Bacterial strain for producing low-molecular-weight polyhydroxyalkanoate and application of bacterial strain

Abstract

The invention relates to a recombinant Halomonas (Halomonas) for producing polyhydroxyalkanoate (PHA). The recombinant Halomonas (Halomonas) overexpresses polyhydroxyalkanoate synthetase (PhaC) from Cupriavidus necator (Cupriavidus necator). The recombinant Halomonas (Halomonas) is used for producing the polyhydroxyalkanoate (PHA). The present invention also relates to a method of adjusting the molecular weight of a polyhydroxyalkanoate (PHA), the method comprising adjusting the molecular weight of the PHA product synthesized in the recombinant halomonas by adjusting the level of overexpression of the PhaC. According to the method, PHB with the molecular weight of 200,000 Da or below and P34HB with the molecular weight of 150,000 Da or below can be obtained, so that the application requirements in the field of medicines are particularly met.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and more particularly to a strain for producing low-molecular-weight polyhydroxyalkanoate and its application. Background Art

[0002] Polyhydroxyalkanoates (PHA) are a generic term for intracellular high-molecular-weight polyesters synthesized by microorganisms. PHAs are biodegradable and therefore considered environmentally friendly. PHAs are diverse in type and performance, and have broad application prospects. Their excellent biocompatibility has led to their widespread use in the medical and cosmetic fields.

[0003] The molecular weight of PHA used in the pharmaceutical field must be kept low. Generally, it is below 150,000-200,000 for in vivo stents and below 100,000 for injectable microspheres. Currently, it is difficult to obtain PHA products with such low molecular weights through fermentation. Therefore, to expand its medical applications, it is necessary to process existing PHA products through chemical processing techniques or to modify existing PHA-producing strains through gene editing techniques to produce low-molecular-weight PHA products through fermentation.

[0004] Currently, there are two main methods for preparing low-molecular-weight PHA. The first involves dissolving crude PHA in a solvent such as chloroform and degrading it under heating. After a specified degradation period, the PHA is extracted from the solution using a solvent such as an alcohol. Once the precipitate stabilizes and does not increase, the precipitate and supernatant are separated, washed 3-5 times with purified water, and then dried to obtain a low-molecular-weight PHA product. The second method involves enzymatic hydrolysis of the powder using lipases, proteases, or under alkaline conditions to reduce the molecular weight.

[0005] A large number of organic solvents, such as chloroform, are volatile during use, which not only pollutes the environment but also endangers operators. Using this method to degrade the molecular weight of crude products is complicated and consumes a lot of electricity and manpower. The powder needs to be dried again, which reduces the recovery rate and makes the purification step more expensive. The enzymatic hydrolysis method has requirements for the initial molecular weight of the powder. If the molecular weight is too high, the enzymatic hydrolysis cannot reduce the molecular weight. In addition, the degradation efficiency of lipase and protease is not high in actual experimental operations, and the usage scenarios are limited.

[0006] Therefore, there is still a need to develop a simpler, more convenient, greener and controllable method to obtain low molecular weight PHA. Summary of the Invention

[0007] In response to the above-mentioned problems existing in the prior art, the inventors have used gene editing technology to transform the existing PHA production strains to obtain low-molecular-weight PHA products through fermentation production, which is simpler, more convenient and green. Polyhydroxyalkanoate synthase (PhaC) is a key enzyme in PHA biosynthesis, catalyzing the polymerization of PHA monomers into PHA, and determining the yield, monomer type and composition, and molecular weight of PHA. The core invention of this application is to overexpress the polyhydroxyalkanoate synthase gene phaC derived from Cupriavidus necator (hookworm copper bacteria) H16 strain in Halomonas bluephagenesis, and to regulate the molecular weight of the synthesized PHA by integrating expression on the genome, or / and expressing it using a recombinant plasmid, and expressing it through promoters of different strengths, thereby effectively reducing the molecular weight of PHA.

[0008] Therefore, according to one aspect of the present invention, a recombinant Halomonas for producing polyhydroxyalkanoate (PHA) is provided, which overexpresses polyhydroxyalkanoate synthase (PhaC) from Cupriavidus necator.

[0009] In one embodiment, the overexpression is constitutive expression under the control of a constitutive promoter or inducible expression under the control of an inducible promoter, preferably the inducible promoter is selected from Mmp1 promoter, lux promoter, lac promoter, trp promoter, tac promoter, hypoxia-inducible promoter, bacterial quorum sensing-inducible promoter, temperature-sensitive promoter or a combination thereof, preferably the constitutive promoter is selected from the wild-type porin gene Pporin promoter or a mutant thereof, the wild-type PHA particle binding protein phaP1 gene promoter or an essential gene promoter, wherein the mutant is selected from Pporin1, Pporin3, Pporin42, Pporin51, Pporin58, Pporin68, Pporin140, Pporin141, Pporin183, Pporin192, Pporin194, Pporin203, Pporin221, Pporin226, Pporin259 and Pporin278. These Pporin promoters and mutants thereof are disclosed in, for example, CN117143793B.

[0010] In another embodiment, the overexpression is on a plasmid and / or genome.

[0011] In another embodiment, the PhaC encoding gene is integrated into the genome of the Halomonas by gene editing method (preferably CRISPR / Cas9 gene editing method).

[0012] In another embodiment, the endogenous plasmid has been knocked out in the recombinant Halomonas. Preferably, the knockout of the endogenous plasmid is achieved using CRISPR / Cas9 gene editing.

[0013] In another embodiment, the recombinant Halomonas is a recombinant Halomonas, and / or the expression plasmid is a pSEVA plasmid (preferably pSEVA341, pSEVA321 or pSEVA241), and / or the expression plasmid is a toxin-antitoxin system plasmid, more preferably the toxin-antitoxin system plasmid is pHbPBC.

[0014] In a preferred embodiment, the Halomonas is Halomonas bluephagenesis, Halomonassaydingkolgenesis, Halomonas campaniensis, Halomonas lutescens, Halomonashydrothermalis, Halomonas sp.KM1, Halomonas elongata and Halomonas smyrnensis, and more preferably Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD01 (culture collection number CGMCC No.4353), Halomonas aydingkolgenesis M1 (culture collection number CGMCC No.19880), Halomonas campaniensis LS21 (culture collection number CGMCC No.6593), Halomonasbluephagenesis TD27, Halomonas bluephagenesis TDB141, Halomonas bluephagenesisTDB141ΔAC, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis WZY278 or Halomonas bluephagenesis CYL0307.

[0015] In one embodiment, the PHA comprises at least one of PHB, P4HB, P3HB4HB3HV, PHV, PHBHHX, P34HB, P3HO3HH, PHO, PHP, and PHBVHHX; preferably one or more of PHB, P34HB, PHBHHX, and PHBV.

[0016] In another embodiment, the recombinant Halomonas is Halomonas bluephagenesis, more preferably Halomonas bluephagenesis WZY254; the PhaC is encoded by the nucleotide sequence 1335-3104 of SEQ ID No.1; the overexpression is carried out on the genome and / or plasmid (preferably pSEVA plasmid or pHbPBC), and / or under the control of Pporin promoter (preferably Pporin140 or Pporin141).

[0017] According to another aspect of the present invention, a method for producing polyhydroxyalkanoate (PHA) is provided, which comprises fermenting and culturing the recombinant Halomonas according to the present invention.

[0018] According to another aspect of the present invention, a method for regulating the molecular weight of polyhydroxyalkanoate (PHA) synthesized in recombinant Halomonas is provided, the method comprising: 1) constructing the recombinant Halomonas according to the present invention; and 2) regulating the molecular weight of the PHA product synthesized in the recombinant Halomonas by regulating the level of overexpression of the PhaC (e.g., regulating the promoter strength and / or the copy number of the PhaC encoding gene).

[0019] In a preferred embodiment, the recombinant Halomonas is Halomonas bluephagenesis, more preferably Halomonas bluephagenesis WZY254; the PhaC is encoded by the nucleotide sequence 1335-3104 of SEQ ID No.1 (GenBank Accession WP_013956451.1) (i.e., SEQ ID No.5); the overexpression is carried out on the genome and / or plasmid (preferably pSEVA plasmid or pHbPBC), and / or under the control of the Pporin promoter (preferably Pporin140 or Pporin141).

[0020] In a more preferred embodiment, the recombinant Halomonas is Halomonas bluephagenesis WZY254; the PhaC is encoded by the nucleotide sequence 1335-3104 of SEQ ID No. 1 (GenBank Accession WP_013956451.1) (i.e., SEQ ID No. 5); and the overexpression is carried out under the control of the Pporin140 promoter on the genome (preferably at the G7 site).

[0021] In a more preferred embodiment, the recombinant Halomonas is Halomonas bluephagenesis WZY254; the PhaC is encoded by the nucleotide sequence 1335-3104 of SEQ ID No. 1 (GenBank Accession WP_013956451.1) (i.e., SEQ ID No. 5); the overexpression is carried out under the control of the Pporin140 promoter on the genome (preferably G7 site) and / or on the pHbPBC plasmid under the control of the Pporin140 promoter; and / or the recombinant Halomonas has been knocked out of the endogenous plasmid.

[0022] In a preferred embodiment, the recombinant Halomonas is Halomonas bluephagenesis, more preferably Halomonas bluephagenesis WZY254; the PhaC is encoded by the nucleotide sequence 1335-3104 of SEQ ID No. 1 (GenBank Accession WP_013956451.1 (i.e., SEQ ID No. 5)); the overexpression is carried out on the genome and / or plasmid (preferably pSEVA plasmid or pHbPBC), and / or under the control of promoters of different strengths (preferably Pporin140 or Pporin141); and / or the recombinant Halomonas has been knocked out of the endogenous plasmid.

[0023] In a preferred embodiment, the regulation is to increase the level of overexpression of PhaC, thereby obtaining PHA with a reduced molecular weight, preferably a (weight-average) molecular weight of less than 200,000 Da, more preferably less than 150,000 Da. Preferably, the (weight-average) molecular weight of the homopolymer PHB is reduced to less than 200,000 Da, and the (weight-average) molecular weight of the copolymer P34HB is reduced to less than 150,000 Da. In a particularly preferred embodiment, the molecular weight of the PHA is determined by gel permeation chromatography. Preferably, the molecular weight of the PHA is determined using a Shimadzu LC-20AD gel permeation chromatograph.

[0024] According to another aspect of the present invention, there is provided the use 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, ecological building materials, conductive materials, food or cosmetics.

[0025] The beneficial effects of the technical solution of the present invention include:

[0026] A) Using gene editing technology to modify the Halomonas bluephagenesis WZY254 strain, by expressing exogenous polyhydroxyalkanoate polymerase in the genome or / and recombinant plasmids, and regulating expression through promoters of varying strengths, the molecular weight of PHA was effectively reduced. The molecular weight of the homopolymer PHB was reduced to below 200,000, and the molecular weight of the copolymer P34HB was reduced to below 150,000, meeting the requirements of pharmaceutical applications.

[0027] B) Gene editing avoids the use of large amounts of organic matter and chemical reagents, as well as excessive physical manipulation, and is an efficient, simple, convenient, and environmentally friendly method for reducing PHA molecular weight.

[0028] C) The effects of expressing PhaC on the genome and recombinant plasmids can be superimposed. By combining different expression intensities, PHA products with different molecular weights within a certain range can be obtained. DETAILED DESCRIPTION

[0029] Unless otherwise indicated, the terms used herein have their ordinary technical meanings as understood by those skilled in the art. For definitions and terms in the art, the skilled artisan is particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., 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).

[0030] 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.

[0031] In the present invention, the terms "comprising" or "including" are open-ended expressions, referring to the specific components or steps being described, while not excluding other components or steps that have no substantial impact. When describing a protein or nucleic acid sequence, the sequence may constitute the target molecule in isolation, or may have additional amino acids or nucleotides appended to one or both ends, or undergo protein engineering, while retaining the functional activity described herein.

[0032] The term "and / or" as used herein includes all combinations of the items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B, and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0033] In the present invention, the term "Halophiles" refers to a type of archaea that lives in a high-salinity environment. In a preferred embodiment of the present invention, the halophiles include but are not limited to the genus Halomonas. Preferably, the Halomonas genus includes but is not limited to Halomonas bluephagenesis, Halomonasaydingkolgenesis, Halomonas campaniensis, Halomonas lutescens, Halomonashydrothermalis, Halomonas sp.KM1, Halomonas elongata and Halomonas smyrnensis, and more preferably Halomonas bluephagenesis TD1.0 (described in "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), Halomonasbluephagenesis TD01 (culture collection number CGMCC No.4353), Halomonas aydingkolgenesis M1 (culture collection number CGMCC No.19880), Halomonas campaniensis LS21 (culture deposit number CGMCC No. 6593) (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, YU LP, et al. 2021. Hyperproduction of

[0034] 3-hydroxypropionate by Halomonas bluephagenesis.Nat Commun[J],12:1513.)、Halomonas bluephagenesis TDB141(described in "Yan X,Liu X,Yu L P,etal.2022.Biosynthesis of diverse alpha,omega-diol-derived polyhydroxyalkanoates by engineered Halomonas bluephagenesis.Metab Eng[J],72:275-288.")、Halomonas bluephagenesis TDB141ΔAC(described in "Lizhan Zhang et al.,2022.

[0035] 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 M K,Zheng T R,Wang Z Y,etal.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 Y L,Liu X,Zhang L Z,et al.2025.Cell Sizes Matter for Industrial Bioproduction,a Case of Polyhydroxybutyrate.Advanced Science[J].”).

[0036] In the present invention, the term "expression" may refer to "overexpression", which is defined as a gene expression level higher than the natural state, which may be achieved by an increase in transcriptional level (producing more messenger mRNA) or an increase in translation efficiency (generating more functional proteins). In a specific embodiment, preferably, the expression or overexpression of an exogenous gene can be inserted into the target gene through a genomic non-translational site, or a plasmid is overexpressed. Preferably, the gene is inserted 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). The genome editing sites of Halomonas described in this patent are also described in CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al., Metabolic Engineering, 2018, May: 47: 219-229). The genome editing sites of exemplary Halomonas include G7, G4, G5 and G49 sites. Preferably, the plasmid overexpression is introduced into the chassis strain by electroporation or conjugation transformation. More preferably, the plasmid vector is a pSEVA series vector (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 Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis. Acs Synthetic Biology[J], 13: 61-67), etc.

[0037] In the present invention, the term "toxin-antitoxin system" (i.e., Toxin-Antitoxin, TA system) refers to a system that can maintain the stability of a plasmid by a post-segregation lethal effect (PSK, Postsegregational killing). In a preferred embodiment, the recombinant microorganism of the present invention contains a suitable toxin-antitoxin system / plasmid, so that the plasmid can be naturally (i.e., in the absence of selective pressure such as antibiotics) stably maintained. Eight types of TA systems are known, and the TA system-hok / sok system has been widely used in industrial fermentation to stabilize the plasmid pMJR1750 (Lin et al., EngineeringMicrobiology 3 (2023) 100069). Chinese patent application No. 2023112420281, entitled "A recombinant plasmid expressing toxins and antitoxins, its construction method and application", discloses a TA system derived from a halophilic microorganism and a recombinant plasmid comprising the TA system, and discloses that the recombinant plasmid comprising 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 embodiment, the host cell is a prokaryotic microorganism or a eukaryotic microbial cell, and the toxin-antitoxin is a TA system suitable for prokaryotic microorganisms or eukaryotic microbial cells. In one embodiment, the microorganism is a Gram-negative bacterium or a 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.

[0038] In the present invention, the term "polyhydroxyalkanoate (PHA)" refers to polyhydroxyalkanoate, which can be divided into homopolymers and copolymers according to the 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 whose monomer is C3-C5) or a medium-chain PHA (i.e., a hydroxy fatty acid whose monomer is C6-C18), but is not limited thereto. PHA can be a homopolymer, including but not limited to polyhydroxypropionate (PHP), polyhydroxybutyrate (PHB), polyhydroxyoctanoate (PHO), polyhydroxyvalerate (PHV), etc., for example, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP) or poly-3-hydroxyvalerate (P3HV), etc. PHA can be a copolymer, for example, 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-hydroxyvaleric acid (P(3HB-co-3HV)), a binary copolymer of 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid (P3HO3HH), a ternary copolymer of 3-hydroxybutyric acid, 3-hydroxyvaleric acid and 3-hydroxyhexanoic acid (PHBVHHX), a ternary copolymer of 3-hydroxybutyric acid co-4-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB4HB3HV), etc.

[0039] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0040] The bacteria used in the example is Halomonas bluephagenesis WZY254, which is an outer membrane-defective strain that can produce high amounts of PHB and P3HB4HB in a 7L fermenter, as disclosed in "Ziyu Wang et al., 2022. Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors [J]. Microbial Biotechnology".

[0041] The competent Escherichia coli S17-1 used to construct the plasmid was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.

[0042] For the CRISPR / Cas9 genome editing technology method in the examples, please refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0043] For the genome editing sites described in the examples, refer to the literature CRISPR / Cas9 editing genome of extremophile Halomonas spp (Qin et al, Metabolic Engineering, 2018).

[0044] The promoter sequences described in the examples can be found in the literature Promoter Engineering for Enhanced P (3HB-co-4HB) Production by Halomonas blue phagenesis. (Shen et al, ACS Synthetic Biology, 2018).

[0045] Conjugative transformation method: Electroporate or chemically transform the target plasmid into E. coli S17-1. Grow the donor E. coli S17-1 overnight in LB medium; grow the recipient Halomonas bacteria overnight in LB60 medium. Transfer 2% of the donor and recipient bacteria to 20 ml of culture medium and incubate for 4-6 hours. Remove 1 ml of each suspension by centrifugation, remove the supernatant, resuspend in antibiotic-free LB20, and spread on antibiotic-free LB20 plates. Incubate at 37°C for 6-8 hours. Scrape the bacterial lawn, resuspend, and spread on antibiotic-containing plates. Incubate at 37°C for 24-48 hours. Screen for positive clones for further processing.

[0046] The culture medium formula in the embodiment is:

[0047] LB60 medium: containing 60 g / L NaCl, 10 g / L peptone (purchased from OXIOD, product catalog number LP0042), 5 g / L yeast extract (purchased from OXIOD, product catalog number LP0021), made up to volume with water, and sterilized by high-pressure steam at 121°C.

[0048] MM50 medium (pH 8.0-8.5): 30 g / L glucose, 50 g / L NaCl, 1 g / L yeast extract, 1 g / L urea, 0.2 g / L MgSO₄, 9.65 g / L Na₂HPO₄·12H₂O, 1.5 g / L KH₂PO₄, 10 ml / L trace element solution I, and 1 ml / L trace element solution II. Trace element solution I consists of 5 g / L ammonium ferric citrate and 2 g / L CaCl₂, both prepared in 1 M HCl. Trace element solution II was composed of 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, all prepared in 1 M HCl. The final pH of the culture medium was adjusted to 8.5. These reagents were purchased from Sinopharm Chemical Reagent Company.

[0049] The method for preparing seed solution in the embodiment:

[0050] 1) Bacteria activation

[0051] Take the glycerol tube of bacteria stored in a -80℃ refrigerator, streak it onto an LB60 medium plate, and culture it at 37℃ for 24h.

[0052] 2) First-level seeds

[0053] A single colony was picked from the plate obtained in step 1) and inoculated into 20 mL of liquid LB60 medium, and cultured at 37°C and 200 rpm for 24 h.

[0054] 3) Secondary seeds

[0055] The first-level seed solution obtained in step 2) was inoculated into 20 mL of liquid LB60 medium at a 1% inoculum size, and cultured at 37° C. and 200 rpm with shaking for 8-10 h.

[0056] The method of shake flask culture in the embodiment:

[0057] Prepare a secondary seed solution according to the seed solution preparation method described in the Examples. Inoculate 50 mL of MM50 medium at a 2% inoculum. Add γ-butyrolactone at the desired concentration. Incubate three replicates per experimental group at 37°C, 200 rpm for 48 hours. After the shake flask culture, centrifuge the culture, freeze-dry the cells, weigh them, and calculate the dry weight. The freeze-dried cells were analyzed for PHA content and monomer ratio by GC, and for molecular weight by GPC.

[0058] Freeze-drying method in the embodiment:

[0059] After fermentation, 35 mL of cell culture medium was taken and centrifuged at 8000 g for 15 min. The bacterial precipitate was collected, washed with water, and then freeze-dried (the centrifuge tube containing the bacterial precipitate was first placed at -80°C for 1 h and then placed in a vacuum freeze dryer for 36 h) to obtain a freeze-dried product.

[0060] Method for calculating dry cell weight in the examples:

[0061] Measured as cell dry weight per liter of fermentation system. Cell dry weight is expressed in g / L. Cell dry weight (CDW) = (weight of freeze-dried centrifuge tube - weight of empty centrifuge tube) ÷ 0.035; the weight of the freeze-dried centrifuge tube and the weight of the empty centrifuge tube are both expressed in g. 0.035 represents 0.035 L.

[0062] Method for detecting the content of PHA in the bacteria and the content of each monomer in the embodiment:

[0063] The freeze-dried products were subjected to esterification, and the monomer content was determined by gas chromatography (GC);

[0064] Esterification: Place 60-70 mg of the freeze-dried product in an esterification tube, add 2 mL of chloroform and 2 mL of esterification solution (containing 1 g / L benzoic acid and 3% concentrated sulfuric acid in methanol), mix thoroughly, cover tightly, and esterify in a 100°C metal bath for 4 h. After cooling to room temperature, add 1 mL of distilled water, shake thoroughly, and allow to stand for separation. After the chloroform phase is completely separated from the water, analyze the chloroform phase by gas chromatography.

[0065] 10-100 mg of poly 3-hydroxybutyrate (P3HB) and γ-butyrolactone were taken for esterification reaction as standard samples;

[0066] Gas chromatography (GC) analysis parameters: The analytes were separated using an HP-5 column in a Shimadzu GC-2014 gas chromatograph. The GC analysis temperature program was set as follows: inlet temperature (240°C), detector temperature (250°C), starting temperature and hold time (80°C, 1.5 min), first-stage temperature increase (heating rate 30°C / min), second-stage temperature increase (heating rate 40°C / min, reaching 240°C and holding for 2 min), and a total program time of 8 min.

[0067] The corresponding PHA monomer ratio was calculated by reading the internal standard peak area, the PHA monomer methyl ester peak area of ​​the standard sample, the internal standard peak area of ​​the sample, and the PHA monomer methyl ester peak area of ​​the sample measured by gas chromatography.

[0068] PHA content (wt%) = (mass of 3HB + mass of 4HB) ÷ mass of freeze-dried product × 100%;

[0069] Molar ratio of 3HB (mol%) = number of moles of 3HB ÷ (number of moles of 3HB + number of moles of 4HB) × 100%;

[0070] The calculation method of the molar ratio of 4HB refers to the content of 3HB.

[0071] Method for determining the molecular weight of PHA samples in the examples:

[0072] 1) Standard Sample Preparation: Prepare four clean, numbered 10mL esterified tubes. Sequentially select eight standards (S-6.3 to S-2530) (containing eight molecular weight standards, from highest to lowest), numbered 1#-8#, and assign them to each of the four esterified tubes in pairs, 1#-5#, 2#-6#, 3#-7#, and 4#-8#. Weigh 2mg of each standard, add 2mL of chromatographic-grade chloroform, tighten the cap, and shake well. Allow the standard to stand for 18 hours, then filter through a 0.22µm nylon filter membrane for use as the GPC sample.

[0073] 2) Sample Preparation: Prepare the sample to be tested. Weigh 10 mg of sample into a 10 mL esterification tube. Add 3 mL of chromatographic-grade chloroform, tighten the cap, and shake well. Place the test sample in the esterifier, heat at 100°C for 2 hours, and then allow it to stand for 18 hours. Filter through a 0.22 μm nylon filter membrane before using as the GPC sample.

[0074] 3) Sample Analysis: PHA molecular weight was determined using a Shimadzu LC-20AD gel permeation chromatograph (GPC). The detector was a RID-20A differential refractive index detector. The GPC analysis procedure was as follows: column oven temperature 40°C, mobile phase chloroform, mobile phase flow rate 1 ml / min, and total program time 15 min.

[0075] Example 1

[0076] Genome-wide overexpression of the phaC gene

[0077] 1. Construction of pC01 Plasmid

[0078] The backbone of the pC01 plasmid is pSEVA341 (for the plasmid, see 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). The sequence of the inserted plasmid is SEQ ID No. 1, which is arranged in the following order: sgRNA expression module (nucleotides 1-158), upstream homology arm (nucleotides 159-1158), Pporin140 promoter module (nucleotides 1159-1334), phaC gene (nucleotides 1335-3104, GenBank Accession WP_013956451.1), terminator module (nucleotides 3105-3263), and downstream homology arm (nucleotides 3264-4263).

[0079] 2. Construction of low molecular weight strain 254C1

[0080] The pQ08 plasmid used in the embodiment (plasmid see Qin Q, Ling C, Zhao Y, et al. CRISPR / Cas9 editing genome of extremophile Halomonas spp [J]. Metabolic engineering, 2018, 47: 219-229). Plasmid pQ08 and pC01 plasmid are sequentially transferred into Halomonas blue phagenesis WZY254 by conjugation transformation. PCR and sequencing verification verified that the phaC gene was successfully integrated into the G7 site of the Halomonas blue phagenesis WZY254 genome to obtain 254C1. Then, the strain after successful editing was continuously and repeatedly passaged in liquid culture medium, and streaked on spectinomycin resistance, chloramphenicol resistance and non-resistant plates, respectively, to identify the strain with CRISPR plasmid loss, for the next round of genome editing.

[0081] Production of low molecular weight PHA by strain 3.254C1 in shake flasks

[0082] To investigate whether the recombinant 254C1 strain could produce low-molecular-weight PHA, strains WZY254 and 254C1 were inoculated into 20 mL of LB60 medium, cultured for 12-16 hours, and then transferred to a fresh 20 mL of LB60 medium at a 1% volume ratio. Culture was continued for another 8-12 hours. A 2.5 mL aliquot of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of basal medium for a shake flask experiment. The shaker was maintained at 37°C and 200 rpm. After 48 hours of culture, PHA content and molecular weight were measured. Three replicates were performed for each experiment, and the results were averaged. The results are shown in Table 1.

[0083] The results showed that the dry weight and PHA content of the 254C1 strain remained unchanged compared to those of the WZY254 strain, while the PHA molecular weight decreased from 752,365 kilos to 623,841 kilos, a decrease of 128,524 kilos, or 17.08%. This suggests that overexpressing the phaC gene via a strong promoter can reduce PHA molecular weight to a certain extent.

[0084] Table 1. Production of low molecular weight PHA by strain 254C1

[0085]

[0086] Example 2

[0087] Recombinant plasmid overexpressing phaC gene

[0088] The pHbPBC recombinant plasmid is constructed based on an endogenous plasmid in the Halomonas bluephagenesis strain. It carries the replicon, toxin-antitoxin system, and spectinomycin resistance gene of the endogenous plasmid and can be stably present in an antibiotic-free culture medium. At the same time, the expression of exogenous genes on the plasmid is stronger than that of genomic integration (Ren K, Zhao Y, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-ntitoxin System of Halomonas bluephagenesis [J]. ACS SynthBiol. 2024: 13(1): 61-67). Based on the results of Example 1, the expression of the phaC gene using the pHbPBC recombinant plasmid is expected to increase the expression level of PHA polymerase and more effectively reduce the molecular weight of PHA.

[0089] 1. Knockout of endogenous plasmids in WZY254 strain

[0090] Since the replicon of the pHbPBC plasmid is the same as the endogenous plasmid, the endogenous plasmid in the WZY254 strain must be removed before the plasmid can be transformed. The pLCP-O2-sgE plasmid used to knock out the endogenous plasmid (see Ren K, Zhao Y, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-ntitoxin System of Halomonas bluephagenesis [J]. ACS Synth Biol. 2024: 13 (1): 61-67) expresses the sgRNA and antitoxin of the target endogenous plasmid, which can neutralize the toxicity of the toxin. The pQ08 plasmid and the pLCP-O2-sgE plasmid were sequentially transferred into Halomonas bluephagenesis WZY254 by conjugation transformation. The endogenous plasmid was completely knocked out by PCR to obtain the 254LCP strain. Then, the successfully edited strains were continuously and multiple times passaged in liquid culture medium and streaked on spectinomycin-resistant, chloramphenicol-resistant and non-resistant plates to identify strains that had lost the CRISPR plasmid.

[0091] 2. Construction of low molecular weight production strain 254C2

[0092] The phaC gene was expressed using the pHbPBC plasmid, generating a new recombinant plasmid, pC02. The insert sequence is SEQ ID No. 2, comprising the Pporin140 promoter module (nucleotides 1-176) and the phaC gene (nucleotides 177-1946, GenBank Accession WP_013956451.1). The pC02 plasmid was transformed into the Halomonas sp. 254LCP strain via conjugation to generate the 254C2 strain.

[0093] Production of low molecular weight PHA by strain 3.254C2 in shake flasks

[0094] Strain 254C2 was inoculated into 20 mL of LB60 medium supplemented with spectinomycin. After culturing for 12-16 hours, the culture was transferred to a fresh 20 mL of LB60 medium supplemented with spectinomycin at a 1% volume ratio and cultured for another 8-12 hours. A 2.5 mL aliquot of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 47.5 mL of basal medium for a shake flask experiment. The shaker was maintained at 37°C and 200 rpm. After 48 hours of culture, PHA content and molecular weight were measured. Three replicates were performed for each experiment, and the results were averaged. Strain WZY254 was used as a control. The results are shown in Table 2.

[0095] The results showed that the dry weight and PHA content of the 254C2 strain remained unchanged compared to those of the WZY254 strain. However, the PHA molecular weight decreased from 756,598 kilos to 464,524 kilos, a decrease of 292,074 kilos, or 38.60%. This suggests that enhancing the expression of the phaC gene using a recombinant plasmid can further reduce the molecular weight of PHA.

[0096] Table 2. Production of low molecular weight PHA by strain 254C2

[0097]

[0098] Example 3

[0099] Simultaneous expression of phaC gene by genome and recombinant plasmid

[0100] The endogenous plasmid in the 254C1 strain was knocked out, and the recombinant plasmid pC02 was transformed into the strain to obtain a new recombinant strain 254C3. The 254C3 strain was inoculated into 20 ml of LB60 medium supplemented with spectinomycin. After culturing for 12-16 hours, it was transferred to a new 20 mL of LB60 medium supplemented with spectinomycin at a volume ratio of 1% and continued to be cultured for 8-12 hours. Take 2.5 mL of seed bacterial liquid and inoculate it into a 500 mL conical flask containing 47.5 mL of basal medium for a shake flask experiment. The shaker temperature was 37 ° C and the speed was 200 rpm. After 48 hours of culture, the PHA content and molecular weight were detected. Three parallel experiments were set for each group of experiments, and the results were averaged. The WZY254 strain was used as a control. The results are shown in Table 3.

[0101] The results showed that the dry weight and PHA content of the 254C3 strain remained unchanged compared to those of the WZY254 strain, while the PHA molecular weight decreased from 748,412 kilos to 335,471 kilos, a decrease of 412,941 kilos, or 55.18%. This suggests that the effects of genomic integration and recombinant plasmid expression of the phaC gene on reducing PHA molecular weight are additive.

[0102] Table 3. Production of low molecular weight PHA by strain 254C3

[0103]

[0104] Example 4

[0105] phaC gene expression regulated by the promoter

[0106] To further enhance phaC gene expression, the more potent Pporin141 promoter was proposed for expression. A pC03 plasmid was constructed for genomic integration of Pporin141-phaC, using the pSEVA341 backbone. The sequence inserted into the plasmid is SEQ ID No. 3, consisting of the sgRNA expression module (nucleotides 1-158), upstream homology arm (nucleotides 159-1158), Pporin141 promoter module (nucleotides 1159-1334), phaC gene (nucleotides 1335-3104, GenBank Accession WP_013956451.1), terminator module (nucleotides 3105-3263), and downstream homology arm (nucleotides 3264-4263). A pC04 plasmid was constructed for recombinant expression of Pporin141-phaC, using the pHbPBC backbone. The sequence inserted into the plasmid is SEQ ID No. 4, which is arranged in the order of Pporin141 promoter module (nucleotides 1-176) and phaC gene (nucleotides 177-1946, GenBank Accession WP_013956451.1).

[0107] The WZY254 strain was genome-edited using pQ08 and pC03 plasmids to integrate and express Pporin141-phaC at the G7 locus of the genome. The endogenous plasmid was knocked out and the strain was transformed with the pC02 recombinant plasmid to express Pporin140-phaC, resulting in the 254C4 strain. The endogenous plasmid in the 254C1 strain was knocked out and the strain was transformed with the recombinant plasmid pC04 to express Pporin141-phaC, resulting in the new recombinant strain 254C5. The WZY254 strain was genome-edited using pQ08 and pC03 plasmids to integrate and express Pporin141-phaC at the G7 locus of the genome. The endogenous plasmid was knocked out and the strain was transformed with the pC04 recombinant plasmid to express Pporin141-phaC, resulting in the 254C6 strain.

[0108] Strains 254C4, 254C5, and 254C6 were inoculated into 20 mL of LB60 medium supplemented with spectinomycin. After 12-16 hours of incubation, the cultures were transferred to fresh 20 mL of LB60 medium supplemented with spectinomycin at a 1% volume ratio and incubated for another 8-12 hours. A 2.5 mL aliquot of the seed culture was inoculated into 47.5 mL of basal medium in a 500 mL conical flask for a shake flask experiment. The shaker was maintained at 37°C and 200 rpm. After 48 hours of incubation, PHA content and molecular weight were measured. Three replicates were performed for each experiment, and the results were averaged. Strain WZY254 was used as a control. The results are shown in Table 4.

[0109] The results showed that the dry weight and PHA content of strain 254C4 remained unchanged compared to those of strain WZY254, while the PHA molecular weight decreased by 478,180 kilos, or 62.81%, from 761,344 kilos to 283,164 kilos. The dry weight and PHA content of strain 254C5 remained unchanged compared to those of strain WZY254, while the PHA molecular weight decreased by 515,893 kilos, or 67.76%, from 761,344 kilos to 245,451 kilos. The PHA content of strain 254C6 remained unchanged compared to WZY254, but the dry weight decreased slightly, while the PHA molecular weight decreased by 572,892 kilos, or 75.25%, from 761,344 kilos to 188,452 kilos. This suggests that expressing the phaC gene using a stronger promoter can effectively reduce PHA molecular weight without affecting cell dry weight or PHA content.

[0110] Table 4. Low molecular weight production strains from 254C4, 254C5, and 254C6 strains

[0111]

[0112] Example 5

[0113] Production of P34HB by low molecular weight strains

[0114] To investigate the effect of phaC gene overexpression on the molecular weight of the P34HB copolymer, 5 g / L butyrolactone was added to the shake flask culture medium as a precursor to generate 4HB. Strain 254C6 was inoculated into 20 mL of LB60 medium supplemented with spectinomycin and cultured for 12-16 hours. After culturing, the cells were transferred to 20 mL of fresh LB60 medium supplemented with spectinomycin at a 1% volume ratio and cultured for another 8-12 hours. 2.5 mL of the seed culture was inoculated into 500 mL Erlenmeyer flasks containing 47.5 mL of basal medium and supplemented with 5 g / L butyrolactone for shake flask experiments. The shaker was maintained at 37°C and 200 rpm. After 48 hours of culture, PHA content, 4HB molar ratio, and molecular weight were measured. Three replicates were performed for each experiment, and the results were averaged. Strain WZY254 was used as a control. The results are shown in Table 5.

[0115] Results showed that the dry weight and PHA content of the 254C6 strain were significantly lower than those of the WZY254 strain. Overexpression of PhaC had a modest impact on bacterial growth when supplemented with butyrolactone. The 4HB molar ratio of P34HB produced by the 254C6 strain reached 20.42%, and the molecular weight was reduced to 142,658 kilos. This suggests that overexpression of PhaC significantly reduces the molecular weight of the copolymer PHA.

[0116] Table 5. Production of low molecular weight P34 by strain 254C6

[0117]

[0118] The above examples illustrate that the method of regulating the molecular weight of polyhydroxyalkanoate (PHA) synthesized in recombinant Halomonas sp. of the present invention is applicable not only to the production of PHB, but also to the production of other PHAs such as P34HB.

[0119] Those skilled in the art will appreciate 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 by the present 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 within the scope of the present invention.

Claims

1. A recombinant Halomonas bacterium for producing polyhydroxyalkanoate (PHA), which overexpresses polyhydroxyalkanoate synthase (PhaC) from Cupriavidus necator.

2. The recombinant salt mononas according to claim 1, wherein the overexpression is constitutive expression under the control of a constitutive promoter or inducible expression under the control of an inducible promoter, preferably the inducible promoter is selected from Mmp1 promoter, lux promoter, lac promoter, trp promoter, tac promoter, hypoxia-inducible promoter, bacterial quorum sensing-inducible promoter, temperature-sensitive promoter or a combination thereof, preferably the constitutive promoter is selected from wild-type porin gene Pporin promoter or a mutant thereof, wild-type PHA particle binding protein phaP1 gene promoter or an essential gene promoter, wherein the mutant is selected from Pporin1, Pporin3, Pporin42, Pporin51, Pporin58, Pporin68, Pporin140, Pporin141, Pporin183, Pporin192, Pporin194, Pporin203, Pporin221, Pporin226, Pporin259 and Pporin278.

3. The recombinant Halomonas according to claim 1 or 2, wherein: 1) The overexpression is on a plasmid and / or genome; 2) integrating the PhaC encoding gene into the genome of the Halomonas by a gene editing method (preferably CRISPR / Cas9 gene editing method); 3) The endogenous plasmid has been knocked out in the recombinant Halomonas; and / or 4) The plasmid used in the overexpression is a pSEVA plasmid (preferably pSEVA341, pSEVA321 or pSEVA241), and / or the plasmid is a toxin-antitoxin system plasmid, more preferably the toxin-antitoxin system plasmid is pHbPBC.

4. The recombinant Halomonas according to any one of claims 1 to 3, wherein the Halomonas is Halomonas bluephagenesis, Halomonas aydingkolgenesis, Halomonas campaniensis, Halomonas lutescens, Halomonas hydrothermalis, Halomonas sp.KM1, Halomonas elongata, and Halomonas smyrnensis, and more preferably Halomonas bluephagenesis TD1.0, Halomonas bluephagenesis TD01 (Culture Collection Number CGMCC No. 4353), Halomonas aydingkolgenesis M1 (Culture Collection Number CGMCC No. 19880), Halomonas campaniensis LS21 (Culture Collection Number CGMCC No. 6593), Halomonas bluephagenesis TD27, Halomonas bluephagenesis TDB141, Halomonas bluephagenesis TDB141ΔAC, Halomonas bluephagenesis WZY254, Halomonas bluephagenesis WZY278 or Halomonas bluephagenesis CYL0307.

5. The recombinant Halomonas according to any one of claims 1 to 4, wherein the PHA comprises at least one of PHB, P4HB, P3HB4HB3HV, PHV, PHBHHX, P34HB, P3HO3HH, PHO, PHP, and PHBVHHX; preferably one or more of PHB, P34HB, PHBHHX, and PHBV.

6. The recombinant Halomonas according to any one of claims 1 to 5, wherein the recombinant Halomonas is Halomonas bluephagenesis, more preferably Halomonas bluephagenesis WZY254; the PhaC is encoded by the nucleotide sequence 1335-3104 of SEQ ID No. 1 (i.e., SEQ ID No. 5); and the overexpression is carried out on the genome and / or plasmid (preferably pSEVA plasmid or pHbPBC), and / or under the control of the Pporin promoter (preferably Pporin140 or Pporin141). 7 . A method for producing polyhydroxyalkanoate (PHA), comprising fermenting and culturing the recombinant Halomonas according to claim 1 .

8. A method for regulating the molecular weight of a polyhydroxyalkanoate (PHA) synthesized in recombinant Halomonas sp., the method comprising: 1) constructing the recombinant Halomonas according to any one of claims 1 to 6; and 2) regulating the molecular weight of the PHA product synthesized in the recombinant Halomonas by regulating the level of overexpression of the PhaC (eg, regulating the promoter strength and / or the copy number of the PhaC encoding gene).

9. The method according to claim 8, wherein the regulation is to increase the level of overexpression of the PhaC, thereby obtaining PHA with a reduced molecular weight, preferably a molecular weight below 200,000 Da, more preferably below 150,000 Da.

10. Use of the PHA obtained according to the method of claim 7 or 8 in the preparation of packaging materials, biomedical materials, drug carriers, biodegradable agricultural films, slow-release fertilizers, ecological building materials, conductive materials, food or cosmetics.

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