lin-Ping Yu, Xin-Yi Liu, Guo-QiangChen. Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis. Nature Communications 12, 1513 (2021)”), and the plasmid pWJL55 was introduced into the wild-type TD01 and TD (ΔphaCAB) by conjugation transformation, respectively (for plasmid composition, see Figure 1 , is phaC un and abfT gene combination), and a single clone was selected for shake flask fermentation experiment. The culture medium was MM60, in which valerolactone was added as a precursor at a concentration of 5 g / L. The fermentation culture conditions were 37°C, 220 rpm, and 48 h.

[0154] The final experimental results showed that in the TD (ΔphaCAB) strain, the molar proportion of 5HV increased from less than 40% to more than 99% ( Figure 4 ).

[0155] Example 3

[0156] Introduction of phaC into the genome un and abfT gene

[0157] phaC un and abfT genes were inserted into the G4 site of the TD (ΔphaCAB) genome, and their nucleotide sequences were shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0158] 1) phaC un Construction of abfT gene donor plasmid

[0159] ① Using CRISPR-Cas9 to insert genes, first select 20bp as guide RNA (sequence is ttcacctagctagatgagac (SEQ ID NO: 31)) near the insertion site according to the PAM site, use pQ41 plasmid as template, design the guide RNA sequence in the primer, perform circular PCR with the primer, and then connect the PCR product with T4 ligase to obtain a plasmid containing guide RNA. The primers are:

[0160] sgRNA-F:

[0161] 5'-ttcacctagctagatgagacgttttagagctagaaatagcaagttaaaataaggctagtccg-3' (SEQ ID NO: 32)

[0162] sgRNA-R:

[0163] 5'-gtctcatctagctaggtgaaactagtattatacctaggactgagctagctgtc-3'(SEQ IDNO:33)

[0164] PCR reaction conditions:

[0165] Pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; post-extension at 72°C for 10 minutes.

[0166] ② Select two 1000 bases near the guide RNA as homology arms. Using the TD (ΔphaCAB) genome as a template, use primers F1 and R1 to amplify the upstream homology arm, and use primers F2 and R2 to amplify the downstream homology arm. Use primers F3 and F4 to amplify the P on the pWJL55 plasmid. porin -phaC un -sequence of the abfT operon.

[0167] Among them, primers:

[0168] F1:5'-gtgcgcttgccacagcttcg-3'(SEQ ID NO:34)

[0169] R1:5'-gtgatcgatgtgctcggaaggtg-3'(SEQ ID NO:35)

[0170] F2:5'-ctcaccttcatttccttatgctgaacacgg-3'(SEQ ID NO:36)

[0171] R2:5'-tgttccctaaaacttcgcctccaatttagc-3'(SEQ ID NO:37)

[0172] F3:5'-cttccgagcacatcgatcacctcggtaccaaattccagaaaagaggcc-3'(SEQ ID NO:38) R3:5'-cataaggaaatgaaggtgagattctcaccaataaaaaacgcccggc-3'(SEQ ID NO:39)

[0173] ③ The plasmid containing guide RNA in ① was amplified into a linear fragment using primers F4 and R4, and the fragment was assembled with the three fragments in ② by Gibson ligation to construct plasmid pWJL83: pSEVA341-phaC un -abfT-ki. Transformed into E. coli S17-1, obtained transformants, and sequenced. The correct sequenced transformants were selected and preserved and cultured overnight in LB medium to extract plasmids.

[0174] Among them, primers:

[0175] F4:5'-aggcgaagttttagggaacagcggccgcgtcgtgac-3'(SEQ ID NO:40)

[0176] R4:5'-cgaagctgtggcaagcgcacggtaccccgggttcaaaaaaagcac-3'(SEQ ID NO:41)

[0177] 2) Construction of H. bluephagenesis JL01

[0178] The chloramphenicol-resistant plasmid pQ08 containing Cas9 was transformed into TD (ΔphaCAB) using the conjugation transformation method. The plasmid pWJL83 was then transferred into the bacteria and cultured on a plate containing chloramphenicol and spectinomycin for two days until a single colony grew.

[0179] After confirmation by colony PCR and gene sequencing, a pair of primers WJL592 and WJL593 were designed upstream and downstream of the insertion site for verification by colony PCR and further verification by sequencing.

[0180] Among them, primers:

[0181] WJL592:5'-cggttgtaagtggaacagcacagc-3'(SEQ ID NO:42)

[0182] WJL593:5'-cgccaaacggaaccgatacatttcg-3'(SEQ ID NO:43)

[0183] The strain with the correct sequencing was selected and cultured in a non-antibiotic seed culture medium until the strain completely lost the plasmid and obtained phaC un -abfT insertion strain Halomonas blue phagenesis TD1.0 (ΔphaCAB, G4::phaC un -abfT) (referred to as H.bluephagenesis JL01).

[0184] Example 4

[0185] Knockout of endogenous fadB gene

[0186] Knockout the endogenous fadB gene of H. bluephagenesis JL01 to block its 3HB-CoA from the β-redox pathway and crotonyl-CoA. Figure 6 .

[0187] 1) Construction of fadB gene knockout plasmid

[0188] ① Using CRISPR-Cas9 to insert genes, first select 20bp as guide RNA (sequence is agtgctgttgtggcagagtt (SEQ ID NO: 44)) near the insertion site according to the PAM site, use pQ41 plasmid as template, design the guide RNA sequence in the primer, perform circular PCR with the primer, and then connect the PCR product with T4 ligase to obtain a plasmid containing guide RNA. The primers are:

[0189] sgRNA-F:5'-agtgctgttgtggcagagttgttttagagctagaaatagcaagttaaaataagg-3'(SEQ ID NO:45)

[0190] sgRNA-R:5'-aactctgccacaacagcactactagtattatacctaggactgagctagc-3'(SEQID NO:46)

[0191] PCR reaction conditions:

[0192] Pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; post-extension at 72°C for 10 minutes.

[0193] ② Select two 1000 bases near the guide RNA as homology arms. Using the H. bluephagenesis TD01 genome as a template, use primers F1 and R1 to amplify the upstream homology arm, and use F2 and R2 to amplify the downstream homology arm. Among them, primers:

[0194] F1:5'-atcaagagccggggaaagtg-3'(SEQ ID NO:47)

[0195] R1:5'-tgcgtggtggccgcgctctctccacaag-3'(SEQ ID NO:48)

[0196] F2:5'-gagagcgcggccaccacgcataggagatagaag-3'(SEQ ID NO:49)

[0197] R2:5'-agatctttcaagacaggcaagcc-3'(SEQ ID NO:50)

[0198] ③ The plasmid containing guide RNA in ① was amplified into a linear fragment using primers F3 and R3, and the fragment was assembled with the three fragments in ② by Gibson ligation to construct plasmid pWJL84: pSEVA341-fadB-ko. Transformed into E. coli S17-1, transformed, sequenced, selected and stored, and cultured overnight in LB medium to extract plasmid.

[0199] Among them, primers:

[0200] F3:5'-ttgcctgtcttgaaagatctgtcgtgactgggaaaaccc-3'(SEQ ID NO:51)

[0201] R3:5'-cactttccccggctcttgatcatcccgggttcaaaaaaagc-3' (SEQ ID NO:52)

[0202] 2) Construction of H. bluephagenesis JL02

[0203] The chloramphenicol-resistant plasmid pQ08 containing Cas9 was transformed into H. bluephagenesis JL01 using the conjugation transformation method. The plasmid pWJL84 was then transferred into the bacterium and cultured on a plate containing chloramphenicol and spectinomycin for two days until a single colony grew.

[0204] After confirmation by colony PCR and gene sequencing, a pair of primers WJL1214 and WJL1215 were designed upstream and downstream of the insertion site for verification by colony PCR and further verification by sequencing.

[0205] Among them, primers:

[0206] WJL1214:5'-cggttgtaagtggaacagcacagc-3'(SEQ ID NO:53)

[0207] WJL1215:5'-gtaagtgccattgtcccgtgaggtg-3'(SEQ ID NO:54)

[0208] The strain with the correct sequencing was selected and cultured in a non-antibiotic seed culture medium until the strain completely lost the plasmid and obtained phaC un -abfT-inserted strain based on the fadB gene knockout strain Halomonas bluephagenesis TD1.0 (ΔphaCAB, ΔfadB, G4::phaCun-abfT) (referred to as H.bluephagenesis JL02).

[0209] The shake flask experiment showed that after knocking out the fadB gene, the proportions of DCW and 5HV were significantly increased. The yield of the shake flask experiment was about 6.8g / L, the percentage of PVL (i.e. PHA in the figure) was 45wt%, and the molar ratio of 5HV was higher than 98.4% ( Figure 5 ). Figure 5 The middle control is the corresponding strain H. bluephagenesis JL01 without knockout of fadB gene.

[0210] Example 5

[0211] Verification of PVL production by H.bluephagenesis JL02 in a 7L fermenter

[0212] The recombinant bacterium Halomonas bluephagenesis JL02 was streaked on an LB60 agarose plate for activation. After 24 h, a single clone was selected and inoculated into 20 mL of LB60 medium. After culturing for 12-16 h, the culture was transferred to a new LB60 medium at a volume ratio of 1%, and the culture was continued for 8-12 h. 300 mL of seed solution was prepared as the secondary seed solution for a 7 L bioreactor (NBSBioflo3000, New Brunswick, USA).

[0213] 2.7 L of base culture medium was prepared, the base containing glucose (30 g), sodium chloride (150 g), magnesium sulfate (2 g), urea (12.4 g), potassium dihydrogen phosphate (9.9 g), and disodium hydrogen phosphate dodecahydrate (20.4 g).

[0214] The feed I medium was prepared to contain glucose (30 g), magnesium sulfate (2 g), urea (7.6 g), potassium dihydrogen phosphate (0.79 g), and sodium dihydrogen phosphate dodecahydrate (2.12 g).

[0215] Feed II medium was prepared to contain glucose (30 g) and urea (7.6 g).

[0216] Feed III medium was prepared to contain glucose (100 g) to maintain the glucose concentration in the medium.

[0217] The pH value of the culture medium was automatically adjusted by NaOH.

[0218] 15 g of substrate valerolactone was added every 10 hours.

[0219] During the fermentation process, we adjusted the dissolved oxygen content by stirring and aeration, set the aeration volume to 3L / min, the stirring speed to 800rpm, and set the dissolved oxygen content to 100%, and ensure that the dissolved oxygen content was 30% during the fermentation process. The glucose content in the fermentation medium was monitored by testing the residual glucose concentration with a blood glucose tester every 2 hours, thereby regulating the feed amount.

[0220] The results showed that after 48 hours of fermentation, at the 40th hour, the dry weight of the bacteria was 22 g / L and the PVL yield was 60 wt% ( Figure 7 ). This result indicated that Halomonas bluephagenesis JL02 had the potential to efficiently produce PVL and could be used as a platform strain for industrial production of PVL.

[0221] Example 6

[0222] Purity, molecular weight, thermodynamic and mechanical properties testing of PVL

[0223] The PVL material was extracted from H.bluephagenesis JL02 after 48 hours of fermentation using a Soxhlet extractor. Cells were first collected from the shake flask by centrifugation at 4700 rpm for 30 minutes. The cell pellet was then washed twice with distilled water at 2500 rpm for 5 minutes each time, followed by freeze drying for 12 hours to remove moisture. PHA polymers including PVL were extracted from the freeze-dried cells using a Soxhlet extractor (Soxtec 2050, Foss, Denmark). The extraction process involved dissolving the cells in chloroform and precipitating the polymer with cold anhydrous ethanol.

[0224] After the extraction, about 20 mg of PHA sample was dissolved in deuterated chloroform and analyzed for purity using a nuclear magnetic resonance spectrometer (JNM-ECA600, JEOL). 1 HNMR and 13 C NMR spectroscopy. Chemical shifts are reported in parts per million (ppm) and are calibrated against the signal of a 0.03% v / v tetramethylsilane (TMS) reference standard.

[0225] To determine the molar mass and polydispersity of PVL samples, the samples were dissolved in chromatographically pure chloroform at a concentration of 2 mg / mL. The solution was then filtered using a 0.22 μm nylon membrane syringe filter to remove any undissolved particles. The filtered samples were subsequently analyzed by gel permeation chromatography (LC-20AD, Shimadzu Corporation, Japan). The mobile phase consisted of chromatographically pure chloroform at a temperature of 40 °C at a flow rate of 1 mL / min. The injection volume of each sample was 40 μL. A series of polystyrene standards with different number average molar masses (1 × 10 4 Da,2×10 4 Da,3×10 4 Da,7×10 4 Da,1.5×10 5 Da,3×10 5 Da,7×10 5 Da, and 1×10 6 Da; Sigma-Aldrich, USA) to generate the calibration curve for GPC analysis.

[0226] The thermodynamic properties of the samples were measured using 10 mg of PVL material, including the melting point and glass transition temperature, using a differential scanning calorimeter (DSC-Q20, TA Company, USA). The temperature scan was performed from -80°C to 200°C with a nitrogen flow rate of 50 ml / min. The sample was initially cooled to -80°C and then heated to 180°C at a heating rate of 10°C / min. To ensure the accuracy of the measurement, this cooling and heating cycle was repeated once in the same temperature range.

[0227] The thermal decomposition temperature was tested by a thermogravimetric analyzer (TGA-50, Shimadzu Corporation, Japan). The analysis was carried out in a nitrogen environment, with the temperature rising from room temperature to 600°C at a heating rate of 10°C / min.

[0228] After the PVL material was stretched, its mechanical properties were tested. 1 gram of PVL was dissolved in 30 ml of chloroform and dripped onto a glass petri dish at room temperature in a fume hood. The solution was allowed to evaporate to form a PVL film, which took about 48 hours. A 4 mm wide dumbbell-shaped specimen was precisely cut using an RR / PCP cutter (Ray-Ran, UK). Stress-strain measurements were performed at a speed of 10 mm / min on an Instron tensile strength tester (Instron 3365, Instron, USA). Each sample was tested three times and the average value was taken to ensure the reliability of the data.

[0229] After NMR verification, PVL homopolymer with a purity close to 100% was obtained ( Figure 8 ). According to calculation, the molar percentage composition of the material is 1.58% 3HB-co-98.42% 5HV, and the content of 3HB monomer is not enough to affect its material properties.

[0230] The molecular weight and mechanical properties of PVL obtained by biosynthesis are better than those of PVL obtained by chemical synthesis (trade name polyvalerolactone, product number AP299, batch number 230620TMT-A, commercially available from PolySciTech USA) measured by the same method (Table 2), among which the elongation at break has a significant advantage over the chemically synthesized material.

[0231]

[0232] 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0233] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations. In addition, the various different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An engineered bacterium for producing polyvalerolactone (PVL), which expresses or overexpresses exogenous or endogenous polyhydroxy fatty acid polymerase and CoA ligase of 5-hydroxybutyrate monomer, preferably the engineered bacterium does not have or has been knocked out or knocked down endogenous β-ketothiolase (PhaA) gene, acetoacetyl-CoA reductase (PhaB) gene, PHA polymerase (PhaC) gene and enoyl-CoA hydratase (FadB) gene One or more (preferably all).

2. The engineered bacterium according to claim 1, which is unable or almost unable to produce poly 3-hydroxybutyrate (PHB), for example, in a mineral culture medium with glucose as the carbon source.

3. The engineered bacteria according to claim 1 or 2, wherein the engineered bacteria is selected from halophiles, preferably selected from Halomonas, Pseudomonas, Escherishia, Ralstonia eutropha, Aeromonas, Bacilllus, Alcaligenes latus, Alcaligenes eutropus or a combination thereof, more preferably Halomonas, even more preferably Halomonas bluephagenesis, Halomonasaydingkolgenesis, Halomonas campaniensis or Halomonaslutescens, most preferably Halomonasbluephagenesis TD01 (strain collection number CGMCC No.4353), Halomonas bluephagenesisTD141, Halomonas bluephagenesis TD1.0, Halomonas aydingkolgenesis M1 (Cultivation Number CGMCC No. 19880) or Halomonas campaniensis LS21 (Cultivation Number CGMCC No. 6593).

4. The engineered bacterium according to any one of claims 1 to 3, wherein the polyhydroxyalkanoic acid polymerase is selected from PhaC un , PhaC re , PhaC 4AK4 , PhaC 61-3 , PhaC 1437 , PhaC ac and PhaC ar , and / or the CoA ligase of the 5-hydroxybutyrate monomer is selected from 4-hydroxybutyryl CoA transferase (AbfT), orfZ, pct540 and alkK, preferably the polyhydroxyalkanoic acid polymerase is PhaC un The coenzyme A ligase of the 5-hydroxybutyrate monomer is 4-hydroxybutyryl-CoA transferase (AbfT), and more preferably the polyhydroxyalkanoic acid polymerase (PhaC un ) and the CoA ligase for 5-hydroxybutyrate monomer (4-hydroxybutyryl-CoA transferase, AbfT) are each independently expressed or overexpressed on the genome of the engineered bacteria or on one or more plasmids.

5. The engineered bacteria according to claim 4, wherein the gene encoding 4-hydroxybutyryl-CoA transferase abfT is derived from Clostridium aminobutyricum, and / or the gene encoding polyhydroxyalkanoic acid polymerase phaC un The uncultured bacteria derived from the red forest swamp or a mutant of the gene, preferably the amino acid sequence of the polyhydroxyalkanoic acid polymerase is as shown in SEQ ID NO: 1, more preferably the phaC un The nucleotide sequence of abfT is shown in SEQ ID NO:2, and / or preferably the amino acid sequence of 4-hydroxybutyryl-CoA transferase is shown in SEQ ID NO:3, and more preferably the nucleotide sequence of abfT is shown in SEQ ID NO:

4.

6. The engineered bacteria according to any one of claims 1 to 5, wherein the coding gene of the polyhydroxyalkanoic acid polymerase (PhaCun) and the coding gene of 4-hydroxybutyryl-CoA transferase (AbfT) are each independently expressed under the control of a constitutive or inducible promoter, preferably the inducible promoter is selected from IPTG-inducible Mmp1 promoter, lux promoter, lac promoter, trp promoter, tac promoter or a combination thereof, preferably the constitutive promoter is selected from wild-type Pporin promoter or a mutant thereof, wherein the mutant is selected from porin3, porin 42 、porin 58 、porin 68 、porin 140 、porin 141 、porin 194 、porin 211 、porin 221 、porin 226 、porin 278 , or a combination thereof.

7. The engineered bacterium according to any one of claims 1 to 6, wherein the gene encoding the polyhydroxyalkanoic acid polymerase (PhaCun) and the gene encoding the 4-hydroxybutyryl-CoA transferase (AbfT) are introduced into the engineered bacterium by transformation or conjugation.

8. A method for producing polyvalerolactone (PVL), the method comprising fermenting and culturing the engineering bacteria according to any one of claims 1 to 7.

9. The method according to claim 8, wherein the fermentation culture is: A) carried out using a mineral medium, B) using valerolactone as a substrate, and / or C) carried out under open conditions.

10. A polyvalerolactone, wherein: 1) The purity of the polyvalerolactone is greater than 98 mol%; 2) the polyvalerolactone has an Mw greater than 100 kDa and an Mn greater than 50 kDa; 3) the PDI (Mw / Mn) of the polyvalerolactone is about 2; 4) the elongation at break of the polyvalerolactone is greater than 100%, Preferably the polyvalerolactone is produced by the method according to claim 8 or 9.

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