Recombinant strain of zymomonas mobilis for producing polyhydroxybutyric acid lactate and preparation method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and relates to a recombinant strain of Zymomonas mobilis for producing polyhydroxybutyric acid lactate and a preparation method and application thereof. The application realizes one-step biosynthesis of polyhydroxybutyric acid lactate in the same cell by constructing a 3HB-CoA synthesis module, an LA-CoA synthesis module and a polymerization module in a host cell; further, by using a high-lactic acid background strain ZMB3 and replacing L-lactic acid dehydrogenase, the lactic acid component in the copolymer is effectively increased and regulated to 14.76 mol%. The application can be fermented under anaerobic conditions, reduces energy consumption, and provides a new way for green biological manufacturing of degradable polyhydroxybutyric acid lactate.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a recombinant strain of *Bacillus musculosus* that produces polyhydroxybutyrate lactate, its preparation method, and its application. Background Technology

[0002] Polyhydroxybutyrate lactate (hereinafter referred to as "P(3HB-co-LA)") combines the performance advantages of polyhydroxybutyrate (PHB) and polylactic acid (PLA), exhibiting good thermoplasticity, flexibility, and biodegradability, and showing promising applications in packaging, pharmaceuticals, and agriculture. Existing lactic acid-based polymers mostly employ a two-step process of "fermentation to produce lactic acid + chemical polymerization," which is lengthy and may suffer from issues such as catalyst residue, safety concerns, and insufficient green manufacturing. Therefore, establishing a biosynthetic system capable of completing the copolymerization of 3-hydroxybutyrate (3HB) monomers, lactic acid monomers, and their copolymerization in a single step within microbial cells has become an important development direction in this field.

[0003] Currently, publicly available P(3HB-co-LA) production systems mainly focus on hosts such as *Escherichia coli*, *Corynebacterium glutamicum*, *Vibrio vulnificus*, and *Halomonas*. These systems construct dual precursor supply systems of 3-hydroxyisobutyryl-CoA (3HB-CoA) and lactyl-CoA (LA-CoA) by exogenously introducing genes such as phaA, phaB, phaC, pct, and lactate dehydrogenase. Related studies have confirmed that this type of copolymer can be synthesized in a one-step process. However, existing systems suffer from problems such as high oxygen demand from the host, high energy consumption for aeration and stirring, excessive carbon flow to byproducts, inflexible control of lactate components, and a lack of mature implementation pathways in non-mode facultative anaerobic chassis. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a recombinant strain of *Bacillus mobilis* for the production of polyhydroxybutyrate lactate, which solves or partially solves the problems existing in the existing methods for synthesizing polyhydroxybutyrate lactate.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a recombinant strain for producing polyhydroxybutyrate lactate, comprising the following steps:

[0007] SA-1. The promoter Pgap, pct gene and lactate dehydrogenase encoding gene are sequentially linked to obtain operon fragment I;

[0008] SA-2, Operator fragment I is ligated into the pEZ15A vector to obtain recombinant vector I;

[0009] SA-3. Recombinant vector I was transformed into the starting strain, and positive clones were screened to obtain the recombinant strain.

[0010] Preferably, the nucleotide sequence of the promoter Pgap is shown in SEQ ID No. 6.

[0011] Preferably, the nucleotide sequence of the pct gene is shown in SEQ ID No. 5.

[0012] Preferably, the lactate dehydrogenase encoding gene in step SA-1 is selected from at least one of the dldh gene and the lpldh gene.

[0013] More preferably, the nucleotide sequence of the dldh gene is shown in SEQ ID No. 4.

[0014] More preferably, the nucleotide sequence of the lpldh gene is shown in SEQ ID No. 9.

[0015] Preferably, the method for constructing the starting strain in step SA-3 includes the following steps:

[0016] SB-1. The promoter Pgap, phaA gene, phaB gene and phaC gene are sequentially linked to obtain operon fragment II;

[0017] SB-2. Operator fragment II is ligated into the pL2R vector to obtain recombinant vector II;

[0018] SB-3. Integrate recombinant vector II into the genome of the original strain, and transfer it into the original strain to obtain the starting strain.

[0019] More preferably, the nucleotide sequence of the promoter Pgap is shown in SEQ ID No. 6.

[0020] More preferably, the nucleotide sequences of the phaA gene, phaB gene, and phaC gene are shown in SEQ ID No. 1-3, respectively.

[0021] More preferably, the original strain mentioned in step SB-3 is either *Fermentomonas motilityis* NPΔΔ or *Fermentomonas motilityis* ZMB3.

[0022] More preferably, when the original strain is *Bacillus motility fermentation motility* NPΔΔ, the recombinant vector II is integrated into the ZMO1650 site of the *Bacillus motility fermentation motility* NPΔΔ genome via homologous recombination;

[0023] Alternatively, if the original strain is *Zygomorpha motileis* ZMB3, the recombinant vector II is integrated into the ZMO0959 site of the *Zygomorpha motileis* ZMB3 genome via homologous recombination.

[0024] More preferably, the gRNA sequence targeting the ZMO1650 site is shown in SEQ ID No. 7, and the upstream and downstream arm sequences for integration into the ZMO1650 site are shown in SEQ ID No. 11-12, respectively.

[0025] Alternatively, the gRNA sequence targeting the ZMO0959 site is shown in SEQ ID No. 8, and the upstream and downstream arm sequences for integration into the ZMO0959 site are shown in SEQ ID No. 13-14, respectively.

[0026] Secondly, the present invention also provides a recombinant strain for producing polyhydroxybutyrate lactate prepared by the above preparation method.

[0027] Thirdly, the present invention also provides a method for preparing polyhydroxybutyrate lactate, comprising the following steps: inoculating the above-mentioned recombinant strain into RMG5 medium and carrying out fermentation culture to obtain a fermentation culture broth containing polyhydroxybutyrate lactate.

[0028] Preferably, the RMG5 culture medium contains the following components: 50 g / L glucose, 10 g / L yeast extract, and 2 g / L KH2PO4.

[0029] Preferably, the fermentation conditions include: an inoculum of 10% of the recombinant strain, a fermentation temperature of 25-35 °C, and a rotation speed of 50-150 rpm.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) Using motile fermentation monoclonal bacteria as a host, fermentation can be carried out under low oxygen or even anaerobic conditions. Compared with traditional aerobic hosts, it is more conducive to reducing aeration and stirring costs, and is suitable for developing green and low-energy biomanufacturing processes.

[0032] (2) By combining the design of “chromosomal integration 3HB-CoA synthesis module + plasmid expression lactate supply module”, the genetic stability of the core 3HB-CoA pathway is improved, while the lactate supply module can be quickly replaced, thus realizing modular construction and optimization.

[0033] (3) Using high lactate background strains to construct recombinant strains can significantly improve the extracellular lactate supply capacity;

[0034] (4) Replacing D-lactic acid dehydrogenase with L-lactic acid dehydrogenase to construct a recombinant strain further enhances the lactic acid component of the copolymer. The recombinant strain constructed in this invention can not only achieve the synthesis of P(3HB-co-LA), but also adjust the copolymer composition by changing the stereoconfiguration of the lactic acid precursor, providing a basis for customizing material properties. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram of the synthesis pathway of polyhydroxybutyrate lactate;

[0037] Figure 2 A schematic diagram illustrating the construction process of the recombinant strain LAHB-1;

[0038] Figure 3 This is a schematic diagram of the quantitative analysis process;

[0039] Figure 4 Figure 1 shows the fermentation performance test results of recombinant strain LAHB-1; Figure A is a statistical analysis of biomass; Figure B is a statistical analysis of specific growth rate; Figure C is a statistical analysis of polymer content and lactic acid composition.

[0040] Figure 5 A schematic diagram illustrating the construction process of the recombinant strain LAHB-2;

[0041] Figure 6 Figure 1 shows the fermentation performance test results of recombinant strain LAHB-2; Figure A is a statistical analysis of biomass; Figure B is a statistical analysis of extracellular lactic acid production; Figure C is a statistical analysis of polymer content and lactic acid composition.

[0042] Figure 7 A schematic diagram illustrating the construction process of the recombinant strain LAHB-3;

[0043] Figure 8 Figure 1 shows the fermentation performance test results of recombinant strain LAHB-3; Figure A is a statistical analysis of biomass; Figure B is a statistical analysis of polymer content and lactic acid composition. Detailed Implementation

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with 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.

[0045] The following specific embodiments further illustrate a recombinant *Fermentomonas motilityis* strain for producing polyhydroxybutyrate lactate, its preparation method, and its application. This section further illustrates the invention with reference to specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the techniques used in the embodiments are conventional methods well known to those skilled in the art, such as the conditions described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0046] Zymomonas mobilis, a naturally occurring facultative anaerobic Gram-negative bacterium that produces ethanol, possesses a unique ED metabolic pathway and high sugar fermentation efficiency. It also exhibits ideal characteristics for industrial cell factories, including high ethanol yield, low biomass, strong ethanol tolerance, high osmotic pressure tolerance, and fermentation without the need for additional oxygen. Currently, the production fermentation of products such as PHB, 3-HB, isobutanol, and lactic acid has been achieved using Zymomonas mobilis.

[0047] In the following embodiments, the *Zymomonas mobilis* NPΔΔ was obtained with reference to the contents recorded in the literature ([1] Geng B, Liu S, Chen Y, et al. A plasmid-free Zymomonas mobilis mutant strain reducing reactive oxygen species for efficient bioethanol production using industrial effluent of xylose mother liquor[J]. Frontiers in Bioengineering and Biotechnology, 2022, 10: 1110513.) (the strain ZMNP in the literature [1] is the *Zymomonas mobilis* NPΔΔ recorded in this invention); the *Zymomonas mobilis* ZMB3 was obtained with reference to the contents recorded in the literature ([2] Yan X, Bao W, Wu Y, et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis[J]. Nature Communications, 2024, 15(1): 10441.).

[0048] In the following embodiments, the standard gene synthesis of the target fragment was commissioned to Suzhou Genewiz Biotechnology Co., Ltd.

[0049] In the following embodiments, the recombinant vector is transferred into the recipient strain, comprising the following steps:

[0050] (1) Preparation of competent cells: 100 μL of the recipient strain culture was inoculated into 1 mL of RMG5 solid medium and incubated at 30 °C for activation; after the culture became turbid, it was transferred to 200 mL of RMG5 medium and incubated at 30 °C for further activation; after the culture reached OD 600 After reaching >0.3, the bacterial cells were collected by centrifugation at 100 rpm; the bacterial cells were washed once with sterile water, and then washed twice with 10% glycerol solution; the bacterial cells were resuspended in 2 mL of 10% glycerol solution to obtain competent recipient strain cells (hereinafter referred to as "competent cells"), which were then aliquoted for use.

[0051] (2) Electroporation of recombinant vector: 1 mg of the recombinant vector to be transferred was added to 55 μL of competent cells; after mixing, electroporation was performed using a Neon NxT electroporator (Thermo Scientific Inc., model: N10096) with the following electroporation program: 200 Ω, 25 µF, 1.6 KV; after electroporation, 1 mL of RMG5 medium was added, mixed, and incubated at 30 °C for 5 h.

[0052] (3) Validation of the target strain: After incubation, the target strain is selected by screening with RMG5-Spe solid plates. The clone that is positive and correctly sequenced is the target strain.

[0053] Furthermore, the RMG5 medium contains the following components: 50 g / L glucose, 10 g / L yeast extract, and 2 g / L KH2PO4; the RMG5 solid medium is RMG5 medium with an additional 3 g / L agar powder; and the RMG5-Spe solid plate is RMG5 solid medium with an additional 100 μg / mL spectinomycin.

[0054] In the following embodiments, the genes involved, the encoded proteins, and the relevant primer sequences used are shown in Table 1.

[0055] Table 1: Sequences of genes, encoded proteins, and related primers in the embodiments of the present invention

[0056]

[0057] Example 1: Exogenous introduction of polyhydroxybutyrate lactate biosynthesis system

[0058] This embodiment uses *Bacillus niger* NPΔΔ as the original strain and introduces genetic engineering techniques such as β-ketothionase (encoded by the phaA gene), acetyl-CoA reductase (encoded by the phaB gene), and PHA synthase (encoded by the phaC gene) required for 3HB-CoA synthesis, as well as lactate dehydrogenase (encoded by the dldh gene) and propionyl-CoA transferase (encoded by the pct gene) required for LA-CoA synthesis. Figure 1As shown in the figure, the key pathway for the synthesis of P(3HB-co-LA) by engineered strains was gradually modified to achieve one-step biosynthesis of P(3HB-co-LA) within the same cell. The nucleotide sequences of the phaA, phaB, and phaC genes are shown in SEQ ID No. 1-3, respectively, and are all derived from Ralstonia eutropha H16; the nucleotide sequence of the dldh gene is shown in SEQ ID No. 4, and is derived from Leuconostoc mesenteroides; the nucleotide sequence of the pct gene is shown in SEQ ID No. 5, and is derived from Clostridium propionicum.

[0059] 1. Construct the starting strain NPPg

[0060] (1) The phaA gene, phaB gene, phaC gene and promoter Pgap (as shown in SEQ ID No. 6) were synthesized using standard gene synthesis technology (Suzhou Genewise Biotechnology Co., Ltd.).

[0061] (2) The promoter Pgap, phaA gene, phaB gene and phaC gene are sequentially linked to obtain the Pgap-phaCAB operon fragment; the Pgap-phaCAB operon fragment is linked to the pL2R vector to obtain the pL2R-Pgap-phaCAB recombinant vector; the above operations are performed in accordance with the method described in the existing published patent (CN113528409A, an engineered bacterium containing nCas3 single-stranded endonuclease, preparation method and application).

[0062] (3) The pL2R-Pgap-phaCAB recombinant vector was integrated into the ZMO1650 site of the genome of *Fermentomonas motilityis* NPΔΔ and transformed into *Fermentomonas motilityis* NPΔΔ to obtain the starting strain NPPg. The above operation was performed in accordance with the method described in the existing published patent (CN110358768A, A genome editing method based on the endogenous CRISPR-Cas system of *Fermentomonas motilityis* and its application). The gRNA sequence targeting the ZMO1650 site is shown in SEQ ID No. 7. The upstream arm 1650-UP and downstream arm 1650-Down sequences used to integrate the ZMO1650 site of the *Fermentomonas motilityis* NPΔΔ genome are shown in SEQ ID No. 11-12, respectively.

[0063] 2. Construction of recombinant strain LAHB-1

[0064] Figure 2A schematic diagram of the construction process of recombinant strain LAHB-1 is shown, and the specific steps are as follows:

[0065] (1) The dldh gene, pct gene and promoter Pgap were synthesized by standard gene synthesis technology (Suzhou Genewise Biotechnology Co., Ltd.). The dldh gene and pct gene were tandemly linked by the rbs ribosome binding sequence (as shown in SEQ ID No. 10).

[0066] (2) The promoter Pgap, pct gene and dldh gene were sequentially linked to obtain the Pgap-dldh-pct operon fragment; the Pgap-dldh-pct operon fragment was linked to the pEZ15A vector to obtain the pEZ-Pg recombinant vector; the above operation method and the source of the pEZ15A vector were carried out and obtained in accordance with the method described in the literature ([3] Yang S, Mohagheghi A, Franden MA, et al.Metabolic engineering of Zymomonas mobilis for 2, 3-butanediol production from lignocellulosic biomass sugars[J]. Biotechnology for biofuels, 2016, 9(1): 189.).

[0067] (3) The pEZ-Pg recombinant vector was transformed into the starting strain NPPg to obtain the recombinant strain LAHB-1.

[0068] 3. Fermentation performance test

[0069] 3.1 Fermentation Culture

[0070] The activated recombinant strain LAHB-1 (inoculum size 10%) was inoculated into RMG5 medium and cultured at 30 ℃ and 100 rpm until OD500. 600 >0.3, to obtain seed culture; inoculate the seed culture into RMG5 medium, culture at 30 ℃ and 100 rpm for 12 h, and add spectinomycin to a final concentration of 100 μg / mL.

[0071] 3.2 Quantitative analysis of P(3HB-co-LA)

[0072] Figure 3 A schematic diagram of the quantitative analysis process is shown, and the specific steps are as follows:

[0073] (1) Preparation of esterification solution: Prepare esterification solution with a volume ratio of methanol to sulfuric acid of 17:3, mix thoroughly and store at 4 °C for later use.

[0074] (2) Sample pretreatment: The sample to be tested was centrifuged at 25 ℃ and 4000 rpm to collect the bacterial cells in the sample and washed once with deionized water. After washing, it was placed in an oven and dried at 65 ℃ for 24 h. After drying, it was ground into powder. 15 mg was weighed and 1.5 mL of esterification solution and 1.5 mL of chloroform were added respectively. After mixing, it was reacted in a boiling water bath for 4 h. After the reaction cooled, 1 mL of deionized water was added, and the lower organic phase liquid was taken out for gas chromatography analysis.

[0075] (3) Gas chromatography (GC) analysis: The relevant parameters for GC analysis are shown in Table 2; the analysis programs for HP-5 gas chromatography column and DB-WAX gas chromatography column are shown in Tables 3-4 respectively.

[0076] Table 2: Parameters related to GC analysis

[0077]

[0078] Table 3: HP-5 Gas Chromatography Column Analysis Procedure

[0079]

[0080] Table 4: DB-WAX Gas Chromatography Column Analysis Program

[0081]

[0082] Figure 4 The fermentation performance test results of recombinant strain LAHB-1 are shown in the figure. Figure A is a statistical analysis of biomass; Figure B is a statistical analysis of specific growth rate (μ); and Figure C is a statistical analysis of polymer content and lactate fraction. The results show that the maximum biomass of recombinant strain LAHB-1 is 3.58, which is lower than the 5.22 of the original strain NPΔΔ. Figure 4 (Figure A in the original text); the specific growth rate of the recombinant strain LAHB-1 was 0.14±0.02, significantly lower than that of the original strain NPΔΔ (0.35±0.01). Figure 4 (See Figure B in the original text). The results showed that after introducing the P(3HB-co-LA) synthesis pathway, extracellular lactic acid affected cell growth by lowering the pH of the culture medium. Furthermore, the original strain NPΔΔ had zero polymer content and zero lactic acid content, indicating that it lacked the ability to synthesize P(3HB-co-LA); while in the recombinant strain LAHB-1, the P(3HB-co-LA) content was 2.80 ± 0.03 wt%, and the lactic acid content was 7.52 ± 0.81 mol%. Figure 4Figure C in the diagram shows that after introducing the P(3HB-co-LA) synthesis pathway, the recombinant strain LAHB-1 can synthesize P(3HB-co-LA) containing lactic acid components.

[0083] Example 2: Increasing the yield of D-lactic acid, a precursor of polyhydroxybutyrate lactate.

[0084] With changes in the proportion of lactic acid units, the appearance and mechanical properties of copolymer P(3HB-co-LA) changed significantly: when the lactic acid content was low, the material was generally opaque; however, as the proportion of lactic acid gradually increased, the transparency of the sample improved, and this trend was significant. Furthermore, due to the relative reduction of hydroxybutyric acid (HbA1c) in the copolymer P(3HB-co-LA) system, the brittleness of the material was alleviated, the toughness was improved, and the elongation at break also showed an increasing trend.

[0085] ZMB3, a key intermediate chassis strain with restricted ethanol metabolism, exhibits significant advantages in lactic acid production. Its core advantage lies in effectively limiting ethanol production by knocking out genes related to ethanol production, allowing carbon flux to be redirected towards the synthesis of other target compounds, particularly 2,3-butanediol (2,3-BDO). Furthermore, this strain demonstrates strong redox equilibrium and excellent growth and fermentation capabilities. Therefore, this embodiment uses ZMB3 as the original strain to further increase the yield of D-lactic acid, a precursor of the copolymer P(3HB-co-LA).

[0086] 1. Construct the starting strain ZMB3Pg

[0087] The Pgap-phaCAB operon fragment was ligated into the pL2R vector, and the resulting recombinant vector was integrated into the ZMO0959 site of the ZMB3 genome of *Z. motility fermentation mononuclear*, and transformed into *Z. motility fermentation mononuclear* ZMB3 to obtain the starting strain ZMB3Pg. The above operations were performed according to the method described in Section 1 of Example 1. The gRNA sequence targeting the ZMO0959 site is shown in SEQ ID No. 8, and the upstream arm 0959-UP and downstream arm 0959-Down sequences used for integration into the ZMO0959 site of the *Z. motility fermentation mononuclear* ZMB3 genome are shown in SEQ ID Nos. 13-14, respectively.

[0088] 2. Construction of recombinant strain LAHB-2

[0089] Figure 5A schematic diagram of the construction process of recombinant strain LAHB-2 is shown. The specific steps are as follows: the Pgap-dldh-pct operon fragment is ligated into the pEZ15A vector, and the resulting recombinant vector is transformed into the starting strain ZMB3Pg to obtain recombinant strain LAHB-2; the above operations are performed in accordance with the method described in Section 2 of Example 1.

[0090] 3. Fermentation performance test

[0091] Fermentation culture and quantitative analysis were performed according to the method described in Section 3 of Example 1. Figure 6 The fermentation performance test results of recombinant strain LAHB-2 are shown in the figure. Figure A is a statistical analysis of biomass; Figure B is a statistical analysis of extracellular lactic acid production (LA-Production); and Figure C is a statistical analysis of polymer content and lactate fraction. The results show that the growth curve of recombinant strain LAHB-2 is relatively delayed, reaching a maximum biomass of 3.37 after 36 h. Figure 6 (See Figure A in the original text). The results showed that, compared to recombinant strain LAHB-1, the growth rate of recombinant strain LAHB-2 was inhibited in the initial stage because it used part of its carbon source for lactic acid synthesis. Furthermore, recombinant strain LAHB-2 showed improved lactic acid synthesis: extracellular lactic acid production increased from 3.43±0.15 g / L in recombinant strain LAHB-1 to 11.45±1.2 g / L (…). Figure 6 (Figure B in the table); the P(3HB-co-LA) content decreased from 2.80±0.03 wt% in the recombinant strain LAHB-1 to 2.43±0.08 wt% (Figure B in the table). Figure 6 (See Figure C in the original text); the proportion of lactic acid component increased significantly, from 7.52±0.81 mol% in recombinant strain LAHB-1 to 12.62±1.25 mol% (Figure C in the original text). Figure 6 (Figure C in the diagram).

[0092] Example 3: Replacing the polyhydroxybutyrate lactate precursor with L-lactic acid

[0093] The lactic acid content of P(3HB-co-LA) is closely related to the formation efficiency of lactyl-CoA. The supply of lactic acid monomers directly restricts the accumulation ratio of lactic acid in the copolymer and the polymerization efficiency, which is crucial for the synthesis of copolymers with high lactic acid content. Generally, when D-lactic acid is used as a precursor, although engineered strains can synthesize P(3HB-co-LA), the lactic acid content is low. This is related to the lactic acid synthesis throughput and the substrate recognition efficiency of specific stereoisomers. On the other hand, the stereoisomers of lactic acid (L-form and D-form) not only affect the polymer composition but also extensively influence the crystallization behavior and material properties of polylactic acid (PLA) and its copolymers. The crystal structure formed by the stereocombination of poly(L-lactic acid) and poly(D-lactic acid) stereoisomers can significantly change the thermal and mechanical properties of the polymer, indicating that different chiral monomers have a key influence on polymer behavior.

[0094] This embodiment is based on the starting strain ZMB3Pg prepared in Example 2. The D-lactate dehydrogenase encoded by the dldh gene is replaced with the L-lactate dehydrogenase encoded by the lpldh gene. This alters the L-lactate monomer supply and stereochemical environment to further increase the lactic acid content in the polymer. The nucleotide sequence of the lpldh gene, as shown in SEQ ID No. 9, is derived from *Lactobacillus plantarum*.

[0095] 1. Construct recombinant strain LAHB-3

[0096] Figure 7 A schematic diagram of the construction process of recombinant strain LAHB-3 is shown, and the specific steps are as follows:

[0097] (1) The lpldh gene, pct gene and promoter Pgap were synthesized by standard gene synthesis technology (Suzhou Genewise Biotechnology Co., Ltd.). The lpldh gene and pct gene were tandemly linked by the rbs ribosome binding sequence.

[0098] (2) The promoter Pgap, pct gene and lpldh gene are sequentially linked to obtain the Pgap-lpldh-pct operon fragment.

[0099] (3) The Pgap-lpldh-pct operon fragment was ligated into the pEZ15A vector, and the resulting recombinant vector was transformed into the starting strain ZMB3Pg to obtain the recombinant strain LAHB-3; the above operations were performed in accordance with the method described in Section 2 of Example 1.

[0100] 2. Fermentation performance test

[0101] Fermentation culture and quantitative analysis were performed according to the method described in Section 3 of Example 1. Figure 8The fermentation performance test results of recombinant strain LAHB-3 are shown in the figure; Figure A is a statistical analysis of biomass; Figure B is a statistical analysis of polymer content and lactate fraction. The results show that the maximum biomass of recombinant strains LAHB-2 and LAHB-3 were 3.37 and 3.11, respectively, and both showed a tendency to plateau growth after 36 h. Figure 8 (See Figure A in the original text); the P(3HB-co-LA) content decreased from 2.43±0.08 wt% in recombinant strain LAHB-2 to 1.93±0.09 wt%. The lactic acid content in the polymer increased from 12.62±1.25 mol% in recombinant strain LAHB-2 to 14.76±0.42 mol%. Figure 8 (See Figure B in the diagram). The results indicate that switching the precursor type is beneficial for regulating the composition of copolymer P(3HB-co-LA).

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a recombinant strain for producing polyhydroxybutyrate lactate, characterized in that, Includes the following steps: SA-1. The promoter Pgap, pct gene and lactate dehydrogenase encoding gene are sequentially linked to obtain operon fragment I; The nucleotide sequence of the promoter Pgap is shown in SEQ ID No. 6; The nucleotide sequence of the pct gene is shown in SEQ ID No. 5; SA-2, Operator fragment I is ligated into the pEZ15A vector to obtain recombinant vector I; SA-3. Recombinant vector I was transformed into the starting strain, and positive clones were screened to obtain the recombinant strain.

2. The preparation method according to claim 1, characterized in that, The lactate dehydrogenase encoding gene mentioned in step SA-1 is selected from at least one of the dldh gene and the lpldh gene; The nucleotide sequence of the dldh gene is shown in SEQ ID No. 4; The nucleotide sequence of the lpldh gene is shown in SEQ ID No.

9.

3. The preparation method according to claim 1, characterized in that, The method for constructing the starting strain described in step SA-3 includes the following steps: SB-1. The promoter Pgap, phaA gene, phaB gene and phaC gene are sequentially linked to obtain operon fragment II; The nucleotide sequence of the promoter Pgap is shown in SEQ ID No. 6; The nucleotide sequences of the phaA, phaB, and phaC genes are shown in SEQ ID No. 1-3, respectively; SB-2. Operator fragment II is ligated into the pL2R vector to obtain recombinant vector II; SB-3. Integrate recombinant vector II into the genome of the original strain, and transfer it into the original strain to obtain the starting strain.

4. The preparation method according to claim 3, characterized in that, The original strain mentioned in step SB-3 is either *Morphozoa motifosum* NPΔΔ or *Morphozoa motifosum* ZMB3.

5. The preparation method according to claim 4, characterized in that, When the original strain is *Bacillus motilityis* NPΔΔ, the recombinant vector II is integrated into the ZMO1650 site of the *Bacillus motilityis* NPΔΔ genome via homologous recombination. Alternatively, if the original strain is *Zygomorpha motileis* ZMB3, the recombinant vector II is integrated into the ZMO0959 site of the *Zygomorpha motileis* ZMB3 genome via homologous recombination.

6. The preparation method according to claim 5, characterized in that, The gRNA sequence targeting the ZMO1650 site is shown in SEQ ID No. 7, and the upstream and downstream arm sequences for integration into the ZMO1650 site are shown in SEQ ID No. 11-12, respectively. Alternatively, the gRNA sequence targeting the ZMO0959 site is shown in SEQ ID No. 8, and the upstream and downstream arm sequences for integration into the ZMO0959 site are shown in SEQ ID No. 13-14, respectively.

7. A recombinant bacterial strain for producing polyhydroxybutyrate lactate, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. A method for preparing polyhydroxybutyrate lactate, characterized in that, Includes the following steps: The recombinant strain as described in claim 7 was inoculated into RMG5 medium and fermented to obtain a fermentation broth containing polyhydroxybutyrate lactate.

9. The method as described in claim 8, characterized in that, The RMG5 culture medium contains the following components: 50 g / L glucose, 10 g / L yeast extract, and 2 g / L KH2PO4.

10. The method as described in claim 8, characterized in that, The fermentation conditions include: an inoculum size of 10% for the recombinant strain, a fermentation temperature of 25-35 ℃, and a rotation speed of 50-150 rpm.

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