A novel PHA synthase (PhaC) for biosynthesis of epsilon-poly caprolactone (PCL)

Abstract

The application discloses a novel polyhydroxyalkanoate (PHA) polymerase (PhaC) which can efficiently polymerize the activated monomer 6-hydroxyhexanoate-CoA (6HHx-CoA) of 6-hydroxyhexanoate (6HHx). The PhaC is co-expressed with a CoA ligase AlkK, and can synthesize poly-epsilon-caprolactone (PCL) from the direct precursor sodium 6-hydroxyhexanoate in a halomonas sp. Halomonas which has deleted endogenous beta-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), PhaC and enoyl-CoA hydratase (FadB).

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology, microbial engineering, and biopolymer materials, specifically relating to a novel PHA synthase PhaC constructed through chimeric and semi-rational design. au And a method for biosynthesizing poly(ε-caprolactone) PCL in Halomonas using this enzyme. Background Technology

[0002] Poly(ε-caprolactone) (PCL) has attracted much attention due to its excellent low-temperature mechanical properties, controllable degradation behavior, and good biocompatibility (Mohamed and Yusoh 2016). Its glass transition temperature is approximately -60°C, melting point is 56–64°C, and it exhibits good thermoplastic processing properties, making it suitable for various processes such as melt spinning and 3D printing. The long methylene units in its molecular chain contribute to its high flexibility, with an elongation at break reaching 800%, but its tensile strength is relatively low (Mohamed and Yusoh 2016). PCL degrades gradually under natural conditions through hydrolysis or enzymatic degradation over periods ranging from several months to several years, making it suitable for applications requiring long-term structural integrity. Its degradation product, ε-hydroxyhexanoic acid, can be metabolized by the body, exhibits no cytotoxicity, and meets the safety standards for medical materials (Archer, Torretti and Madbouly 2020, Malikmammadov et al. 2018, Behl et al. 2020). In biomedical engineering, PCL is an ideal material for tissue engineering scaffolds, promoting cell adhesion and proliferation. Through 3D printing, surface modification, or compounding with natural polymers, it can be customized to match complex anatomical structures and enhance repair efficacy (Shah et al. 2025, Kirmanidou et al. 2024, Liang et al. 2024). In drug delivery, PCL can be fabricated into microspheres or nanoparticles for sustained release over weeks to months, suitable for long-acting therapies. In environmentally friendly materials, PCL combines biodegradability with thermoplastic processability, making it suitable for biodegradable packaging, agricultural films, etc. Blending with PLA or natural fibers can regulate mechanical and degradation behavior, promoting the development of sustainable materials (Ngwenya et al. 2025).

[0003] 嗜盐蓝藻菌This halophilic bacterium not only tolerates high temperatures and highly alkaline environments but also naturally possesses the ability to synthesize polyhydroxy fatty acids (primarily poly-3-hydroxybutyric acid, PHB), making it a leading chassis strain for next-generation industrial biotechnology (Tan et al. 2011). This bacterium can achieve open, continuous fermentation, offering irreplaceable advantages in energy conservation and cost reduction, highlighting its unique technological competitiveness and market application prospects. Furthermore, the genome annotation system and gene editing technology related to halophilic bacteria are becoming increasingly mature, giving this strain the potential to develop diverse target products (Qin et al. 2018, Zhao et al. 2017, Ma et al. 2020, Wang et al. 2022).

[0004] 嗜盐蓝藻菌 Natural synthesis of PHB depends on endogenous β-ketothiolysis enzyme (PhaA), acetyl-CoA reductase (PhaB), and polyhydroxy fatty acid synthase (PhaC), but no studies on PCL biosynthesis have been published for this strain (Yan et al. 2025). Meanwhile, due to the extremely strong polymerization activity of its endogenous PHA synthase for 3-hydroxybutyrate (3HB) monomer, current research utilizes… 嗜盐蓝藻菌 Synthesized polyhydroxyalkanoates (PHAs) almost always contain a high proportion of 3HB monomers, making it difficult to directly obtain homopolymer PCL or other non-natural PHA copolymers. Given the strict specificity and catalytic activity of enzymes towards their substrates, screening or constructing enzymes capable of... 嗜盐蓝藻菌 The specific recognition of 6-hydroxyhexanoic acid monomers by coenzyme A ligase (CoA ligase) and the polymerization of 6-hydroxyhexanoyl-CoA by PHA synthase are the primary steps in the biosynthesis of PCL in this bacterium. Furthermore, due to… 嗜盐蓝藻菌 Bacteria naturally possess a strong ability to synthesize PHB, and all known PHA synthases can recognize and polymerize 3HB-CoA. Therefore, it is necessary to inhibit the endogenous bacterial metabolic pathway from glucose to acetyl-CoA via glycolysis, which then synthesizes PHB. Even if this pathway is blocked, other sources of 3HB-CoA may still exist. Similar to the production of polyδ-valerolactone, to further increase the proportion of 6HHx monomer in the polymer, it is also necessary to knock out genes related to the 3HB-CoA pathway from sources other than glycolysis in bacteria (Wang et al. 2025).

[0005] Traditionally, PCL is prepared via metal-catalyzed ring-opening polymerization (ROP) of ε-caprolactone, typically carried out at 100–180°C under strictly anhydrous and oxygen-free conditions, using metal catalysts such as stannous octoate (Mohamed and Yusoh 2016). While this method allows for molecular weight control, it suffers from biosafety risks due to metal residues, a broadened molecular weight distribution due to transesterification side reactions, and dependence on petroleum-based feedstocks. Enzyme-catalyzed ring-opening polymerization (eROP), as a green alternative, can be carried out at 40–80°C under metal-free conditions, but it usually relies on non-aqueous organic media such as toluene, has a slow reaction rate, is sensitive to conditions, and produces products with relatively low molecular weights (approximately 10). 4 Immobilized enzymes are limited by their high cost and easy activity decay (on the order of g / mol), which restricts their large-scale sustainable application (Zhang et al. 2018, Zhao 2018, Veneral et al. 2018).

[0006] In contrast, microbial synthesis of PCL is still in its early stages: natural microorganisms lack endogenous pathways to synthesize ε-caprolactone or its activated CoA form, and the catalytic efficiency of natural PHA synthases for medium-chain ω-hydroxy monomers (such as 6HHx-CoA) is far lower than their activity for 3HB-CoA, making it difficult to obtain high-purity PCL homopolymers. Current research mainly focuses on PHA copolymers containing 6HHx units (such as P3HB-...). co PCL can be synthesized using chimeric enzymes or consensus design, but the proportion of 6HHx incorporation is low and it is highly dependent on precursors. Even if PCL biosynthesis is achieved through chimeric enzymes or consensus design, the yield is still extremely low (about 25 mg / L PCL, with a monomer conversion rate of only about 0.6%), and there is still a significant metabolic bottleneck between precursor activation and polymerization steps (Yanagawa et al. 2026).

[0007] Therefore, there is an urgent need to construct a novel PHA synthase to... 嗜盐蓝藻菌 To achieve efficient biosynthesis of PCL.

[0008] References Archer, E., Torretti, M., and Madbouly, S. (2023). Biodegradable polycaprolactone (PCL)-based polymers and composites. Physical Sciences Reviews 8, 4391–4414. Behl, A., Parmar, V.S., Malhotra, S., and Chhillar, A.K. (2020).Biodegradable diblock copolymeric PEG-PCL nanoparticles: Synthesis,characterization and applications as anticancer drug delivery agents. Polymer207, 122901. Hozumi, Y., Hachisuka, S.I., Tomita, H., Kikukawa, H., and Matsumoto,K. (2024). Engineering of the long-main-chain monomer-incorporatingpolyhydroxyalkanoate synthase PhaCAR for the biosynthesis of poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate]. Biomacromolecules 25, 2973–2979. Jiang, X.R., Yan, X., Yu, L.P., Liu, X.Y., and Chen, G.Q. (2021).Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis. NatureCommunications 12, 1513. Kirmanidou, Y., Chatzinikolaidou, M., Michalakis, K., and Tsouknidas,A. (2024). Clinical translation of polycaprolactone-based tissue engineeringscaffolds fabricated via additive manufacturing: A review of theircraniofacial applications. Biomaterials Advances 162, 213902. Liang, H.Y., Lee, W.K., Hsu, J.T., Shih, J.Y., Ma, T.L., Vo, T.T.T.,Lee, C.W., Cheng, M.T., and Lee, I.T. (2024). Polycaprolactone in bone tissueengineering: A comprehensive review of innovations in scaffold fabricationand surface modifications. Journal of Functional Biomaterials 15, 213. Ma, H., Zhao, Y., Huang, W., Zhang, L., Wu, F., Ye, J., and Chen,G.Q. (2020). Rational flux-tuning of Halomonas bluephagenesis for co-production of bioplastic PHB and ectoine. Nature Communications 11, 3313. Malikmammadov, E., Tanir, T.E., Kiziltay, A., Hasirci, V., andHasirci, N. (2018). PCL and PCL-based materials in biomedical applications.Journal of Biomaterials Science, Polymer Edition 29, 863–893. Mohamed, R.M., and Yusoh, K. (2016). A review on the recent researchof polycaprolactone (PCL). Advanced Materials Research 1134, 249–255. Ngwenya, M., Gumede, T.P., Pérez Camargo, R.A., and Motloung, B.(2025). Nanocellulose-reinforced poly(lactic acid) and poly(ε-caprolactone)bio-nanocomposites: A review and future outlook for poly(lactic acid) / poly(ε-caprolactone) blend systems. Materials18, 5172. Qin, Q., Ling, C., Zhao, Y., Yang, T., Yin, J., Guo, Y., and Chen,G.Q. (2018). CRISPR / Cas9 editing genome of extremophile Halomonas spp.Metabolic Engineering 47, 219–229. Shah, S.R., Modi, C.D., Singh, S., Mori, D.D., Soniwala, M.M., andPrajapati, B.G. (2025). Recent advances in additive manufacturing ofpolycaprolactone-based scaffolds for tissue engineering applications: Acomprehensive review. Regenerative Engineering and Translational Medicine 11,112–131. Tan, D., Xue, Y.S., Aibaidula, G., and Chen, G.Q. (2011). Unsterileand continuous production of polyhydroxybutyrate by Halomonas TD01.Bioresource Technology 102, 8130–8136. Veneral, J.G., de Oliveira, D., Ferreira, S.R.S., and Oliveira, J.V.(2018). Continuous enzymatic synthesis of polycaprolactone in packed bedreactor using pressurized fluids. Chemical Engineering Science 175, 139–147. Wang, J.L., Yan, X., Zheng, S., Wen, R., Chen, Y.L., Yang, W.N.,Sheng, J.T., Wu, Q., Wu, F.Q., and Chen, G.Q. (2025). Biosynthesis of poly(δ-valerolactone) (PVL) by Halomonas bluephagenesis. Chemical EngineeringJournal 519, 163311. Wang, L.J., Jiang, X.R., Hou, J., Wang, C.H., and Chen, G.Q. (2022).Engineering Halomonas bluephagenesis via small regulatory RNAs. MetabolicEngineering 73, 58–69. Yan, X., Wang, J.L., Wen, R., Chen, X.Y., and Chen, G.Q. (2025). Thehalo of future bio-industry based on engineering Halomonas. MetabolicEngineering90, 16–32. Yanagawa, K., Hachisuka, S.I., Kusumoto, H., Yamamoto, K., Furukawa, S., Sasaki, M., Iseki, K., Nakagawa, N., Nakano, S., Kikukawa, H., and Matsumoto, K. (2026). Biosynthesis of poly(6-hydroxyhexanoate) [poly(ε-caprolactone)] using engineered polyhydroxyalkanoate synthetic system in Escherichia coli. Journal of Bioscience and Bioengineering. Zhang, Y.Y., Lu, P.Y., Sun, Q.H., Li, T., Zhao, L.J., Gao, X., Wang, F.Y., and Liu, J.H. (2018). Lipase-mediated direct in situ ring-opening polymerization of epsilon-caprolactone formed by a chemo-enzymatic method. Journal of Biotechnology 281, 74–80. Zhao, H. (2018). Enzymatic ring-opening polymerization (ROP) of polylactones: Roles of non-aqueous solvents. Journal of Chemical Technology and Biotechnology 93, 9–19. Zhao, H., Zhang, H.M., Chen, X., Li, T., Wu, Q., Ouyang, Q., and Chen, G.Q. (2017). Novel T7-like expression systems used for Halomonas. Metabolic Engineering 39, 128–140. Summary of the Invention

[0009] To address the problems of low biosynthesis efficiency, poor product purity, and low molecular weight of PCL in existing technologies, the inventors have achieved, for the first time, the efficient biosynthesis of high-purity PCL using 6-hydroxyhexanoic acid (6HHx) as a precursor in halophilic bacteria through systematic screening and engineering of coenzyme A ligase and PhaC, combined with host metabolic pathway remodeling, plasmid stability optimization, and fermentation process regulation. The resulting PCL material exhibits significantly better performance than chemically synthesized PCL, which has profound implications for the industrial production of biodegradable polyesters using biological methods.

[0010] Therefore, the present invention provides an engineered PHA synthase comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion is derived from Aeromonas vaginalis (…). 豚鼠气单胞菌 PHA synthase mutant PhaC ac (N149D) and contains 1, 2, 3, 4 or 5 (preferably 4) α-helical domains, the C-terminal portion of which originates from uncultured bacteria from red forest swamps ( 未培养细菌 PHA synthase PhaC un PHA synthase does not maintain the exact same sequence in all strains, but its α / β hydrolase folding structure and core functional roles (especially the catalytic triplet residues in the active site) are highly conserved.

[0011] In one specific embodiment, the PhaC ac The amino acid sequence of (N149D) is shown in SEQ ID NO: 4 and the PhaC un The amino acid sequence is shown in SEQ ID NO: 6.

[0012] In one specific embodiment, the engineered PHA synthase is made using PhaC. ac The segment from the N-terminus of (N149D) to the end of its 1st, 2nd, 3rd, 4th, or 5th (preferably 4th) α-helical domain (i.e., containing a segment from the N-terminus to the end of the 1st, 2nd, 3rd, 4th, or 5th (preferably 4th) α-helical domain) replaces PhaC. un The corresponding part (i.e., replacing PhaC) un It is obtained by starting from the N-terminus and ending at the 1st, 2nd, 3rd, 4th or 5th (preferably the 4th) α-helical domain.

[0013] In one specific embodiment, the coding nucleotide sequence of the engineered PHA synthase is shown in SEQ ID NO: 7, 8, 9, 10 or 11, and most preferably it is shown in SEQ ID NO: 10.

[0014] In one specific embodiment, the amino acid sequence of the engineered PHA synthase is shown in SEQ ID NO: 13, and optionally includes mutations or combinations of mutations selected from the following: D394A; L395Y; N419A; D394A+L395Y; D394A+N419A; L395Y+N419A; and D394A+L395Y+N419A, preferably including L395Y+N419A.

[0015] Another aspect of the present invention provides a gene encoding an engineered PHA synthase according to the present invention or a recombinant vector containing the gene.

[0016] Another aspect of the present invention provides a recombinant bacterium containing and / or expressing the gene according to the present invention or containing a recombinant vector, preferably, the recombinant bacterium is selected from halophilic bacteria, more preferably from the genus *Halomonas*. 嗜盐菌属 ), Pseudomonas spp. 假单胞菌属 ), Escherichia coli ( 埃希氏菌属 ), Roche's et al. 真养产碱菌 Aeromonas spp. 气单胞菌属 ), Bacillus spp. 芽孢杆菌属 ), Alcaligenes megaterium ( 产碱菌属 侧耳属 ), Alkali-producing bacteria ( 嗜碱产碱菌 ) or combinations thereof, more preferably halomonas ( 嗜盐菌属 ), and even better as 嗜盐蓝藻菌 , 艾丁湖嗜盐菌、 坎帕尼亚嗜盐菌 or 微黄嗜盐菌 The best option 嗜盐蓝藻菌 TD01 (CGMCC No. 4353) 嗜盐蓝藻菌 TD1.0 嗜盐菌属 艾丁湖嗜盐菌 M1 (CGMCC No. 19880) or 坎帕尼亚嗜盐菌 LS21 (CGMCC No. 6593).

[0017] In one specific embodiment, the recombinant bacteria have the following endogenous gene deleted or knocked out in their genome: β-ketothiolate gene. phaA acetyl-CoA reductase gene phaB Polyhydroxy fatty acid polymerase gene phaC and / or esteracyl-CoA hydratase gene fadB Preferred 嗜盐蓝藻菌 (Δ phaCAB ), 嗜盐菌属 蓝藻菌 (Δ phaCAB , Δ fadB )or嗜盐蓝藻菌 (Δ phaCAB , Δ fadB , ΔEnP).

[0018] In one specific embodiment, the recombinant bacteria are further introduced with a gene encoding the CoA ligase AlkK, preferably the gene encoding the CoA ligase AlkK originating from *Pseudomonas oleophila* (…). 食油假单胞菌 (More preferably, its amino acid sequence is shown in SEQ ID NO: 15, and most preferably, its encoding nucleotide sequence is shown in SEQ ID NO: 14.)

[0019] In one specific embodiment, the gene for the engineered PHA synthase and the gene encoding the CoA ligase AlkK are each independently expressed on the genome of the recombinant bacteria or on one or more plasmids, and are expressed under the control of a constitutive promoter or an inducible promoter.

[0020] In one specific embodiment, the plasmid is a pSEVA series plasmid, and / or the plasmid contains... hbpB / hbpC The plasmid of the toxin-antitoxin operon (SEQ ID NO: 30), preferably pHbPBC. The pSEVA series plasmids include pSEVA341, pSEVA241, pSEVA321, etc. (see, for example, Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpCToxin-Antitoxin System of Halomonas bluephagenesis). ACS合成生物学 . 2023, 13(1): 61-67.).

[0021] In one specific embodiment, the inducible promoter is selected from the 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 P porin Promoter or mutant thereof, wherein the mutant is selected from 孔蛋白 3 , 孔蛋白 42 , 孔蛋白 58 , 孔蛋白 68 , 孔蛋白 140 , 孔蛋白 141 ,孔蛋白 194 , 孔蛋白 211 , 孔蛋白 221 , 孔蛋白 226 , 孔蛋白 278 Or combinations thereof. These P porin The promoter and its mutants are disclosed, for example, in CN117143793B.

[0022] In one specific embodiment, the recombinant bacteria is recombinant halomonas, and the endogenous plasmid has been knocked out.

[0023] Another aspect of the present invention provides a method for producing poly-ε-caprolactone (PCL), comprising fermenting a recombinant strain according to the present invention, and obtaining poly-ε-caprolactone from the fermentation product.

[0024] In one specific embodiment, the fermentation culture medium is a mineral-based medium or Luria-Bertani (LB) medium, and / or includes the substrate sodium 6-hydroxyhexanoate (6HHx-Na).

[0025] The beneficial effects of this invention include: (1) PhaC through chimeric design ac The four α-helical domains at the N-terminus of (N149D) are related to PhaC. un The C-terminal catalytic domain was fused to construct PhaC au The chimeric enzyme, combined with the L395Y and N419A double-site mutations, significantly improved the enzyme's catalytic efficiency and substrate selectivity for 6HHx-CoA through the synergistic effect of the double mutations. This increased the molar percentage of 6HHx in the polymer from 3% in the existing technology to 44% (shake flask fermentation), laying the enzyme engineering foundation for the subsequent production of high-purity PCL. (2) By simultaneously knocking out the halophilic bacteria chassis phaA , phaB , phaC and fadB Four endogenous genes block the PHB synthesis pathway (glycolysis → acetyl-CoA → 3HB-CoA) and the 3HB-CoA production pathway (fatty acid β-oxidation) from the source, making 6HHx-CoA absolutely dominant in the intracellular CoA activated monomer pool, effectively eliminating the competitive incorporation of 3HB monomers, and further increasing the 6HHx molar percentage from 44% to 90%. (3) By adopting a portable hbpB / hbpCThe low-copy stable plasmid pHbPBC of the toxin-antitoxin system, along with the knockout of the host endogenous plasmid, utilizes the high stability of the toxin within the cell to exert selective pressure on plasmid-deficient cells, effectively maintaining the genetic stability of the plasmid during long-term fermentation. This increases cell dry weight from approximately 5 g / L to 11 g / L and PHA content from approximately 30% to 52%, solving the problem of low yield caused by plasmid loss. (4) By adopting LB60 nutrient-rich medium, controlling the glucose concentration at 5 g / L, uniformly adding 6HHx-Na (2 g / h) and maintaining the glucose concentration above 10 g / L, the fermentation process was designed to promote cell growth and metabolism, limit the flux of endogenous 3HB-CoA generation, and allow exogenous 6HHx-CoA to dominate the intracellular metabolic pool. In a 7-L bioreactor, a high level of production was achieved with a cell dry weight of 22 g / L, PHA content >44%, 6HHx molar ratio >98%, and 3HB incorporation <2 mol%. Compared with the existing technology, the yield (25 mg / L) was increased by about 400 times, which verified the feasibility of industrial scale-up. (5) The weight-average molecular weight (M) of the obtained biosynthetic PCL w Greater than 480 kDa, number-average molecular weight (M n The value is greater than 200 kDa, significantly higher than that of chemically synthesized PCL (M). w Typically <100 kDa); thermal decomposition temperature ( T d ) greater than 370℃, higher than chemically synthesized PCL ( T d Typically <350℃); Young's modulus greater than 600 MPa, tensile strength greater than 8 MPa, mechanical properties superior to chemically synthesized PCL, and no metal residue, providing a new path for the industrial production of bio-based biodegradable plastics. Attached Figure Description

[0026] Figure 1 : Halomonas 嗜盐菌属 Schematic diagram of the metabolic pathway of PCL synthesis in China (wherein) fadD The gene encodes a long-chain acyl-CoA synthase; fadE The gene encodes acyl-CoA dehydrogenase; fadB The gene encodes a β-oxidative multifunctional enzyme; fadA The gene encodes 3-ketoacyl-CoA thiolytic enzyme; phaA The gene encodes β-ketothiolase; phaB The gene encodes acetyl-CoA reductase. phaC The gene encodes endogenous PhaA synthase. alkK The gene encodes a medium-chain acyl-CoA synthase. phaC au(L395Y / N419A) gene encodes a novel engineered PHA synthase. Figure 2 : Plasmid map of PHA synthase PhaC and CoA ligase / transferase overexpression; Figure 3 Different PHA synthases (PhaC) in 嗜盐蓝藻菌 Comparison of the catalytic polymerization capacity of 6HHx in the medium. (A. Cell dry weight (DCW) (g / L) and PHA content (wt%) in the shake flask experiment. B. Molar ratio of 6HHx in the shake flask experiment (mol%). Figure 4 Different CoA ligases in 嗜盐蓝藻菌 China and PhaC ar Effect of (NDFY) combination on the polymerization ability of 6HHx (A. Cell dry weight (DCW) (g / L) and PHA content (wt%) in shake flask experiments. B. Molar ratio of 6HHx in shake flask experiments (mol%)). Figure 5 Different N-terminal chimeric PhaC (PhaC acun1 ~PhaC acun6 Comparison of the polymerization capacity of 6HHx monomers in shake-flask fermentation (control: PhaC) ar (NDFY); PhaC acun4 (PhaC au (This increases the 6HHx molar ratio from 3% to 5%) (A. Cell dry weight (DCW) (g / L) and PHA content (wt%) in the shake flask experiment. B. 6HHx molar ratio (mol%) in the shake flask experiment); Figure 6 :PhaC au Molecular docking analysis with 6HHx-CoA and alanine scanning results of amino acid residues in the active pocket (three key sites were screened out: D394A, N395A, and L419A, with the 6HHx molar ratio increasing to 5%, 7%, and 15%, respectively) (A. 6HHx ratio (mol%) in shake-flask experiments. B. Cell dry weight (g / L) and PHA content (wt%) in shake-flask experiments); Figure 7Shake-flask screening results of saturated mutants at key amino acid sites (D394, L395, N419) (optimal mutant forms: D394A (12%), L395Y (33%), N419A (11%)) (A and B: screening of D394 saturated mutants; C and D: screening of L395 saturated mutants; E and F: screening of N419 saturated mutants. Cell dry weight (DCW) (g / L) and PHA content (wt%) in ACE shake-flask experiments. Molar ratio of 6HHx (mol%) in BDF shake-flask experiments; Figure 8 Effects of single-point optimal mutations (D394A, L395Y, N419A) and combined mutations on the 6HHx molar ratio (the double mutation L395Y / N419A showed the best performance, with a 6HHx molar ratio of 44%) (A. Cell dry weight (DCW) (g / L) and PHA content (wt%) in shake-flask experiments. B. 6HHx molar ratio (mol%) in shake-flask experiments); Figure 9 : In knocking out endogenous phaCAB and fadB The effect of gene-mediated enhancement on the 6HHx molar ratio in chassis strains (in) H. 蓝藻菌 (Δ phaCAB , Δ fadB Overexpression of pWJL130-CM3 in the cells, with a 6HHx molar ratio of 90% (A. Cell dry weight (DCW) and PHA content (wt%) in shake-flask experiments. B. 6HHx molar ratio (mol%) in shake-flask experiments). Figure 10 The effects of different plasmid stabilization strategies on cell dry weight and PHA yield (including...) hbpB / hbpC The low-copy plasmid pHbPBC (pWJL230) of the toxin-antitoxin system performed best, with a cell dry weight of 11 g / L, a PHA content of 52%, and a 6HHx molar ratio of approximately 87% (A. Cell dry weight (DCW) (g / L) and PHA content (wt%) in shake-flask experiments. B. 6HHx molar ratio (mol%) in shake-flask experiments). Figure 11Results of PCL production by adding different concentrations of 6HHx-Na (5–15 g / L) to nutrient-rich LB60 medium (6HHx molar ratio in each group reached over 97%, close to homopolymer PCL) (A. Cell dry weight (DCW) (g / L) and PHA content (wt%) in shake flask experiments. B. 6HHx molar ratio (mol%) in shake flask experiments); Figure 12 Fermentation curves for PCL production in a 7-L bioreactor using LB60 as substrate and fed-batch 6HHx-Na and glucose (final cell dry weight 22 g / L, PHA content >44%, 6HHx molar ratio >98%, 3HB incorporation <2 mol%) (A: MM60 mineral medium; B: LB60 nutrient-rich medium. The horizontal axis represents fermentation time (h). Cell dry weight (DCW) (g / L) and PHA content (wt%) were measured every 4 hours, and the 6HHx molar ratio (mol%) was calculated.

[0027] Figure 13 Nuclear magnetic resonance (NMR) spectrum of biosynthesized PCL (AB: ¹H NMR; CD: ¹³C NMR, showing that the product is high-purity PCL, with a 6HHx molar ratio of approximately 99% and a 3HB molar ratio of approximately 1%).

[0028] Explanations of the attached tables Table 1: Comparison of Some English Abbreviations with Their Full Chinese and English Names Table 2: Names and characteristic descriptions of important strains in this invention Table 3: MM60 culture medium formulation Table 4: Comparison of properties between biosynthesized PCL and commercially synthesized PCL

[0029] Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] 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).

[0032] 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.

[0033] The term "comprising" or "including" as used in this invention is an open-ended description, encompassing all specified components or steps described, as well as other specified components or steps that do not substantially affect the meaning; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described in this invention.

[0034] 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".

[0035] In this invention, the term "Haloxymonas spp." 嗜盐菌属 ")" includes but is not limited to 嗜盐菌属 蓝藻菌 , 艾丁湖嗜盐菌、坎帕尼亚嗜盐菌 , 嗜盐菌属 微黄嗜盐菌 , 嗜盐栖热菌 , 嗜盐菌属 sp. KM1、 嗜盐栖热菌 and Halomonas smyrnensis Even better Halomonas bluephagenesis TD1.0 ( Halomonas bluephagegenesis TD1.0 is in Halomonas bluephagenesis Inserted on the basis of TD01 drawer For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. et al. (2017) Novel T7-1ike expression systems used for Halomonas. Metab Eng 39, 128-140), Halomonas bluephagegenesis 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).

[0036] 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. Preferably, plasmid overexpression is introduced into the chassis strain via electroporation or conjugation transformation. When the host is *Halomonas*, the plasmid vector can be a pSEVA series vector (Martinez-Garcia E...). et al. ,SEVA 2.0: an update of the Standard European Vector Architecture for de- / re-construction ofbacterial functionalities. Nucleic Acids Res2015, 43 (Database issue): D1183-1189.) or toxin-antitoxin plasmid pHbPBC vector (Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis). ACS Synth. Biol 2023, 13 (1): 61-67).

[0037] As used in this article, the term "mineral (MM) basal medium" is a synthetic basal medium prepared from a variety of inorganic mineral salts to provide the mineral nutrients required for microbial growth and to maintain osmotic pressure and physiological environment.

[0038] This invention provides an engineered bacterium for producing P3HB6HHx copolymers, which expresses or overexpresses exogenous or endogenous PhaC and coenzyme A ligation (transfer) enzymes. Preferably, the engineered bacterium does not possess or has had its endogenous PHA synthase (PhaC) gene knocked out or knocked down. The polyhydroxy fatty acid polymerase can be selected from non-laboratory cultures (…). uncultured bacteria PhaC, the red forest swamp un (Genbank Accession No. AXB72506), derived from *Evodia roximatelyum* ( Cupriavidus nector ) of PhaC re (Genbank Accession No. WP_011615085), derived from Pseudomonas ( Psedomonas ) of PhaC 1437 (Genbank Accession No. VVC85840) and PhaC 61-3 (Genbank Accession No. BAA36200), derived from Aeromonas hydrophila ( Aeromonas hydrophila ) of PhaC 4AK4 (Genbank Accession No. AAM19085), derived from Aeromonas vaginalis ( Aeromonas caviae ) of PhaC ac (Genbank Accession No. ADZ05402), derived from *Methylcystis pyrenoidosa* ( Methylocystis parvus ) of PhaC mp (Genbank Accession No. WP_016919401.1) and PhaCar (NDFY) (Hozumi et al. 2024), and their chimeras and mutants, preferably derived from the parent PhaC ac and PhaC un Constructed chimeric enzyme PhaC au Or its mutants.

[0039] The coenzyme A ligase of the 6-hydroxyhexanoic acid monomer can be selected from Pseudomonas oleophila (…). Pseudomonas oleovorans Acyl-CoA synthase AlkK (Genbank Accession No. CAB69080) and coenzyme A transferase can be selected from Clostridium propionate ( Clostridium propionicum The CaiB / BaiF CoA transferase family protein Act (Genbank Accession No. WP_304509132), derived from *Clostridium gamma-aminobutyricum* (GABA). Clostridium aminobutyricum Acetyl-CoA transferase AbfT (Genbank Accession No. WP_206580961), derived from Clostridium propioni ( Clostridium propionicum Propionate-CoA transferase Pct540 (Genbank Accession No. ALJ75581), and from Clostridium klufernum ( Clostridium kluyveri ) of acetyl-CoA transferase OrfZ (Genbank Accession No. WP_012103361), preferably AlkK.

[0040] In one specific embodiment, the polyhydroxy fatty acid polymerase is PhaC. ar (NDFY) or from parent PhaC ac and PhaC un Constructed chimeric enzyme PhaC au Or a mutant thereof, and the coenzyme A ligase of the 6-hydroxyhexanoic acid monomer is AlkK.

[0041] In one specific embodiment, the polyhydroxy fatty acid polymerase is a chimeric enzyme PhaC. au It is from Aeromonas caviae PhaC ac The N-terminal α-helical domain and the domain derived from PhaC un Chimeric enzymes formed by the fusion of the C-terminal catalytic domain, preferably PhaC acun4 (named PhaC) auMore preferably, the polyhydroxy fatty acid polymerase is a mutant PhaC. au (L395Y / N419A), relative to PhaC au It contains two mutations at the L395Y and N419A sites.

[0042] In one specific embodiment, the engineered bacteria are selected from halophilic bacteria, preferably from the genus *Halomycium*. Halomonas ), Pseudomonas spp. Pseudomonas ), Escherichia coli ( Escherichia ), Roche's et al. Ralstonia eutropha Aeromonas spp. Aeromonas ), Bacillus spp. Bacillus ) or combinations thereof, more preferably halomonas ( Halomonas ), and even better as Halomonas bluephagenesis , Halomonas aydingkolgenesis , Halomonas campaniensis or Halomonas lutescens The best option Halomonas bluephagenesis TD01 (CGMCC No. 4353) and its derived strains H. bluephagegenesis (Δ phaCAB ), H. bluephagegenesis (Δ phaCAB , Δ fadB )or H. bluephagegenesis (Δ phaCAB , Δ fadB , ΔEnP).

[0043] In one specific embodiment, the coenzyme A ligase is AlkK, and its encoding gene is derived from *Pseudomonas oleophila* (…). Pseudomonas oleovorans (or other sources.)

[0044] In one specific embodiment, the polyhydroxy fatty acid polymerase and coenzyme A ligase are each independently expressed or overexpressed on the genome of the engineered bacteria or on a plasmid. Preferably, the plasmid is a stable plasmid containing a toxin-antitoxin (TA) system, for example derived from... H. bluephagegenesis pHbPBC modified with endogenous plasmids (Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonasbluephagenesis. ACS Synth. Biol2023, 13 (1): 61-67), which carries hbpB / hbpC Toxin-antitoxin expression cassette. More preferably, the endogenous plasmids of the engineered bacteria have been knocked out.

[0045] In one specific embodiment, the coding gene is introduced into engineered bacteria via conjugation transfer or transformation.

[0046] Another aspect of the present invention provides a method for producing PCL or copolymers with high 6HHx content, the method comprising fermenting an engineered bacterium according to the present invention. The fermentation culture employs a mineral medium (e.g., MM60) or a nutrient-rich medium (e.g., LB60), using sodium 6-hydroxyhexanoate (6HHx-Na) as a substrate (precursor), optionally with the addition of glucose as an auxiliary carbon source. The fermentation can be carried out in a shake flask or a bioreactor (e.g., a 7-L reactor), preferably under open conditions (when the engineered bacterium is *Halomonas*).

[0047] In one specific embodiment, a strategy of uniformly adding 6HHx-Na is employed during fermentation, for example, adding 6HHx-Na at a rate of, for example, 2 g / h during fermentation for 12-42 hours, while maintaining a glucose concentration above 10 g / L. Preferably, LB60 nutrient-rich medium is used as the substrate, resulting in a final cell dry weight of over 22 g / L, a PHA content exceeding 44%, a 6HHx molar ratio in the copolymer exceeding 98%, and 3HB incorporation below 2 mol%.

[0048] Another aspect of the present invention provides a biosynthetic polycaprolactone (PCL) having one or more (preferably all) of the following properties: The purity of the polycaprolactone is greater than 97 wt%, preferably greater than 98 wt% or greater than 99 wt%, more preferably close to 100%; the molar percentage of 6HHx in the copolymer is greater than 97 mol%, preferably greater than 98 mol% or greater than 99 mol%, and the molar percentage of 3HB monomer is less than 3 mol%, preferably less than 2 mol% or less than 1 mol%. The weight-average molecular weight (M) of the polycaprolactone w ) greater than 400 kDa, preferably greater than 450 kDa or greater than 480 kDa; number average molecular weight (M n (Greater than 150 kDa, preferably greater than 180 kDa or greater than 200 kDa;) Polydispersity (PDI, M) w / M n The value is greater than 2.0, preferably greater than 2.2, greater than 2.3, and / or less than 3.0; Glass transition temperature (T) gThe melting temperature is -65℃ to -60℃, and the melting temperature (T) is... m The thermal decomposition temperature is 50-60℃, and the thermal decomposition temperature (T) is... d Temperatures greater than 350℃, preferably greater than 370℃; The Young's modulus is greater than 500 MPa, preferably greater than 600 MPa; the compressive strength is greater than 7 MPa, preferably greater than 8 MPa; and the elongation at break is greater than 5%, preferably greater than 6%.

[0049] Preferably, the above properties are measured by the methods described in this application specification, particularly Example 9, with molecular weight measured by gel permeation chromatography (GPC), thermodynamic properties measured by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), and mechanical properties measured by a tensile strength tester.

[0050] In one specific embodiment, the biosynthesized polycaprolactone is produced by the method described in this invention.

[0051] This invention, for the first time, utilizes synthetic biology methods to develop a polycaprolactone-producing bacterium (halophilic bacterium) using 6-hydroxyhexanoic acid (6HHx) as a substrate, through enzyme engineering (chimeric enzyme PhaC). au and mutant L395Y / N419A), metabolic engineering (knockout) phaCAB , fadB Through the optimization of endogenous plasmids, plasmid stabilization (toxin-antitoxin system) and fermentation process (LB60 nutrient-rich medium, fed-batch strategy), high-level production with cell dry weight of 22 g / L, PHA content >44%, and 6HHx molar ratio >98% was achieved in a 7-L bioreactor. The resulting PCL exhibited significantly better molecular weight, thermal stability, and mechanical properties than chemically synthesized PCL, providing a novel pathway for the industrial production of bio-based biodegradable plastics.

[0052] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0053] 1. Halomonas bluephagenesis TD01 (CGMCC No. 4353): The applicant selected a salt- and alkali-tolerant Gram-negative halophilic bacterium that naturally produces PHB. This bacterium can accumulate high levels of polyhydroxy fatty acids, showing promising prospects for industrial production applications. This is documented in "Tan Dan, Wu Qiong, Chen, Jin-Chun and Chen GQ. Engineering". HalomonasThe article “TD01 for Low Cost Production of Polyhydroxyalkanoates. Metabolic Engineering 26 (2014) 34–47” has been published in CN102120973A and is available to the public from the applicant. It can only be used to repeat the experiments of this invention.

[0054] 2. Culture medium formulation: LB medium contains: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and the remainder is water. Adjust the pH to 7.0-7.2 and autoclave.

[0055] LB60 medium is LB medium containing 60 g / L NaCl, and the other components and preparation conditions are the same as LB medium.

[0056] The formulation of MM60 medium is shown in Table 3. The optimal pH for the halophilic monocytogenes used in the experiment is around 8-9, and the pH is adjusted using NaOH.

[0057]

[0058] 3. pSEVA321 plasmid in the example: The record is found in the following literature: "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. Available to the public from the applicant and may only be used for repeating experiments of this invention.

[0059] The pSEVA321 plasmid sequence is shown in SEQ ID NO: 1 (containing P proin - phaC au - alkK Representation boxes (sequences are represented in lowercase): P porin The promoter sequence is shown in SEQ ID NO: 2: cggccgctgagacctgccagttgcccatgggttccttaaaaaaatgcaaatcgtaaaaaaacactgtttttttctattgcgttcactggaatcccagtatagagtttga cctgcgagcattggactataacaaggcttagttgaggacatgccgcataccaccgggaaaaaccggaggatggcataaagagcatggcccgcaagccagctgcagactt 4. Unless otherwise specified, the gene editing technology used in this invention is CRISPR-Cas9 gene editing technology, and the specific operation method is described in the following literature: "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”. Available to the public from the applicant and may only be used for repeating experiments of the present invention.

[0060] 5. Parental PhaC sequence used to construct chimeric PhaC in the embodiments: phaC ac The nucleotide sequence of (N149D) is shown in SEQ ID NO: 3: PhaC ac The amino acid sequence of (N149D) is shown in SEQ ID NO: 4: MSQPSYGPLFEALAHYNDKLLAMAKAQTERTAQALLQTNLDDLGQVLEQGSQQPWQLIQAQMNWWQDQLKLMQHTLLKSAGQPSEPVITPERSDRRFKAEAWSEQPIYDYLKQSYLLTARHLLASVDALEGVPQKSRERLRFFTRQYVDAMAPSNFLATNPELLKLTLESDGQNLVRGLALLAEDLERSADQLNIRLTDESAFELGRDLALTPGRVVQRTELYELIQYSPTTETVGKTPVLIVPPFINKYYIMDMRPQNSLVAWLVAQGQTVFMISWRNPGVAQAQIDLDDYVVDGVIAALDGVEAATGEREVHGIGYCIGGTALSLAMGWLAARRQKQRVRTATLFTTLLDFSQPGELGIFIHEPIIAALEAQNEAKGIMDGRQLAVSFSLLRENSLYWNYYIDSYLKGQSPVAFDLLHWNSDSTNVAGKTHNSLLRRLYLENQLVKGELKIRNTRIDLGKVKTPVLLVSAVDDHIALWQGTWQGMKLFGGEQRFLLAESGHIAGIINPPAANKYGFWHNGAEAESPESWLAGATHQGGSWWPEMMGFIQNRDEGSEPVPARVPEEGLAPAPGHYVKVRLNPVFACPTEEDAA phaC un The nucleotide sequence of is shown in SEQ ID NO: 5: PhaC un has the amino acid sequence shown in SEQ ID NO: 6: MASKDSFGKTGDLWSSMFNWMSGTMTAAAQIQQANMRAFAQSMELATSAYARMWGQPVEQVVPADRRFKDEAWTENMAADLLKQSYLITSQWLMEIADGWQAIDPDLHERTRFWTQQLVDATSPANFAMTNPVVMQEIARTGGMNLIQGAQNLLKDAQSGRLTQVPEDAFEVGKDLAITPGKVVYRNRLIELIQYTPATETVHEIPILVVPPWINKYYVMDMQPENSLFKYLVDAGFTLFTISWKNPDETVLDLEWDDYLDLGTLEALRMVKEIMGVEQVNLVGYCLGGIISQVTLAYLAATGDDAQINSATYFTTHQDFSDAGEISVFISRLDVMFLEWLMKISGGYLDGRNLAATFNMLRANDLLWNYVVHNYLLGQEPASFDLLYWNNDGTRVPGKVHSFLLREFFLDNKLKEPEGIQVKGVGIDLGKITTPTYVVTADRDHIVPWRGAFLVRQLQSGPVRFILSGGGHIAGVISPPTKNRGFWINEEEKDDADAWLAGATKHDGSWWVDWIPWLEERSGRRVKPPTAAGSDEFKPLMDAPGTYVLEK phaC acun1 has the nucleotide sequence shown in SEQ ID NO: 7: phaC acun2 The nucleotide sequence is shown in SEQ ID NO: 8: phaC acun3 The nucleotide sequence is shown in SEQ ID NO: 9: PhaC au ( phaC acun4 The nucleotide sequence of ) is shown in SEQ ID NO: 10: phaC acun5 The nucleotide sequence is shown in SEQ ID NO: 11: phaC acun6 The nucleotide sequence is shown in SEQ ID NO: 12: PhaC au (phaC acun4 ) has the amino acid sequence shown in SEQ ID NO: 13: MSQPSYGPLFEALAHYNDKLLAMAKAQTERTAQALLQTNLDDLGQVLEQGSQQPWQLIQAQMNWWQDQLKLMQHTLLKSAGQPSEPVITPERSDRRFKAEAWSEQPIYDYLKQSYLLTARHLLASVDALEGVPQKSRERLRFFTRQYVDAMAPSNFAMTNPVVMQEIARTGGMNLIQGAQNLLKDAQSGRLTQVPEDAFEVGKDLAITPGKVVYRNRLIELIQYTPATETVHEIPILVVPPWINKYYVMDMQPENSLFKYLVDAGFTLFTISWKNPDETVLDLEWDDYLDLGTLEALRMVKEIMGVEQVNLVGYCLGGIISQVTLAYLAATGDDAQINSATYFTTHQDFSDAGEISVFISRLDVMFLEWLMKISGGYLDGRNLAATFNMLRANDLLWNYVVHNYLLGQEPASFDLLYWNNDGTRVPGKVHSFLLREFFLDNKLKEPEGIQVKGVGIDLGKITTPTYVVTADRDHIVPWRGAFLVRQLQSGPVRFILSGGGHIAGVISPPTKNRGFWINEEEKDDADAWLAGATKHDGSWWVDWIPWLEERSGRRVKPPTAAGSDEFKPLMDAPGTYVLEK CoA ligase gene for producing PCL alkK The nucleotide sequence is shown in SEQ ID NO: 14: The amino acid sequence of the CoA ligase AlkK used for PCL production is shown in SEQ ID NO: 15: MIGQMMRQPLIISSLIEHAARYHGNTEVVSVETSGEVTRAYWSGVALRARKLASALGKMGLTQSDRCATIAWNNVRHLEIYFGVSGAGMVCHTINPRLFIEQIAYVINHAEDKVIFFDETFLPIIAGLDGLLTTAK AFVLMGCANLKASAKIPGLLAYEDLIGQGDDDYVWPDVDEKEASSLCYTSGTTGNPKGVLYSHRSTVLHSMTTAMPDTLNLSARDTILPVVPMFHVNAWGIPHSAAMVGAKLVLPGPALDGASLAKLIDQERVSIAL GVPVIWQGLLASLAESGSKAESLTRTVVGGSACPPSMIKEFNDLYGVEVIHAWGMTELSPFGTANTPLGHQVNLPWDEKLALRQSQGRPPYGVELKIVDDEGVTLSEDGVTKGNLMVRGHWVIKDYFRSTIGTTLS NGWFSTGDVATIDQDGFMTICDRAKDIIKSGGEWISTVELEGVAIAHPAIVDAAVIAVKHKKWDERPLLVAVRAPNSVLTDDELLSYFEGKVAKWQIPDAAIFIDELPRNGAGKILKNRLREKYADILSCNDSSALE 6. Other coenzyme A ligase gene and hydroxy fatty acid polymerase gene sequences used for screening in the examples (codon-optimized for suitability) Halomonas TD01) abfT The nucleotide sequence is shown in SEQ ID NO: 16: orfZ The nucleotide sequence is shown in SEQ ID NO: 17: pct540 The nucleotide sequence is shown in SEQ ID NO: 18: act The nucleotide sequence is shown in SEQ ID NO: 19: phaC re The nucleotide sequence is shown in SEQ ID NO: 20: phaC 4AK4 The nucleotide sequence is shown in SEQ ID NO: 21: phaC 61-3 The nucleotide sequence is shown in SEQ ID NO: 22: phaC 1437 The nucleotide sequence is shown in SEQ ID NO: 23: phaC ar The nucleotide sequence of (NDFY) is shown in SEQ ID NO: 24: phaC mp The nucleotide sequence is shown in SEQ ID NO: 25: 7. The endogenous gene sequence knocked out in the examples (the knockout method can be found in Jiang, XR, Yan, X., Yu, LP, Liu, XY, and Chen, GQ (2021). Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis . Nature Communications 12, 1513) phaA The nucleotide sequence is shown in SEQ ID NO: 26: phaB The nucleotide sequence of is shown in SEQ ID NO: 27 as follows: phaC The nucleotide sequence of atggccaatcaagcccccgtcgcctgggtaactggtggaactggtggaatcggaacgtcaatttgtcattcattggcggatgcggggtatttggtagtcgcggggtatcataatcctgagaaagccaaaacctggctggaaactcagcaagccgccggttatgacaacatcgcgttatcgggtgttgatctttccgaccacaatgcctgcctagaaggtgctcgcgaaatccaagaaaaatatgggcctgtcagcgtgttggttaactgcgctggcatcactcgtgatggcactatgaagaagatgtcctatgagcagtggcatcaagtgatcgataccaaccttaatagcgtcttcaacccctgccgcagtgtaattgaaatgatgctggagcagggttacggtcgcatcatcaatatttcttctatcaacggtcgcaaaggccagtttgggcaggtgaactatgcggctgccaaagcaggtatgcatgggttgaccatgtcgctggcgcaagaaacggcaaccaaaggcattacggtgaataccgtatcgccaggctacattgccaccgatatgatcatgaaaattcccgaacaggtgcgtgaagccattcgtgaaaccattccagtgaagcgttacggtacaccggaggagattggtcgtctggtcacgttcttagcagataaagagtcaggttttattactggcgccaatattgatattaacggcggtcagtttatgggctaa is shown in SEQ ID NO: 28 as follows: fadB The nucleotide sequence is shown in SEQ ID NO: 29: 8. The methods used in the examples to maintain stable plasmid expression hbpB / hbpC The toxin-antitoxin operon sequence is shown in SEQ ID NO: 30: Example 1 In H. bluephagenesis Construction of 6-hydroxyhexanoic acid polymerization pathway and screening of key enzyme combinations This embodiment verifies that in H. bluephagenesis Copolymer P(3HB-) was produced in strain TD01 co The feasibility of 6HHx was investigated, and by screening different combinations of CoA ligases and PHA synthases, an enzyme system that could initially achieve trace incorporation of 6HHx monomers was determined.

[0061] 1. Preparation of the precursor 6HHx-Na Considering the relatively stable structure of ε-caprolactone (six-membered ring) and its low reactivity in ring-opening reactions, to improve the availability of the precursor, ε-caprolactone was first subjected to alkaline hydrolysis with 1.5 times its volume of NaOH (10 M), and then the pH of the solution was adjusted to neutral with hydrochloric acid (6 M) to generate sodium 6-hydroxyhexanoate (6HHx-Na), which was directly used as a precursor for the polymerization reaction.

[0062] 2. Construction of expression plasmids and transformation of bacterial strains Using pSEVA321 as the backbone plasmid, a series of expression plasmids were constructed. Each plasmid contains a combination of different CoA ligase (transfer) genes driven by the Porin promoter and different PHA synthase (PhaC) genes (see [link to documentation]). Figure 2 The above plasmid was introduced via conjugation transfer. H. bluephagenesis Recombinant strains were obtained from the host bacteria.

[0063] 3. Screening of different PhaC enzymes Using 6HHx-Na as a precursor, and with AlkK as the CoA ligase, the effects of the following PhaC enzymes on the polymerization efficiency of 6HHx monomers were tested: PhaC un 、PhaC re 、PhaC 1437 、PhaC 4AK4 、PhaC 61-3 、PhaC ac 、PhaC mp and PhaC ar (NDFY). Fermentation conditions were MM60 mineral medium with the addition of 20 g / L glucose and 5 g / L 6HHx-Na, and cultured in shake flasks at 37°C and 200 rpm for 48 hours. After fermentation, the dry weight (DCW) of the cells and the molar percentage of 6HHx in the copolymer were determined.

[0064] 4. Screening of different CoA ligases Based on the above screening results, PhaCar (NDFY), which has the strongest polymerization ability, was selected as the immobilization synthase. The effects of different CoA ligases (transferases) on 6HHx polymerization were further compared, including Act, AbfT, Pct540, OrfZ, and AlkK. Fermentation conditions were the same as above.

[0065] 5. Experimental Results In different combinations of PhaC enzymes, PhaCar (NDFY) plays a role in... H. bluephagenesis TD01 exhibited the strongest polymerization ability for 6HHx-CoA. In MM60 medium (20 g / L glucose, 5 g / L 6HHx-Na), this combination synthesized the copolymer with the highest molar proportion of 6HHx, reaching 3% (see [link to TD01]). Figure 3 ).

[0066] In the screening of different CoA ligases, the AlkK group showed the highest 6HHx molar ratio (see [link to study]). Figure 4 ).

[0067] 6. Conclusion This embodiment demonstrates... H. bluephagenesis AlkK and PhaC are expressed in ar The combination of (NDFY) and 6HHx monomers has for the first time achieved trace polymerization (6HHx molar ratio of 3%). Although the current incorporation ratio is low, this result lays the foundation for further improvements in 6HHx polymerization capacity through enzyme engineering (such as chimeric enzyme design and site-directed mutagenesis) and metabolic pathway remodeling. Meanwhile, the method for preparing the precursor 6HHx-Na provides a simple and effective processing technology for subsequent scale-up production.

[0068] Example 2: PhaC engineered enzyme based on N-terminal chimerism and its application in improving 6HHx polymerization ability 1. Design and construction of chimeric enzymes From Aeromonas caviae PhaC ac (with a broad substrate spectrum) and derived from PhaC un (Capable of polymerizing 5-hydroxyvaleryl-CoA) as the parent enzyme. First, PhaC was analyzed using ESPript software (https: / / espript.ibcp.fr / ESPript / ESPript / ). ac With PhaC unThe amino acid sequences of the two enzymes were compared, and the results showed that the overall sequence identity was 41.25%, indicating high structural homology. Further analysis using Clustal Omega software (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) combined with known secondary structure information revealed that the two enzymes were highly conserved in the α / β hydrolase folding core region, while differences existed in the N-terminal α-helix region.

[0069] Based on the above secondary structure boundary information, multiple intercalation truncation sites were designed at the end of the α-helix to incorporate PhaC. ac PhaC with α-helices of different lengths at the N-terminus un A series of chimeric enzymes were constructed from the corresponding N-terminal region and uniformly named PhaC. acun (N), where N represents the source from PhaC ac The number of N-terminal α-helices. The specific naming rules are as follows: PhaC acun1 This indicates that the N-terminal has been replaced with PhaC. ac The first α-helix ends at this position; the remaining sequences are derived from PhaC. un ; and so on, constructing PhaC acun2 、PhaC acun3 、PhaC acun4 、PhaC acun5 、PhaC acun6 The amino acid sequence of the chimeric enzyme is modularly fused based on structural boundaries to introduce PhaC while maintaining overall folding stability. ac Its substrate compatibility advantage.

[0070] 2. Construction of co-expression plasmids and preparation of host strains The encoding genes of each of the above chimeric PhaC types and the encoding gene of the CoA ligase AlkK were cloned into the expression plasmids in the same example 1 (see Example 1). Figure 2 This allows for the co-expression of both genes on the same plasmid. A conjugation transfer method is used to introduce the constructed recombinant plasmid into a host strain whose endogenous PhaC gene has been knocked out. H. bluephagenesis TD01(Δ phaC Recombinant engineered strains were obtained from [the sample].

[0071] 3. Shake-flask fermentation experiment and product analysis Single colonies of the above recombinant strains were picked, inoculated into LB medium, and cultured overnight. Then, the culture was performed according to the initial OD value. 600The culture medium was transferred at a concentration of 0.05 g / L to a mineral fermentation medium containing 20 g / L glucose and 5 g / L 6HHx-Na, and shake-flask fermentation was carried out at 37 °C and 200 rpm. After 48 hours of fermentation, the cells were collected and the dry weight (DCW) of the cells was determined. The extracted PHA polymer was analyzed by gas chromatography to determine the total polymer content and the molar fraction of 6HHx monomer in the copolymer.

[0072] 4. Experimental Results The catalytic performance results of different chimeras are as follows (see Figure 5 ): PhaC acun6 It completely lost its ability to synthesize PHA, suggesting that its N-terminal substitution length was too long, affecting the overall folding stability of the enzyme or the formation of its catalytic conformation.

[0073] PhaC acun4 While maintaining a certain level of polymerization activity, the proportion of 6HHx monomer incorporated into the copolymer was increased. Compared with the control PhaC... ar(NDFY) In comparison, PhaC acun4 The molar fraction of 6HHx monomer in the copolymer was increased from 3% to 5% of the control level. At the same time, the dry weight of the cells and the total PHA content did not decrease significantly, indicating that its overall catalytic ability was maintained.

[0074] PhaC demonstrates superior performance in both polymerization capacity and monomer incorporation ratio. acun4 It has been identified as the base template for subsequent optimization and modification. To simplify naming, PhaC... acun4 Noted as PhaC au .

[0075] Therefore, this embodiment successfully constructed a chimeric PHA synthase PhaC that can enhance the polymerization ability of 6HHx monomers. au (i.e., PhaC) acun4 This provides an effective enzyme engineering modification scheme for enhancing the biopolymerization of medium-chain hydroxy fatty acid monomers.

[0076] Example 3: Improving PhaC Based on Molecular Docking and Alanine Scanning Mutation au Methods for assessing the polymerization ability of 6HHx-CoA This embodiment describes the chimeric PHA synthase PhaC constructed in Example 2. au (i.e., PhaC) acun4 Based on this, a method is further provided to improve the polymerization ability of 6HHx monomers by molecular docking to analyze the active pocket and combining alanine scanning mutation.

[0077] 1. PhaC au Establishment of the three-dimensional structural model and preparation for docking with the ligand 6HHx-CoA Using AlphaFold2 to target PhaC au The three-dimensional structure of the monomer was predicted. 6HHx-CoA was used as a PhaC... au Substrate ligands that can be identified and catalyzed for esterification reactions were plotted using ChemDraw software, and their protonation state was optimized using Open Babel software to obtain ligand conformations for molecular docking.

[0078] 2. Molecular docking analysis active pocket PhaC was analyzed using AutoDock Vina software. au Molecular docking was performed with 6HHx-CoA. During docking, the center of the grid box was positioned near the catalytic triplet (Cys-His-Asp), ensuring the grid box covered amino acid residues within a 5 Å radius around the catalytic center, thus fully encompassing the key catalytic region within the search space. After docking, all amino acid residues within 5 Å of 6HHx-CoA were screened and defined as the predicted active pocket region. This region contains a total of 36 amino acid residues (including the catalytic triplet). See [link to relevant documentation]. Figure 6 .

[0079] 3. Alanine scanning mutation design Based on the active pocket region (excluding the catalytic triplet) obtained from the above molecular docking prediction, alanine substitution mutants were designed for each of the remaining amino acid residues within the pocket. It should be noted that previous studies have demonstrated that substitution of any residue in the catalytic triplet (Cys-His-Asp) leads to complete inactivation of PhaC; therefore, this embodiment does not perform an alanine scan on the catalytic triplet.

[0080] Expression plasmids for each mutant were constructed using conventional molecular cloning methods. The mutant expression plasmids were co-expressed with the CoA ligase AlkK on the same plasmid and then transferred to a host strain with endogenous PhaC knockout according to the method described in Example 1. H. bluephagenesis TD01 (Δ phaC In the process, recombinant strains of each mutant were obtained.

[0081] 4. Shake-flask fermentation and product analysis Each mutant recombinant strain was cultured in shake flasks under the fermentation conditions described in Example 1. The fermentation medium was MM60 mineral medium supplemented with 20 g / L glucose, 0.5 g / L urea, and 5 g / L 6HHx-Na. After fermentation at 37°C and 200 rpm for 48 hours, the cells were collected, and the dry weight (DCW) was determined. Gas chromatography was used to analyze the molar fraction of 6HHx monomer in the copolymer of the extracted PHA polymer.

[0082] 5. Experimental Results With unmutated PhaC au As a control (under the same batch conditions, the 6HHx molar percentage in the synthesized polymer was 3%), shake-flask fermentation results of the alanine scanning mutant were shown (see...). Figure 6 ): Mutant D394A: 6HHx molar percentage increased to 5%; Mutant N395A: 6HHx molar percentage increased to 7%; The mutant L419A: 6HHx molar percentage was significantly increased to 15%.

[0083] The cell dry weight and total PHA content of the three mutant strains at the end of fermentation were similar to those of the wild-type PhaC strain. au Compared to no significant change, this indicates that it maintains overall polymerization activity while improving the 6HHx incorporation capacity.

[0084] 6. Conclusion This embodiment identifies PhaC through molecular docking. au The active pocket region of 6HHx-CoA was identified, and three key site mutants, D394A, N395A, and L419A, were obtained through alanine scanning. These mutation sites provide a clear structural basis for further amino acid optimization and combinatorial mutagenesis to improve the polymerization efficiency of 6HHx.

[0085] Example 4: Further improving PhaC based on saturation mutation and combinatorial mutation au Methods for polymerizing 6HHx This embodiment, based on the three key amino acid sites (D394, L395, N419) identified in Example 3, further provides a method to significantly improve the chimeric PHA synthase PhaC through saturation mutation and combination mutation. au A method for polymerizing 6HHx monomers.

[0086] 1. Construction of saturation mutant libraries at three key sites The chimeric enzyme PhaC constructed in Example 1 au Using templates, site-directed saturation mutant primers were designed for the D394, L395, and N419 sites, respectively. Saturation mutant libraries for each site were constructed using overlap extension PCR or whole-plasmid large primer PCR. The mutant libraries were ligated into expression vectors and co-expressed with the CoA ligase AlkK on the same plasmid. The vectors were then transferred to host strains with endogenous PhaC knockout according to the method described in Example 1. H. bluephagenesis TD01 (Δ phaC In the process, clones are spread on selective plates and randomly selected for subsequent screening.

[0087] 2. Shake-flask screening of saturated mutants and determination of the optimal mutation form Single clones from each saturated mutant library were selected and inoculated into 96-well plates or shake flasks containing MM60 mineral medium for initial fermentation. Fermentation conditions were: MM60 medium, 20 g / L glucose, 0.5 g / L urea, 5 g / L 6HHx-Na, incubated at 37℃ and 200 rpm for 48 hours. Cells were collected, and cell dry weight (DCW) was determined. The PHA content and the molar percentage of 6HHx in the copolymer were determined by gas chromatography. Unmutated PhaC... au As a control (under the same batch conditions, the 6HHx molar ratio is 3%).

[0088] The results of the shake flask screening showed (see) Figure 7 ): Site D394: The optimal mutant form is D394A, which synthesizes P(3HB- co The molar percentage of 6HHx in the (-6HHx) copolymer reaches 12%; Site L395: The optimal mutation form is L395Y, with 6HHx molar percentage reaching 33%; Site N419: The optimal mutation form is N419A, with a 6HHx molar percentage of 11%.

[0089] See the above results Figure 7 Among them, the L395Y mutant showed the most significant increase in the 6HHx incorporation ratio, indicating that the increase in side chain volume and the change in hydrophobicity at this site play a key regulatory role in the recognition of the substrate 6HHx-CoA; while the mutation effects of D394A and N419A were relatively mild, suggesting that the amino acid side chain charge, hydrophobicity and steric hindrance at different sites have different effects on substrate recognition and incorporation efficiency.

[0090] 3. Construction and functional evaluation of combined mutants To examine the synergistic effect of the three effective mutations mentioned above, the following combined mutants were constructed: Two-site combinations: D394A / L395Y, D394A / N419A, L395Y / N419A; Three-site combination: D394A / L395Y / N419A.

[0091] Using site-directed mutagenesis, with PhaC au Using the gene as a template, corresponding point mutations were introduced sequentially. The expression plasmids of each mutant combination were then transformed into [the gene] using the same method. H. bluephagenesis (Δ phaCThe samples were fermented in shake flasks under the same fermentation conditions (MM60 medium, 20 g / L glucose, 5 g / L 6HHx-Na, 37℃, 48 hours), and the contents of DCW, PHA and 6HHx molar percentage were determined.

[0092] 4. Experimental results of combined mutants Fermentation results (see) Figure 8 )show: Unit point mutation controls: L395Y had a 6HHx molar ratio of 33%; D394A had a 6HHx molar ratio of 12%; and N419A had a 6HHx molar ratio of 11%.

[0093] In dual-site combined mutants: D394A / L395Y: The molar ratio of 6HHx is approximately 35%; D394A / N419A: The molar ratio of 6HHx is approximately 13%; The molar ratio of L395Y / N419A:6HHx reaches 44%, which is higher than any single-point mutation and significantly higher than L395Y (33%).

[0094] The three-site combined mutation D394A / L395Y / N419A:6HHx molar ratio is approximately 40%, which is lower than that of the L395Y / N419A combination.

[0095] None of the mutant combinations showed a significant decrease in cell dry weight and total PHA content at the fermentation endpoint, indicating that the mutant combinations maintained overall polymerization activity while improving substrate incorporation capacity.

[0096] 5. Conclusion In this embodiment, saturation mutations were used to optimize three key sites, D394, L395, and N419, respectively, obtaining their optimal mutant forms: D394A, L395Y, and N419A. Further screening using combined mutations identified the dual-site combined mutant PhaC. au (L395Y / N419A) can synthesize P(3HB-) with a 6HHx molar percentage of 44% under the same fermentation conditions. co The 6HHx copolymer provides an excellent foundation for further increasing the 6HHx polymerization ratio and producing medium-chain hydroxy fatty acid copolymers.

[0097] Example 5 Production of PCL in chassis strains with endogenous PhaCAB and FadB knocked out This embodiment builds upon the PhaC constructed in Example 4. au Based on the (L395Y / N419A) mutant enzyme, a method is further provided to reduce the incorporation of 3HB monomers and increase the proportion of 6HHx monomers through metabolic pathway remodeling.

[0098] 1. Selection and construction of chassis strains by H. bluephagenesis Three engineered strains were constructed using the starting strain as the starting strain: Strain A: H. bluephagenesis TD01 (Δ phaC ), that is, knocking out endogenous phaC The strain containing the gene (same as in Example 1); Strain B: H. bluephagenesis TD1.0 (Δ phaCAB ), that is, knocking out endogenous phaC , phaA and phaB Strains containing genes; Strain C: H. bluephagenesis TD1.0 (Δ phaCAB , Δ fadB ), named H. bluephagenesis WJL05, that is, in TD1.0 (Δ phaCAB Further knockout on the basis of ) fadB Gene.

[0099] fadB Genes are coding β - A multifunctional enzyme in the oxidation pathway; knocking out this gene can inhibit the flux of 3HB monomer production.

[0100] 2. Construction of co-expression plasmids The plasmid pWJL130-CM3 was constructed, and its backbone was the same as that in Example 1 (see Example 1). Figure 2 ), using P porin Startup driver alkK The gene (encoding CoA ligase) and the mutant enzyme gene obtained in Example 4 phaC au (L395Y / N419A) were co-expressed.

[0101] 3. Transformation and fermentation verification of engineered strains The plasmid pWJL130-CM3 was introduced into three engineered bacterial strains via conjugation transfer to obtain recombinant strains. Single colonies of each recombinant strain were picked and cultured in shake flasks according to the fermentation method described in Example 1. The fermentation conditions were: MM60 mineral medium with 20 g / L glucose and 5 g / L 6HHx-Na added, and cultured at 37°C and 200 rpm for 48 hours. After fermentation, the bacterial cells were collected, the dry weight (DCW) was determined, and the molar percentage of 6HHx monomer in the extracted PHA copolymer was analyzed by gas chromatography.

[0102] 4. Experimental Results Under the same fermentation conditions described above, the three engineered strains synthesized P(3HB- co The molar percentage of 6HHx monomer in the (-6HHx) copolymer is as follows (see Figure 9 ): TD01 (Δ phaC The molar ratio of pWJL130-CM3 to 6HHx is 44%. TD1.0 (Δ phaCAB ) / pWJL130-CM3: 6HHx molar ratio increased to 85%; TD1.0 (Δ phaCAB , Δ fadB The molar ratio of 6HHx in pWJL130-CM3 is further increased to 90%.

[0103] The results showed that knocking out endogenous phaCAB Further knockout on the basis fadB The gene effectively eliminates the formation of 3HB monomer in the copolymer, thereby greatly increasing the incorporation ratio of 6HHx monomer, making the 6HHx molar ratio in the copolymer reach 90%, close to the composition of pure polycaprolactone (PCL) precursor. This demonstrates that by blocking... β - The oxidation pathway and the endogenous PHA synthesis pathway can significantly enhance the polymerization efficiency of medium-chain hydroxy fatty acid monomers.

[0104] 5. Conclusion This embodiment constructs... H. bluephagenesis WJL05 chassis strain, combined with overexpression plasmid pWJL130-CM3 (carrying alkK and phaC au (L395Y / N419A)) successfully P(3HB- co The molar proportion of 6HHx monomer in the (-6HHx) copolymer is increased to 90%, providing an effective engineering platform for the biological production of PCL or copolymers with high 6HHx content.

[0105] Example 6: A method to improve host cell dry weight and PHA yield by optimizing plasmid stability and toxin-antitoxin system. This embodiment uses the engineered strain constructed in Example 5. H. bluephagenesis Based on WJL05, a method is further provided to improve cell dry weight (DCW) and PHA yield by optimizing the genetic stability of expression plasmids.

[0106] 1. Construction of expression plasmids This embodiment employs two plasmid optimization strategies: Strategy 1: Use sources H. bluephagenesisA stable low-copy plasmid pHbPBC was obtained by artificially modifying the endogenous plasmid (EnP) pHbCP. (Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpCToxin-Antitoxin System of Halomonas bluephagenesis.) ACS Synth. Biol 2023, 13 (1): 61-67). This plasmid carries hbpB / hbpC The expression cassette encodes a toxin protein (HbpC) and an antitoxin protein (HbpB), respectively. Because the toxin protein is more stable than the antitoxin protein within the cell, when a cell loses the plasmid, the original antitoxin rapidly degrades, while the relatively stable toxin continues to exert its effect, leading to cell death due to plasmid deletion, thus achieving stable plasmid inheritance. The PCL-based expression cassette P... porin Startup driver phaC au (L395Y / N419A) and alkK The gene was directly cloned into the pHbPBC plasmid to obtain the recombinant plasmid pWJL230.

[0107] Strategy 2: Insertion into the high-copy plasmid pSEVA321 hbpB / hbpC The toxin-antitoxin element (SEQ ID NO:30) was used to obtain the recombinant plasmid pSEVA321-TA. Then, the same PCL expression cassette (P...) was synthesized. porin - phaC au (L395Y / N419A)- alkK The plasmid was cloned into pWJL231. This strategy aims to take advantage of the high copy number while maintaining plasmid stability through a toxin-antitoxin system.

[0108] As a control, the plasmid pWJL130-CM3 used in Example 5 (carrying the same expression cassette but without) was retained. hbpB / hbpC element).

[0109] 2. Host strain modification: knockout of endogenous plasmids To eliminate the interference of host endogenous toxin-antitoxin plasmids on the plasmid maintenance system, and to make the added... hbpB / hbpC The system can effectively exert screening pressure and use CRISPR / Cas9 technology to knock out strains. H. bluephagenesisEndogenous plasmid deletion strains were obtained from the endogenous plasmid (EnP) in WJL05. H. bluephagenesis (Δ phaCAB , Δ fadB , ΔEnP), named H. bluephagenesis WJL06. Knockout H. bluephagenesis For the method using endogenous plasmids, see Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonasbluephagenesis. ACS Synth. Biol 2023, 13 (1): 61-67.

[0110] 3. Construction and fermentation of recombinant strains The plasmids constructed above were introduced into host strains via conjugation transfer to obtain the following recombinant strains: Group 1: In H. bluephagenesis (Δ phaCAB , Δ fadB Overexpression of pWJL130-CM3 (excluding TA) in [the sample / sample] Group 2: In H. bluephagenesis (Δ phaCAB , Δ fadB Overexpression of pWJL230 in ΔEnP (low copy stable plasmid strategy) Group 3: In H. bluephagenesis (Δ phaCAB , Δ fadB Overexpression of pWJL231 in ΔEnP (high copy + TA module strategy) Fermentation conditions: MM60 mineral medium, supplemented with 15 g / L glucose, 1 g / L urea, and 5 g / L 6HHx-Na, was cultured in shake flasks at 37℃ and 200 rpm for 48 hours. After fermentation, the cells were collected, DCW was measured, and the PHA content and the molar percentage of 6HHx in the copolymer were determined by gas chromatography.

[0111] 4. Experimental Results Results of shake-flask fermentation (see) Figure 10 )as follows: Group 1 (pWJL130-CM3): lower cell dry weight, lower PHA content, and 6HHx molar ratio of approximately 87% (comparable to the results in Example 5, but without a significant increase in dry weight and PHA content).

[0112] Group 2 (pWJL230): Performed best. Cell dry weight reached 11 g / L, PHA content was approximately 52% (as a percentage of cell dry weight), and the molar percentage of 6HHx in the copolymer was approximately 87 mol%. Compared with Group 1, both dry weight and PHA accumulation levels were significantly improved.

[0113] Group 3 (pWJL231): Cell growth and PHA production were also improved compared to Group 1 and Group 2, but slightly lower than Group 2.

[0114] The above results indicate that hbpB / hbpC Toxin-antitoxin elements can effectively maintain the genetic stability of plasmids in host cells, thereby increasing the proportion of cells expressing recombinases in the population, and thus promoting cell growth and PHA accumulation. Among them, using the low-copy endogenous stable plasmid pHbPBC containing the TA system yields the best dry weight and product yield.

[0115] 5. Conclusion This embodiment involves using a host strain with an endogenous plasmid knocked out. H. bluephagenesis In WJL06, a portable hbpB / hbpC The stable plasmid pHbPBC overexpression of the toxin-antitoxin system over the PCL synthesis module (P) porin - phaC au (L395Y / N419A)- alkK This study successfully increased cell dry weight to 11 g / L and PHA content to 52%, while maintaining a high 6HHx molar ratio of approximately 87%. This strategy solves the previous problems of low dry weight and yield caused by plasmid instability, and provides a more efficient engineered strain and plasmid system for the biological production of PHA copolymers with high 6HHx content.

[0116] Example 7: Method for synthesizing pseudo-homogeneous PCL in LB60 enriched culture medium A method for achieving extremely high 6HHx molar ratio and near-homogeneous PCL synthesis in nutrient-rich LB60 medium. This embodiment uses the host strain constructed in Example 6. H. bluephagenesis TD1.0(Δ phaCAB , Δ fadB , ΔEnP) or H. bluephagenesis Based on WJL06 and its stable expression system, a method for producing copolymers with high 6HHx content, or even close to homopolymer polycaprolactone (PCL), in nutrient-rich culture media is further provided.

[0117] 1. Expression plasmids and host strains The same expression plasmid pWJL230 as in Example 6 was used, which carries the expression plasmid generated by P porinStartup driver phaC au (L395Y / N419A) and alkK Gene (PCL synthesis expression cassette), and contains hbpB / hbpC Toxin-antitoxin stabilizing element. The above plasmid was introduced into the host strain via conjugative transfer. [[ID=......]]H. bluephagenesis Recombinant strains were obtained from WJL06. H. bluephagenesis WJL06 / pWJL230.

[0118] 2. Fermentation medium and conditions Fermentation was performed using LB60 enriched medium. LB60 medium contains high levels of organic nitrogen sources and amino acids (the specific formulation is conventional LB medium supplemented with appropriate components to achieve the corresponding total nitrogen source concentration). Based on this medium, 5 g / L glucose was added as an auxiliary carbon source, and different concentrations of 6HHx-Na were added as precursors for PCL synthesis, with concentration gradients of 5 g / L, 7.5 g / L, 10 g / L, and 15 g / L.

[0119] Recombinant strains were ordered according to their initial OD. 600 0.05 mg was inoculated into the above culture medium and cultured in a shake flask at 37°C and 200 rpm for 48 hours.

[0120] 3. Product Analysis Methods After fermentation, the cells were collected and their dry weight (DCW) was determined. Gas chromatography was used to analyze the content of the extracted intracellular PHA polymer (the percentage of PHA in the cell dry weight) and the molar percentage of 6HHx monomer in the copolymer.

[0121] 4. Experimental Results Under the above conditions of 6HHx-Na concentration, the fermentation results (see...) Figure 11 It exhibits the following characteristics: Dry weight of cells: There was little difference in the dry weight of cells among different concentration groups, and they were all stable in the range of 7-8 g / L.

[0122] PHA content: The PHA content in each group reached approximately 50% (as a percentage of cell dry weight).

[0123] 6HHx molar ratio: In all experimental groups, the molar percentage of 6HHx in the copolymer reached over 97%, approaching the level of homopolymer PCL. Different 6HHx-Na concentrations (5–15 g / L) had no significant effect on this high proportion.

[0124] 5. Mechanism Analysis and Conclusions Under LB60 nutrient-rich medium conditions, cells preferentially grow and metabolize, requiring only 5 g / L glucose supplementation. This significantly limits the flux of endogenous 3HB-CoA production, reducing the supply of 3HB monomers at the source. Simultaneously, with sufficient exogenous 6HHx-Na, the 6HHx-CoA generated after AlkK activation dominates the intracellular metabolic pool, effectively inhibiting the incorporation of residual 3HB-CoA at the substrate competition level.

[0125] Therefore, this embodiment successfully utilized LB60 nutrient-rich medium, an optimized JL06 host, and the stable plasmid pWJL230 to synthesize P(3HB-) without significantly increasing glucose supply. co The molar proportion of 6HHx in the (-6HHx) copolymer was increased to over 97%, achieving near-homogeneous biosynthesis of PCL. This method is simple to operate, and produces stable dry weight and PHA content, providing a feasible fermentation process for the biological production of high-performance PCL materials.

[0126] Example 8: Method for scale-up production of PCL using a 7-L bioreactor Based on Examples 6 and 7, this embodiment further provides a method for producing high-purity PCL on a 7-L bioreactor scale, verifying the feasibility of scaling up this biosynthetic pathway from the shake-flask level to industrial scale.

[0127] 1. Fermentation strains and plasmids Using host strains H. bluephagenesis (Δ pha CAB , Δ fadB , ΔEnP) or H. bluephagenesis WJL06 was expressed and overexpressed using the plasmid pWJL230. This plasmid carries the expression of P... porin Startup driver phaC au (L395Y / N419A) and alkK Gene (PCL synthesis expression cassette), see Example 6 for specific construction method.

[0128] 2. Fermentation conditions in a 7-L bioreactor Two different culture media were selected as the fermentation substrate: MM60 mineral medium: used as a control for inorganic salt medium (first batch). Figure 12 A); LB60 nutrient-rich medium: as an optimized medium (second batch), Figure 12 B).

[0129] The fermentation volume is 7L (working volume), and the inoculum size is based on the initial OD. 600Approximately 0.05. The fermentation temperature is controlled at 37℃, and the pH is controlled at 8.0 by automatic acid / alkali replenishment. Dissolved oxygen is maintained above 30% by adjusting the stirring speed and aeration rate.

[0130] Key replenishment strategies: Precursor addition: During fermentation from 12 h to 42 h, 6HHx-Na was added at a constant rate of 2 g / h as a precursor for PCL synthesis.

[0131] Glucose supplementation: After 12 hours of fermentation, the glucose concentration in the fermentation broth is maintained above 10 g / L through glucose concentration feedback control to ensure the continuous cell growth and polymerization reaction.

[0132] The total fermentation cycle is 48 to 60 hours.

[0133] 3. Product Analysis Methods During fermentation, samples were taken at regular intervals to determine the content of DCW and PHA (gas chromatography) and the molar percentage of 6HHx monomer in the copolymer.

[0134] 4. Experimental Results (see...) Figure 12 ) Under MM60 medium conditions: the final DCW was only 15 g / L, the PHA content was 15%, and the 6HHx molar ratio was 94%. Process analysis showed that cell growth essentially stopped after about 24 hours. It is speculated that as the concentration of the fed-batch precursor gradually increased, it may have exerted metabolic stress or toxic effects on the cells, inhibiting further growth and product accumulation.

[0135] Under LB60 medium conditions: the final cell dry weight reached approximately 22 g / L, with a PHA content exceeding 44% (percentage of cell dry weight), of which the molar proportion of 6HHx was higher than 98%. Fermentation process analysis showed that cell dry weight and PHA content continuously increased throughout the fermentation process without significant inhibition. The incorporation proportion of 3HB monomer in the final product was less than 2 mol%, indicating that the polymer was primarily homopolymer PCL.

[0136] The above results are consistent with the patterns observed in shake-flask experiments: LB60 nutrient-rich medium is more conducive to the production of PCL with a high 6HHx ratio, and stable scale-up can be achieved in reactors by using a strategy of uniformly adding precursors and maintaining a high glucose concentration.

[0137] 5. Conclusion In this embodiment, in a 7-L bioreactor, with (Δ phaCAB , Δ fadB , ΔEnP) or H. bluephagenesisWJL06 was used for chassis overexpression of pWJL230. Employing an LB60 substrate combined with a uniformly fed-batch strategy of 6HHx-Na and maintaining a glucose concentration above 10 g / L, a polymer with a cell dry weight of 22 g / L, PHA content >44%, 6HHx molar ratio >98%, and 3HB incorporation below 2 mol% was successfully obtained, approaching the level of homopolymer PCL. This method validates the feasibility of scale-up PCL polymerization modules based on direct precursor supply in fermenters, providing reliable technical parameters for the subsequent industrial production of high-purity bio-based polycaprolactone.

[0138] Example 9 Material properties of biosynthesized PCL PCL material was extracted using a Soxhlet extractor. Cells were first collected 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 lyophilization for 12 hours to remove moisture. PHA polymers, including PCL, were extracted from the lyophilized 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.

[0139] After the material was extracted, approximately 20 mg of PHA sample was dissolved in deuterated chloroform to analyze its purity, and nuclear magnetic resonance (NMR) was performed using a JNM-ECA600 nuclear magnetic resonance spectrometer (JEOL Ltd.). 1 H NMR and 13 C10 NMR spectroscopy analysis. Chemical shifts were recorded in parts per million (ppm), with the signal from a 0.03% v / v tetramethylsilane (TMS) reference standard used as a calibration reference (see [link]). Figure 13 ).

[0140] To determine the molar mass and polydispersity of the PCL 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, and the process was carried out at 40 °C at a flow rate of 1 mL / min. The injection volume for each sample was 40 μL. A series of polystyrene standards with different number-average molar masses (1 × 10⁻⁶) were used. 4 Da, 2 × 10 4 Da, 3 × 10 4 Da, 7 × 10 4 Da, 1.5 × 10 5 Da, 3 × 10 5 Da, 7 × 105 Da and 1 × 10 6 Da (Sigma-Aldrich, USA) is used to generate calibration curves for GPC analysis.

[0141] The thermodynamic properties of the samples, including melting point and glass transition temperature, were determined using 10 mg of PCL material and measured using a differential scanning calorimeter (DSC-Q20, TA Instruments, USA). Temperature scans were performed from -80 °C to 200 °C at a nitrogen flow rate of 50 mL / min. The samples were initially cooled to -80 °C and then heated to 180 °C at a heating rate of 10 °C / min. To ensure accuracy, this cooling and heating cycle was repeated once within the same temperature range.

[0142] The thermal decomposition temperature was measured using a thermogravimetric analyzer (TGA-50, Shimadzu Corporation, Japan). The analysis was conducted in a nitrogen atmosphere, with the temperature increased from room temperature to 600°C at a heating rate of 10°C / min.

[0143] After the PCL material was spread into a film, its mechanical properties were tested. One gram of PCL was dissolved in 30 ml of chloroform and dropped onto a glass petri dish at room temperature in a fume hood, allowing the solution to evaporate and form a PVL film, which took approximately 48 hours. Dumbbell-shaped specimens, 4 mm wide, were precisely cut using an RR / PCP cutter (Ray-Ran, UK). Stress-strain measurements were performed at 10 mm / min using an Instron tensile strength tester (Instron 3365, Instron, USA). Each specimen was tested three times, and the average value was taken to ensure data reliability.

[0144] NMR verification yielded a PCL homopolymer with a purity close to 100%. Figure 13 The calculated molar percentage composition of the material is 1.00% 3HB- co 请注意,原文中“[[ID=......]]H. bluephagenesis ”这里的“......”部分不太明确,我按照原样保留了。如果这是一个错误,请你检查并提供正确内容,以便我能更准确地翻译。 The content of 99.00% 6HHx and 3HB monomers is insufficient to affect its material properties.

[0145] The PCL obtained by biosynthesis has a higher molecular weight and mechanical properties than that of chemically synthesized PCL (trade name poly-ε-caprolactone, catalog number A020626, batch number PBDKSD1H, commercially purchased from Anaiji Chemical, China) (Table 4).

[0146]

[0147] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0148] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, and should also be regarded as the content disclosed by the present invention.

Claims

1. An engineered PHA synthase comprising an N-terminal portion and a C-terminal portion, wherein the N-terminal portion is derived from Aeromonas vaginalis ( ). Aeromonas caviae PHA synthase mutant PhaC ac (N149D) and contains 1, 2, 3, 4 or 5 (preferably 4) α-helical domains, the C-terminal portion of which originates from uncultured bacteria from red forest swamps ( Uncultured bacterium PHA synthase PhaC un PhaC is preferred. ac The amino acid sequence of (N149D) is shown in SEQ ID NO: 4 and the PhaC un The amino acid sequence is shown in SEQ ID NO:

6.

2. The engineered PHA synthase according to claim 1, wherein it is made with PhaC ac The segment from the N-terminus of (N149D) to the end of its 1st, 2nd, 3rd, 4th or 5th (preferably 4th) α-helical domain replaces PhaC. un The corresponding part is obtained, preferably the encoding nucleotide sequence shown in SEQ ID NO: 7, 8, 9, 10 or 11, and most preferably the encoding nucleotide sequence shown in SEQ ID NO:

10.

3. The engineered PHA synthase according to claim 1 or 2, wherein the amino acid sequence is shown in SEQ ID NO: 13, and optionally comprises a mutation or combination of mutations selected from: D394A; L395Y; N419A; D394A+L395Y; D394A+N419A; L395Y+N419A; and D394A+L395Y+N419A, The preferred formulation includes L395Y+N419A.

4. A gene encoding an engineered PHA synthase according to any one of claims 1 to 3, or a recombinant vector containing the gene.

5. A recombinant bacterium comprising the gene or recombinant vector according to claim 4, preferably, the recombinant bacterium is selected from halophilic bacteria, more preferably from the genus *Halomycium*. Halomonas ), Pseudomonas spp. Pseudomonas ), Escherichia coli ( Escherishia ), Roche's et al. Ralstonia eutropha Aeromonas spp. Aeromonas ), Bacillus spp. Bacilllus ), Alcaligenes megaterium ( Alcaligenes latus ), Alkali-producing bacteria ( Alcaligenes eutropus ) or combinations thereof, more preferably halomonas ( Halomonas ), and even better as Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis or Halomonaslutescens The best option Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas bluephagenesis TD1.0 Halomonas aydingkolgenesis M1 (CGMCC No. 19880) or Halomonas campaniensis LS21 (CGMCC No. 6593).

6. The recombinant bacteria according to claim 5, wherein the genome of the recombinant bacteria lacks the following endogenous gene: β-ketothiolate gene. phaA acetyl-CoA reductase gene phaB Polyhydroxy fatty acid polymerase gene phaC and / or esteracyl-CoA hydratase gene fadB Preferred Halomonas bluephagenesis (Δ phaCAB ), Halomonas bluephagenesis (Δ phaCAB , Δ fadB )or Halomonas bluephagenesis (Δ phaCAB , Δ fadB , ΔEnP).

7. The recombinant bacteria according to claim 5 or 6, wherein the recombinant bacteria further incorporates a gene encoding the CoA ligase AlkK, preferably the gene encoding the CoA ligase AlkK originating from *Pseudomonas oleophila* (…). Pseudomonas oleovorans (More preferably, its amino acid sequence is shown in SEQ ID NO: 15, and most preferably, its encoding nucleotide sequence is shown in SEQ ID NO: 14.) 8. The recombinant bacteria according to any one of claims 5 to 7, wherein: 1) The engineered PHA synthase gene and the gene encoding the CoA ligase AlkK are each independently expressed on the genome of the recombinant bacteria or on one or more plasmids, and are expressed under the control of a constitutive promoter or an inducible promoter; 2) The plasmid is a pSEVA series plasmid, and / or the plasmid contains... hbpB / hbpC The plasmid for the toxin-antitoxin operon, preferably the plasmid is pHbPBC; 3) 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 its mutants, wherein the mutants are selected from... porin 3 , porin 42 , porin 58 , porin 68 , porin 140 , porin 141 , porin 194 , porin 211 , porin 221 , porin 226 , porin 278 or combinations thereof; and / or 4) The recombinant bacteria is recombinant halomonas, and the endogenous plasmid has been knocked out.

9. A method for producing poly(ε-caprolactone) (PCL), comprising fermenting the recombinant bacteria according to claim 8, and obtaining poly(ε-caprolactone) from the fermentation product.

10. The method according to claim 9, wherein the fermentation culture medium is a mineral-based medium or Luria-Bertani (LB) medium, and / or includes the substrate sodium 6-hydroxyhexanoate (6HHx-Na).

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