Strain for efficiently synthesizing pha from acetic acid and application thereof

Abstract

This invention relates to a recombinant Halomonas strain that produces polyhydroxyalkanoates (PHA) by overexpressing endogenous enzymes encoding ADP-dependent acetyl-CoA synthase (ADP-ACS). acd This gene not only enhances the recombinant bacterium's tolerance to acetic acid but also enables it to efficiently convert acetic acid or acetate substrates into acetyl-CoA, thereby participating in the tricarboxylic acid cycle (TCA cycle) and the synthesis of PHA. The present invention also relates to a method for producing PHA using the aforementioned recombinant Halomonas bacterium employing a pH-stat strategy.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology. More specifically, this invention relates to a strain that efficiently synthesizes PHA using acetic acid and its applications. Background Technology

[0002] Polyhydroxyalkanoates (PHA) are a class of natural bio-based biodegradable polymers synthesized by microorganisms under conditions of carbon surplus. Their backbone is composed of hydroxy fatty acid monomers linked by ester bonds, exhibiting excellent biocompatibility and environmental friendliness. They can be completely degraded into carbon dioxide and water in environments such as soil, seawater, and compost, leaving no microplastic residue.

[0003] Acetic acid is primarily produced through two main routes: traditional petrochemical byproducts and emerging methods involving the electrolysis of carbon dioxide waste and water. In the petrochemical route, acetic acid is mainly produced via methanol carbonylation, which involves reacting petroleum-derived methanol with carbon monoxide (CO) in the presence of a catalyst. This process dominates global acetic acid production. Another method, acetaldehyde oxidation, relies on the oxidation of ethylene to produce acetaldehyde, which is then converted to acetic acid. In contrast, green chemistry utilizes carbon dioxide waste as a carbon source, directly synthesizing acetic acid through electrochemical reduction reactions (such as the electrolysis of carbon dioxide and water in the presence of a catalyst). This method not only reduces carbon emissions but also achieves resource recycling. These two routes respectively reflect the development trends of fossil fuel dependence and sustainable chemical engineering. Therefore, acetic acid is inexpensive and suitable as a substrate for microbial fermentation.

[0004] However, using acetic acid as a microbial fermentation substrate to produce PHA still faces various challenges: (1) low utilization efficiency of acetic acid; (2) lack of strains with high tolerance to acetic acid. Summary of the Invention

[0005] To address the aforementioned problems, the inventors, through in-depth research and bioinformatics methods, have identified an endogenous encoding of ADP-dependent acetyl-CoA synthase (ADP-ACS) in Halomonas. acd The gene was then used to genetically engineer Halomonas bacteria to overexpress this gene. acd This gene not only enhances the recombinant bacterium's tolerance to acetic acid but also enables it to efficiently convert acetic acid or acetate substrates into acetyl-CoA, thereby participating in the tricarboxylic acid cycle (TCA cycle) and the synthesis of PHA. The present invention also relates to a method for producing PHA using the aforementioned recombinant Halomonas bacterium employing a pH-stat strategy.

[0006] In Halomonas ( Halomonas spp.In organisms like Halomonas, total sugars (such as glucose) can be used as a carbon source to synthesize PHA. A key step involves glucose undergoing glycolysis and the TCA cycle to produce acetyl-CoA, which further synthesizes PHA monomers. Finally, PHA synthases synthesize various types of PHA. Therefore, in order to construct a metabolic pathway for PHA synthesis using acetic acid in Halomonas, a crucial step is to convert acetic acid into acetyl-CoA.

[0007] Unconstrained by any theoretical limitations, through bioinformatics research, the inventors hypothesize that *Halomonas* may possess two metabolic pathways for the conversion of acetyl-CoA into acetyl-CoA. The first pathway is the AMP-dependent acetyl-CoA synthase (AMP-ACS) pathway. This pathway irreversibly converts acetyl or acetate into acetyl-CoA. AMP-ACS first binds ATP to acetyl or acetate, generating acetyl-AMP and releasing pyrophosphate; subsequently, it binds to CoA, converting acetyl-AMP into acetyl-CoA and releasing AMP. The other pathway utilizes α... cd The encoded ADP-dependent acetyl-CoA synthase (ADP-ACS, or ACD) not only catalyzes the breakdown of acetyl-CoA into acetic acid or acetate with the generation of ATP, but also catalyzes its reverse reaction, namely the activation pathway of acetic acid or acetate.

[0008] The inventors first attempted the first approach, the AMP-ACS approach, using bioinformatics to discover the endogenous ACS-coding gene in *Halomonas*. acs2 The study investigated the effect of overexpression of the compound in *Haloxylon ammodendron* on the conversion of acetic acid as a carbon source. However, the results showed that overexpression... acs2 The CDW and PHB contents of the recombinant halomonas were not increased compared with the control group of empty vector, suggesting that this pathway cannot effectively improve the utilization of acetic acid carbon source by halomonas.

[0009] Therefore, the inventors continued to explore a second approach, using bioinformatics to discover the gene encoding the endogenous ADP-dependent acetyl-CoA synthase in Halomonas (…). acd The drug was overexpressed in *Haloxylon ammodendron* to assess its effect on the conversion of acetic acid as a carbon source. Compared to the first pathway, overexpression... acd The recombinant Halomonas strain not only tolerates high concentrations of acetic acid but also efficiently utilizes acetic acid or its salts as a carbon source, with significantly increased CDW and PHB contents compared to the control. Furthermore, this invention also achieves high-yield PHA synthesis from acetic acid through optimization of fermentation conditions.

[0010] Therefore, according to one aspect of the present invention, a recombinant Halomonas strain for producing polyhydroxyalkanoates (PHA) is provided, which overexpresses an endogenous enzyme encoding ADP-dependent acetyl-CoA synthase.acd Gene.

[0011] In one embodiment, the overexpression is constitutive expression controlled by a constitutive promoter or inducible expression controlled by an inducible promoter selected from the Mmp1 promoter, lux promoter, lac promoter, trp promoter, tac promoter, or combinations thereof, and the constitutive promoter is selected from the wild-type Pporin gene promoter or a mutant thereof, wherein the mutant is selected from Pporin1, Pporin3, Pporin42, Pporin51, Pporin58, Pporin59, Pporin68, Pporin140, Pporin141, Pporin183, Pporin192, Pporin194, Pporin203, Pporin221, Pporin226, Pporin259, and Pporin278. These Pporin promoters and their mutants are disclosed, for example, in CN117143793B; see also Ye et. al. Stimulus response-based fine tuning of polyhydroxyalkanoate pathway in Halomonas. Metabolic Engineering. 57(2020) 85-95.

[0012] In one embodiment, the starting strain of the recombinant Halomonas (i.e., overexpressing) acd The host bacteria of the gene) is Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata or Halomonas smyrnensis .

[0013] In a preferred embodiment, the starting strain of the recombinant halometa is: Halomonas bluephagenesis TD1.0, strain preservation number CGMCC No. 4353 Halomonas bluephagenesis TD01, strain with CGMCC No. 19880 Halomonas aydingkolgenesis M1, strain with preservation number CGMCC No. 6593 Halomonas campaniensis LS21 Halomonas bluephagenesis TD27 Halomonas bluephagenesis TDB141 Halomonas bluephagenesis TDB141ΔAC、 Halomonas bluephagenesisWZY254 Halomonas bluephagenesis WZY278 or Halomonas bluephagenesis CYL0307.

[0014] In one embodiment, the amino acid sequence of the ADP-dependent acetyl-CoA synthase is shown in SEQ ID No. 1.

[0015] In one implementation, the acd The gene sequence is shown in SEQ ID No. 2.

[0016] In one embodiment, the starting strain of the recombinant halometa is: Halomonas bluephagenesis .

[0017] In one implementation, the acd The gene is overexpressed on the plasmid and / or genome in the recombinant Halomonas.

[0018] In one implementation, the gene editing method (preferably CRISPR / Cas9 gene editing method) is used to... acd The gene is integrated into the genome of the halomonas.

[0019] In one embodiment, the endogenous plasmid has been knocked out in the recombinant halomonas.

[0020] In one embodiment, the plasmid used in the overexpression is a pSEVA series plasmid (preferably pSEVA341, pSEVA321 or pSEVA241) or a toxin-antitoxin system plasmid (preferably pHbPBC).

[0021] In a preferred embodiment, the starting strain of the recombinant halometa is: Halomonas bluephagenesis TD1.0, and the recombinant Halomonas strain is overexpressed under the control of the Mmp1 promoter or the Pporin59 promoter. acd Gene.

[0022] In a preferred embodiment, the starting strain of the recombinant halometa is: Halomonas bluephagenesis TD1.0, and the recombinant Halomonas halophila comprises recombinant plasmid pAC1 (i.e., pSEVA321-Pmmp1-acd) or pAC2 (i.e., pHbPHC-Pporin59-acd), i.e., overexpression of the recombinant Halomonas halophila in plasmid pSEVA321 under the control of the Mmp1 promoter or in plasmid pHbPHC under the control of the Pporin59 promoter, respectively. acd Gene.

[0023] In one embodiment, the PHA includes at least one of PHB, P4HB, P3HB4HB3HV, PHV, PHBHHX, P34HB, P3HO3HH, PHO, PHP, and PHBVHHX.

[0024] According to another aspect of the present invention, a method for producing polyhydroxyalkanoates is provided, the method comprising fermenting and culturing the recombinant halomonas strain according to the present invention.

[0025] In one embodiment, the fermentation culture utilizes either i) or ii) as the sole carbon source to synthesize the polyhydroxy fatty acid ester:

[0026] i) A mixture of glucose and acetic acid or acetate;

[0027] ii) Acetic acid or acetate.

[0028] Because of the introduction acd The gene and recombinant Halomonas strains have improved tolerance to acetic acid and their utilization rate as a carbon source for PHA synthesis. Therefore, acetic acid can be added during fermentation, serving as both a pH adjuster and a supplementary carbon source.

[0029] Therefore, in one embodiment, during the fermentation process, carbon source feeding is performed by supplementing with acetic acid and maintaining the pH at 7.5 to 8.5 (e.g., 8.0). In this embodiment, a small amount of acetic acid or acetate can be initially added as an initial carbon source at the start of fermentation, and subsequent carbon sources rely entirely on supplemented acetic acid (e.g., fed-on acetic acid (water) solution) while maintaining a constant pH. This optimized fermentation scheme completely eliminates acetate feeding, achieving a dual function of pH regulation and carbon source supply solely through acetic acid from the acid pump (pH-stat strategy). This strategy not only reduces salt ion concentration and alleviates osmotic stress but also simplifies feeding operations and reduces process costs.

[0030] According to another aspect of the present invention, the application of PHA obtained by the method according to the present invention in the preparation of packaging materials, biomedical materials, drug carriers, biodegradable agricultural films, slow-release fertilizers, eco-friendly building materials, conductive materials, food or cosmetics is provided.

[0031] The beneficial effects that the technical solution of the present invention can produce include:

[0032] A) A metabolic pathway for the efficient use of acetic acid was validated in Halomonas, enabling efficient and low-cost synthesis of PHA (e.g., PHB);

[0033] B) Recombinant halomonas bacteria can be used to achieve low-cost and high-efficiency synthesis of PHA (e.g., PHB) without sterilization;

[0034] C) By utilizing the pH-stat strategy to optimize the fermentation process, acetate feeding can be completely eliminated. Relying solely on acetic acid from the acid pump, the dual functions of pH regulation and carbon source supply can be integrated. This not only reduces salt ion concentration and alleviates osmotic pressure stress, but also simplifies feeding operations and reduces process costs. Attached Figure Description

[0035] 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, wherein:

[0036] Figure 1 The shake-flask demonstration showed the efficiency of recombinant ACS expressing endogenous AMP type in synthesizing PHB using acetic acid and the utilization of acetic acid.

[0037] Figure 2 The shake-flask demonstration showed the expression of endogenous ADP-dependent acetyl-CoA synthase. acd The utilization of acetic acid by recombinant bacteria when synthesizing PHB from glucose and acetic acid; and

[0038] Figure 3 The pH-feedback strategy in the fermenter is used to verify the synthesis of PHB.

[0039] Sequence List Description (Sequences used in the illustrative and example versions of this invention)

[0040] SEQ ID No. 1 – Amino acid sequence of ADP-dependent acetyl-CoA synthase

[0041] SEQ ID No. 2 – acd nucleotide sequence of a gene

[0042] SEQ ID No. 3 – Mmp1 promoter

[0043] SEQ ID No. 4 – Pporin59 promoter

[0044] SEQ ID No. 5 – Amino acid sequence of AMP-type ACS

[0045] SEQ ID No. 6 – Gene sequence of AMP-type ACS Detailed Implementation

[0046] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this art, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0047] In this invention, the singular articles “a” and “the” cover a plurality of indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by reference in their entirety.

[0048] In this invention, the terms "comprising" or "including" are open-ended expressions, referring to a specific component or step described, while not excluding other components or steps that do not substantially affect the meaning. When describing protein or nucleic acid sequences, the sequences may constitute the target molecule on their own, or may have additional amino acids or nucleotides added to one or both ends, or may be engineered to resemble proteins, while retaining the functional activity described in this invention.

[0049] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0050] In this invention, the term "Halomonas" includes, but is not limited to, […]. Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Even better Halomonas bluephagenesisTD1.0 ((see "Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD1.0, Tan et al., Bioresource Technology, 2011 Volume 102, Issue 17, September 2011, Pages 8130-8136"), Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) Halomonas campaniensis LS21 (CGMCC No. 6593) (All of the above strains have been deposited with the China General Microbiological Culture Collection Center (CGMCC) under the Budapest Treaty and have been disclosed in previous patent applications. For example, CGMCC No. 4353 has been disclosed in CN102120973A, CGMCC No. 19880 has been disclosed in CN111593006A, and CGMCC No. 6593 has been disclosed in CN102925382A.) Halomonas bluephagenesis TD27 (described in JIANG XR, YAN X, YUL P, et al. 2021. Hyperproduction of 3-hydroxypropionate by Halomonasbluephagenesis. Nat Commun [J], 12: 1513.) Halomonas bluephagenesis TDB141 (documented in "Yan X, Liu X, Yu LP, et al. 2022. Biosynthesis of diverse alpha,omega-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis .Metab Eng [J], 72: 275-288."), Halomonas bluephagenesis TDB141ΔAC (recorded in "Lizhan Zhang") et al., 2022. Effective production of Poly(3-hydroxybutyrate-co-4hydroxybutyrate) by engineered Halomonas bluephagenesis grown on glucose and 1,4-Butanediol [J]. Bioresource Technology”)、 Halomonas bluephagenesis WZY254 (described in "Wang Z, Zheng Y, Ji M, et al. 2022b. Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors. Microb Biotechnol [J], 15: 1586-1597.")、 Halomonas bluephagenesis WZY278 (described in "Ji MK, Zheng T R, Wang Z Y, et al. 2023. PHB production from food waste hydrolysates by harboring PHB operon linked with an essential gene. Metabolic Engineering [J], 77: 12-20.") or Halomonas bluephagenesis CYL0307 (described in "Chen YL, Liu X, Zhang L Z, et al. 2025. Cell Sizes Matter for Industrial Bioproduction, a Case of Polyhydroxybutyrate. Advanced Science [J].")。

[0051] In this invention, the term "expression" can refer to "overexpression," which is defined as a gene expression level higher than the natural state, possibly achieved through increased transcription levels (producing more messenger mRNA) or improved translation efficiency (generating more functional proteins). In one specific embodiment, preferably, the expression or overexpression of the exogenous gene can be achieved by inserting the target gene into a non-translation site in the genome, or by plasmid overexpression. Preferably, gene insertion is performed using the CRISPR / Cas9 method (Qin Q, Ling C, Zhao Y, et al. 2018. CRISPR / Cas9 editing genome of extremophile Halomonas spp. Metab Eng [J], 47: 219-229). Preferably, plasmid overexpression is introduced into the chassis strain via electroporation or conjugation transformation. More preferably, the plasmid vector is a pSEVA series vector (preferably pSEVA341, pSEVA321, or pSEVA241, e.g., see Martinez-Garcia E, Aparicio T, Goni-Moreno A, et al. 2015. SEVA 2.0: an update of the Standard European Vector Architecture for de- / re-construction of bacterial functionalities. Nucleic Acids Res [J],43: D1183-1189) or a toxin-antitoxin plasmid pHbPBC vector (Ren K, Zhao YQ, Chen GQ, et al.2023. Construction of a Stable Expression System Based on the EndogenoushbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis. Acs Synthetic Biology [J], 13: 61-67), etc.

[0052] In this invention, the term "toxin-antitoxin system" (i.e., toxin-antitoxin, TA system) refers to a system that maintains plasmid stability through post-segregational killing (PSK). In a preferred embodiment, the recombinant microorganism of this invention contains a suitable toxin-antitoxin system / plasmid, thereby enabling the plasmid to be stably maintained naturally (i.e., in the absence of selective pressures such as antibiotics). Eight types of TA systems are known. hok / sok The system has been widely used in industrial fermentation to stabilize plasmid pMJR1750 (Lin et al., Engineering Microbiology 3 (2023) 100069). WO1999025870 discloses plasmid pMUT2 containing the TA system, which is stably maintained in Escherichia coli. Chinese Patent Application No. 2023112420281, entitled "A recombinant plasmid expressing toxins and antitoxins, its construction method and application", discloses a TA system derived from halophilic microorganisms and a recombinant plasmid including the TA system, and discloses that the recombinant plasmid containing the TA system can be naturally and stably maintained in halophilic microorganisms, etc. Typical TA systems include the following gene families: ccdAB, mazEF, vapBC, phdd / doc, parDE, higBA, and relBE (Gerdes K et al., Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes, Nucleic Acids Res., 2005, 33(3): 966-976). In one specific embodiment, the host cell is a prokaryotic or eukaryotic microbial cell, and the toxin-antitoxin is a TA system suitable for prokaryotic or eukaryotic microbial cells. In one specific embodiment, the microorganism is a Gram-negative or Gram-positive bacterium. In a preferred embodiment, the recombinant microorganism of the present invention is Halomonas, and / or the toxin-antitoxin system plasmid used therein is pHbPBC (Ren K, Zhao YQ, Chen GQ, et al. 2023. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis . Acs Synthetic Biology[J], 13: 61-67).

[0053] In this invention, the term "polyhydroxy fatty acid ester (PHA)" refers to polyhydroxy fatty acid esters, which can be classified into homopolymers and copolymers based on their monomer composition. Depending on the number of carbon atoms in the monomer, it can be a short-chain PHA (i.e., a hydroxy fatty acid monomer of C3-C5) or a medium- to long-chain PHA (i.e., a hydroxy fatty acid monomer of C6-C18), but is not limited to these. PHA can be a homopolymer, including but not limited to polyhydroxypropionate (PHP), polyhydroxybutyrate (PHB), polyhydroxyoctanoate (PHO), polyhydroxyvalerate (PHV), etc., such as poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP), or poly-3-hydroxyvalerate (P3HV), etc. PHA can be a copolymer, such as a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid (P34HB), a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHBHHX), a copolymer of 3-hydroxybutyric acid and 3-hydroxyvalerate (P(3HB-co-3HV)), a binary copolymer of 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid (P3HO3HH), a terpolymer of 3-hydroxybutyric acid, 3-hydroxyvalerate and 3-hydroxyhexanoic acid (PHBVHHX), a terpolymer of 3-hydroxybutyric acid, 4-hydroxybutyric acid, and 3-hydroxyvalerate (P3HB4HB3HV), etc. Example

[0054] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all performed in triplicate, and the results were averaged.

[0055] LB medium: containing 10 g / L NaCl, 10 g / L peptone (OXIOD, LP0042), and 5 g / L yeast extract (OXIOD, LP0021), diluted with water to a final volume, and autoclaved at 120°C.

[0056] LB60 medium: contains 60 g / L NaCl, and the other components are the same as LB medium.

[0057] Basic culture medium: 20-40 g / L glucose, 55-70 g / L sodium chloride, 1-10 g / L yeast extract, 3-6 g / L urea, 1.5-5.2 g / L potassium dihydrogen phosphate, 0.2-0.4 g / L magnesium sulfate, 8.5-10 g / L disodium hydrogen phosphate, 7-15 ml / L component III, and 1-5 ml / L component IV.

[0058] Component III: 5 g / L ferric ammonium citrate, 2 g / L calcium chloride dihydrate, 41.7 ml concentrated hydrochloric acid (12 mol / L), and water to a final volume of 1000 ml.

[0059] Component IV: 100 mg / L zinc sulfate heptahydrate, 30 mg / L manganese chloride tetrahydrate, 300 mg / L boric acid, 200 mg / L cobalt chloride hexahydrate, 10 mg / L anhydrous copper sulfate, 20 mg / L nickel chloride hexahydrate, and 30 mg / L sodium molybdate dihydrate.

[0060] MM medium (pH 8.0-8.5): contains 1 g / L yeast extract, 0.5 g / L urea, 0.2 g / L MgSO4; 9.65 g / L Na2HPO4·12H2O, 1.5 g / L KH2PO4. Preferably, it can be replaced with 14 g / L K2HPO4·3H2O, 5.2% KH2PO4 to provide a stronger buffering effect. Trace element solution I is added at a volume ratio of 1:100, and trace element solution II is added at a volume ratio of 1:100. Trace element solution I contains 5 g / L ferric ammonium citrate and 2 g / L CaCl2 (prepared with 1 M HCl). Trace element solution II contains 100 mg / L ZnSO4·7H2O, 30 mg / L MnCl2·4H2O, 300 mg / L H3BO3, 200 mg / L CoCl2·6H2O, 10 mg / L CuSO4·5H2O, 20 mg / L NiCl2·6H2O, and 30 mg / L NaMoO4·2H2O (prepared with 1 M HCl). Preferably, the final pH is adjusted to 8.5 using 5 M NaOH solution.

[0061] MM30 medium (pH 8.0-8.5): contains 30 g / L NaCl, and the other components are the same as MM medium.

[0062] All of the above culture media can be prepared using standard preparation methods.

[0063] Halomonas Halomonas bluephagenesis Cultured on LB60 medium.

[0064] Halomonas bluephagenesis TD1.0See "Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD1.0, Tan et al. , Bioresource Technology, 2011 Volume 102, Issue 17, September 2011, Pages 8130-8136”.

[0065] The plasmid pSEVA321 (GenBank accession No. JX560332.1) mentioned in the example can also be found in the literature: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41. D1 (2012): D666-D675.

[0066] The plasmid pSEVA341 mentioned in the example (GenBank accession No. JX560334.1) can also be found in the literature: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture(SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41. D1 (2012): D666-D675).

[0067] Conjugation transformation method: Electroconversion or chemical transformation of the target plasmid to... E. coli S17-1 (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.). Donor bacteria. E. coli S17-1 was cultured overnight in LB medium; the recipient *Haloxymonas* was cultured overnight in LB60 medium. Donor and recipient bacteria were each inoculated at a 2% inoculum into 20 ml of medium and cultured for 4-6 h. 1 ml of bacterial culture was collected from each culture, centrifuged, and the supernatant was removed. The bacteria were resuspended in antibiotic-free LB medium, mixed, and spread onto antibiotic-free LB plates, then incubated at 37°C for 6-8 h. Bacterial colonies were scraped, resuspended, and spread onto plates containing antibiotics, then incubated at 37°C for 24-48 h. Positive clones were screened for further processing.

[0068] Acetic acid detection method: When detecting acetic acid using high-performance liquid chromatography (HPLC), a reversed-phase column (e.g., C18 column, 250 mm × 4.6 mm, 5 μm) is typically used. The mobile phase is a mixture of phosphate buffer (pH 2.5-3.0) and methanol or acetonitrile (volume ratio usually 95:5 or 90:10), and the flow rate is set to 0.8-1.0 mL / min. An ultraviolet (UV) detector is selected, with the detection wavelength set to approximately 210 nm (acetic acid exhibits terminal absorption at low wavelengths), and the column temperature is controlled at 30-40℃. The sample needs to be filtered through a 0.22 μm microporous membrane or centrifuged, and the injection volume is generally 10-20 μL. To improve sensitivity, derivatization methods (e.g., reaction with 2-nitrophenylhydrazine) can be used to convert acetic acid into a derivative with stronger UV absorption. This method requires the establishment of an external standard curve using acetic acid standards for quantification. The linear range is typically 0.1–100 μg / mL, and the limit of detection (LOD) can reach 0.05 μg / mL. During the analysis, it is important to adjust the mobile phase pH to suppress the dissociation of acetic acid and improve peak shape, while avoiding column overload or solvent effects.

[0069] Seed culture preparation: Glycerol tubes stored at -80℃ were inoculated onto LB60 plates and incubated at 37℃ for 24 h. Single colonies were picked and inoculated into 20 mL of liquid LB60 and incubated at 37℃ and 200 rpm for 24 h to obtain the primary seed culture. A 1% inoculum was then transferred to 20 mL of liquid LB60 and incubated at 37℃ and 200 rpm for 8–10 h to obtain the secondary seed culture, which was used for subsequent shake-flask experiments.

[0070] Shaking bottle experiment:

[0071] Seed solution: Prepared according to the method described above.

[0072] Shake flask medium: MM30, with glucose and / or sodium acetate added as needed.

[0073] Sample processing in shake flasks: After 48 hours, collect 40 ml of culture medium, centrifuge at 10000 g for 10 min, collect the bacterial cells, add water, shake and wash, centrifuge again to collect the bacterial cells, pre-freeze the bacterial cells at -80℃ for 1 h, and freeze-dry under vacuum for 12 h. Obtain the cell dry weight CDW (g / L).

[0074] PHA content determination: 30-40 mg of lyophilized product was added to 2 mL of chloroform and 2 mL of esterification solution (containing 1 g / L benzoic acid as an internal control and a methanol solution of 3% concentrated sulfuric acid), and reacted at 100℃ for 4 h. After cooling, 1 mL of water was added, and the chloroform phase was taken for GC analysis. Gas chromatography detection was performed using a Shimadzu GC-2014 with an HP-5 column. The gas chromatography program was set to maintain at 80℃ for 1.5 min, then ramp to 140℃ at a rate of 30℃ / min; ramp to 140℃ at a rate of 40℃ / min and maintain for 2 min; the injection port temperature was 240℃; and the detector temperature was 250℃. Using the internal standard method, a standard curve was plotted by comparing the actual mass of the standard with the ratio of the peak area of ​​methyl ester to the peak area of ​​benzoic acid in the PHA monomer standard as measured by gas chromatography. Substituting the measured ratio of the peak area of ​​methyl ester in the sample to the peak area of ​​the internal standard into the standard curve, the actual mass of the PHA monomer could be calculated.

[0075] Calculate PHA content:

[0076] PHA content (wt%) = (PHA mass) ÷ freeze-dried product mass × 100%

[0077] Example 1: Exploring the synthesis of PHB by recombinant bacteria expressing endogenous AMP-type ACS using acetic acid.

[0078] 1. Construction of plasmid pK19

[0079] The backbone of the pK19 plasmid is pSEVA341. The Mmp1 promoter (SEQ ID No. 3) (see Shen R, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, Chen GQ. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis. ACS SynthBiol. 2018 Aug 17;7(8):1897-1906. doi: 10.1021 / acssynbio.8b00102. Epub 2018Jul 31. PMID: 30024739) and the endogenous encoding of AMP-type ACS... acs2 The gene (SEQ ID No. 6) was inserted into the backbone plasmid pSEVA341, thereby enabling... acs2 Under the control of the Mmp1 promoter, the gene was used to obtain the plasmid pK19, namely pSEVA341-Pmmp1-acs2. The correct plasmid construction was verified by PCR.

[0080] 2. Construction of recombinant bacteria TD-blank and TD-acs2

[0081] The empty vector pSEVA341 and plasmid pK19 were transformed into *Halomycosis fungi* via conjugation. Halomonas bluephagenesis In TD1.0, recombinant strains TD-blank and TD-acs2 were obtained, where TD-blank and TD-acs2 are *Haloxylon ammodendron* strains overexpressing the empty vector and pK19, respectively. PCR verification was performed. acs2 The gene was successfully transferred into Halomonas bluephagenesis In TD1.0.

[0082] 3. Shake-flask verification of the efficiency of PHB synthesis from acetic acid and the utilization of acetic acid.

[0083] Recombinant bacteria TD-blank and TD-acs2 were selected and MM30 medium was prepared, which contained 30 g / L NaCl, 10 g / L glucose and 30 g / L sodium acetate. The medium was induced in a shaker with 10 mg / L IPTG and cultured at 37°C for 48 h. The synthesis of PHB and the utilization of acetic acid were then detected.

[0084] Test results: such as Figure 1 As shown, there was no significant difference in the CDW and PHB content between strain TD-acs2 and the control TD-blank, suggesting that the AMP-dependent acetyl-CoA synthase (AMP-ACS) pathway is not suitable for improving the utilization of acetic acid by recombinant Halomonas to produce PHB.

[0085] Example 2: Exploring the expression of endogenous ADP-dependent acetyl-CoA synthase acd Recombinant bacteria using acetic acid synthesize PHB

[0086] 1. Construction of plasmid pAC1

[0087] The backbone of the pAC1 plasmid is pSEVA321. The mmp1 promoter (SEQ ID No. 3) (see Shen R, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, Chen GQ. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis. ACS SynthBiol. 2018 Aug 17;7(8):1897-1906. doi: 10.1021 / acssynbio.8b00102. Epub 2018Jul 31. PMID: 30024739) and the endogenous promoter encoding ADP-dependent acetyl-CoA synthase... acd The gene (SEQ ID No. 2) was inserted into the backbone plasmid pSEVA321, thus... acd Under the control of the inducible mmp1 promoter, the plasmid pAC1, namely pSEVA321-Pmmp1-acd, was obtained. The correct plasmid construction was verified by PCR.

[0088] 2. Construction of plasmid pAC2

[0089] The pAC2 plasmid backbone is pHbPHC. The Pporin59 promoter (SEQ ID No. 4) and the endogenous enzyme encoding ADP-dependent acetyl-CoA synthase are then used. acd The gene (SEQ ID No. 2) was inserted into the backbone plasmid pHbPHC, making... acd Under the control of the constitutive Pporin59 promoter, the gene was used to obtain the plasmid pAC2, namely pHbPHC-Pporin59-acd. The correct plasmid construction was verified by PCR.

[0090] Example 3 Construction of recombinant strains

[0091] (1) Construction of recombinant bacteria TD-AC01 and TD-AC02

[0092] The empty vector pSEVA321 and plasmid pAC1 were transformed into *Halomonas* via conjugation. Halomonas bluephagenesis In TD1.0, recombinant strains TD-AC01 and TD-AC02 were obtained, where TD-AC01 and TD-AC02 are *Haloxylon ammodendron* strains that overexpressed the empty vector and pAC1, respectively. PCR verification was performed. acd The gene was successfully transferred into Halomonas bluephagenesis In TD1.0.

[0093] (2) Construction of recombinant bacteria TD-AC03 and TD-AC04

[0094] The empty vector pHbPHC and plasmid pAC2 were transformed into *Halomonas* via conjugation. Halomonas bluephagenesis In TD1.0, recombinant strains TD-AC03 and TD-AC04 were obtained, where TD-AC03 and TD-AC04 are *Haloxylon ammodendron* strains that overexpressed the empty vector and pAC2, respectively. PCR verification was performed.

[0095] acd The gene was successfully transferred into Halomonas bluephagenesis In TD1.0.

[0096] Example 4: Shake-flask verification of the efficiency of PHB synthesis from acetic acid and the utilization of acetic acid.

[0097] Recombinant bacteria TD-AC01 and TD-AC02 were selected and MM30 medium was prepared, which contained 30 g / L NaCl, 10 g / L glucose and 30 g / L sodium acetate. The medium was incubated in a shaker at 37°C for 48 h, and the synthesis of PHB and the utilization of acetic acid were detected.

[0098] Results: When different concentrations of IPTG (0, 5, 20, and 200 mg / L) were added to strain TD-AC02, compared with the control TD-AC01 (the first "0" point on the x-axis), the contents of CDW and PHB were significantly increased (Table 1), and the consumption of sodium acetate was also significantly increased. Figure 2 ).

[0099] Table 1

[0100]

[0101] Example 5: Shake-flask verification of the efficiency of PHB synthesis from acetic acid and the utilization of acetic acid.

[0102] Recombinant bacteria TD-AC03 and TD-AC04 were selected and MM30 medium was prepared with 30 g / L NaCl and 40 g / L sodium acetate added. The medium was incubated in a shaker at 37°C for 48 h, and the synthesis of PHB was detected.

[0103] Detection results: As shown in Table 2, compared with the control TD-AC03, the recombinant strain TD-AC04CDW and PHB content were significantly increased.

[0104] Table 2

[0105]

[0106] Example 6: Culture medium optimization and synthesis of PHB using acetic acid fed-batch synthesis.

[0107] The TD-AC04 strain was tested in a 7L fermenter. An optimized fermentation protocol was developed, completely eliminating sodium acetate feeding and relying solely on acetic acid from the acid pump, simultaneously integrating pH control and carbon source supply (pH-stat strategy). This strategy not only reduces sodium ion concentration and alleviates osmotic stress but also simplifies feeding operations and reduces process costs. The experiment used recombinant strain TD-AC04, initially adding 10 g / L sodium acetate to the 7L fermenter to provide the initial carbon source. Acetate was used to replace glucose in the synthesis of polyhydroxyalkanoates (PHA). Subsequent carbon source supply relied entirely on 50% (v / v) acetic acid solution (pH adjusted to 8.0) added via the acid pump. Real-time monitoring of cell growth, PHB synthesis, and substrate consumption dynamics verified the synergistic advantages of this strategy in efficient carbon source conversion and environmental adaptability, aiming to provide a theoretical basis for industrial scale-up.

[0108] The results are as follows Figure 3 As shown. During fermentation, cell growth (OD) 600 It shows a trend of first rising rapidly and then fluctuating slightly. Figure 3 (a) OD 8 hours before culture 600 The concentration increased from 1.27 to 5.3, then entered a rapid growth phase, reaching 240 after 44 hours, peaking at 288 after 54 hours, then slightly decreasing, and finally stabilizing at 271 after 62 hours. Cell dry weight (CDW) and PHB content increased synchronously with culture time. Figure 3 (b) After 62 hours, the concentrations of acetic acid and PHB reached 82.50 g / L and 75.13 wt%, respectively, with a PHB yield of 61.98 g / L and a total conversion rate of 25.46% from acetic acid to PHB. This indicates that the pH-stat strategy significantly improved the fermentation efficiency of acetic acid carbon source by optimizing the metabolic environment.

[0109] Those skilled in the art should understand that although the present invention has been specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught in this invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments obtained therefrom are all within the scope of the present invention.

Claims

1. A recombinant Halomonas strain that produces polyhydroxyalkanoates (PHA) by overexpressing an endogenous enzyme encoding ADP-dependent acetyl-CoA synthase. acd Gene, wherein the starting strain of the recombinant halometa is Halomonas bluephagenesis The amino acid sequence of the ADP-dependent acetyl-CoA synthase is shown in SEQ ID No.

1.

2. The recombinant Halomonas according to claim 1, wherein the overexpression is constitutive expression controlled by a constitutive promoter or inducible expression controlled by an inducible promoter, wherein the inducible promoter is selected from Mmp1 promoter, lux promoter, lac promoter, trp promoter, tac promoter or combinations thereof, and the constitutive promoter is selected from the wild-type porin gene Pporin promoter or its mutants, wherein the mutants are selected from Pporin1, Pporin3, Pporin42, Pporin51, Pporin58, Pporin59, Pporin68, Pporin140, Pporin141, Pporin183, Pporin192, Pporin194, Pporin203, Pporin221, Pporin226, Pporin259 and Pporin278.

3. The recombinant halometabolite according to claim 1 or 2, wherein the starting strain of the recombinant halometabolite is... Halomonas bluephagenesis TD1.0, strain preservation number CGMCC No. 4353 Halomonas bluephagenesis TD01 Halomonas bluephagenesis TD27 Halomonas bluephagenesis TDB141 Halomonas bluephagenesis TDB141ΔAC、 Halomonas bluephagenesis WZY254 Halomonas bluephagenesis WZY278 or Halomonas bluephagenesis CYL0307.

4. The recombinant halomonas according to claim 1 or 2, wherein: 1) The above acd The gene sequence is shown in SEQ ID No. 2; 2) The above acd The gene is overexpressed on the plasmid and / or genome in the recombinant Halomonas; 3) Using gene editing methods to... acd The gene was integrated into the genome of the aforementioned Halomonas bacterium; 4) The endogenous plasmid has been knocked out in the recombinant Halomonas; and / or 5) The plasmids used in the overexpression are pSEVA series plasmids or toxin-antitoxin system plasmids.

5. The recombinant halometabolite according to claim 4, wherein the starting strain of the recombinant halometabolite is... Halomonas bluephagenesis TD1.0, and the recombinant Halomonas strain is overexpressed under the control of the Mmp1 promoter or the Pporin59 promoter. acd Gene.

6. The recombinant halomonas according to claim 1 or 2, wherein the PHA comprises at least one of polyhydroxybutyrate, polyhydroxypropionate, polyhydroxyoctanoate, polyhydroxyvalerate, a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid, a terpolymer of 3-hydroxybutyric acid, 4-hydroxybutyric acid, and 3-hydroxyvalerate, a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoate, a binary copolymer of 3-hydroxyoctanoate and 3-hydroxyhexanoate, and a terpolymer of 3-hydroxybutyric acid, 3-hydroxyvalerate, and 3-hydroxyhexanoate.

7. A method for producing polyhydroxyalkanoates, the method comprising fermenting and culturing recombinant halomonas according to any one of claims 1 to 6.

8. The method according to claim 7, wherein the fermentation culture utilizes either i) or ii) as the sole carbon source to synthesize the polyhydroxy fatty acid ester: i) Acetic acid or acetate; ii) A mixture of acetic acid and either acetate with glucose.

9. The method according to claim 7 or 8, wherein during the fermentation process, acetic acid is added as the sole carbon source while maintaining the pH at 7.5 to 8.5.

Images