A genetically engineered bacterium capable of regulating the content of lactic acid component in polyhydroxyalkanoate and a construction method and application thereof

Abstract

The application discloses a genetically engineered bacterium capable of regulating the content of lactic acid component in polyhydroxyalkanoate, characterized in that the genetically engineered bacterium is a gene pct controlled by an m2 promoter cp The gene is integrated into the genome of the E. coli, and a recombinant strain WJ01 is constructed; then, the recombinant strain WJ01 is used as a starting strain, a pTrc99aABC plasmid and a pBAD-PTrc-lepgt plasmid are introduced into the recombinant strain WJ01 by calcium transformation to obtain a production strain. The application further discloses a construction method of the genetically engineered bacterium and application of the genetically engineered bacterium. The genetically engineered bacterium obtained by the method has little influence on cell growth, and polyhydroxyalkanoate with different contents of lactic acid component can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically, it relates to a genetically engineered bacterium that can regulate the lactic acid content in polyhydroxyalkanoates, its construction method, and its application. Background Technology

[0002] Currently, most commonly used plastics are synthesized from fossil fuels such as oil and natural gas, leading to environmental problems such as global warming and the accumulation of solid waste. These problems urge us to develop and utilize sustainable raw materials to produce bio-based polymers. Polyhydroxy alkanoates (PHA) are polyesters produced by microorganisms, and they have attracted widespread attention from researchers due to their excellent properties such as biodegradability, optical properties, and biocompatibility.

[0003] Polylactic acid (PLA) is a promising biomass-derived polymer because it can replace petroleum-based plastics and possesses several desirable properties, such as biodegradability, biocompatibility, and compostability. PLA itself is non-toxic; however, residual metal catalysts from traditional biochemical production methods can hinder its applications in medicine and other fields. Unfortunately, the lack of natural lactate polymerases currently prevents one-step in vivo production. Seiichi Taguchi et al., utilizing the substrate similarity principle, successfully created a system for the microbial biosynthesis of lactic acid-based polyesters—poly(3-hydroxybutyrate-lactic acid) P(3HB-co-LA)—by mutating PHA synthase.

[0004] Poly(3-hydroxybutyrate lactate) [P(3HB-co-LA)] is a member of the PHA family. This polymer combines the characteristics of polylactic acid (PLA) alone—transparent yet rigid—with poly(3-hydroxybutyrate)—opaque and brittle. Since P(3HB-co-LA) exhibits varying elasticity and transparency depending on its lactic acid content, the lactic acid component in the polymer needs to be carefully controlled.

[0005] P(3HB-co-LA) is synthesized from 3-hydroxybutyryl-CoA (3HB-CoA) and lactyl-CoA (LA-CoA) catalyzed by a mutated PHA synthase with lactate polymerization activity. To further increase the lactate content, researchers attempted anaerobic fermentation to increase the accumulation of lactate in the precursor material. The result was a significant increase in the molar ratio of lactate in the polymer to 47 mol%. However, due to the anaerobic environment's inhibitory effect on cell growth, the polymer dry weight decreased, accounting for only 2 wt% of the bacterial cells. Nduko et al. optimized the carbon source; using xylose as the carbon source, the lactate content in the polymer synthesized by the modified *E. coli* cells reached 34 mol%, a significant increase compared to 26 mol% in glucose. Further optimization of the PHA synthase further increased the lactate content to 60 mol%.

[0006] Previous studies have focused on simply increasing the lactic acid content in polymers. However, the lactic acid content in the resulting polymers is fixed and cannot be flexibly adjusted. This single production strategy is difficult to meet the market demand for polymers with different material properties.

[0007] Chinese patent application CN110295188A discloses a method for increasing the lactic acid content in poly(3-hydroxybutyrate-co-lactic acid) synthesized by *Escherichia coli*, using *E. coli* MG1655 as the host, controlling the recombinant expression of phaA, phaB, and phaCm genes via the trc promoter, and controlling the pct gene expression via the arabinose promoter. cp Recombinant E. coli MG-01 was constructed by recombinant expression of the genes. Based on this, the flavin isopentenyltransferase gene ubiX was knocked out, and / or the D-lactate dehydrogenase gene dld was knocked out, and / or propionyl-CoA transferase pct was expressed instead. cp The promoter is a constitutive ldhA promoter, and it has not been modified from the perspective of dynamically regulating lactate composition. Therefore, it is impossible to directly increase the lactate composition to a very high level. Summary of the Invention

[0008] This invention, from the perspective of dynamically regulating lactate composition, provides a better platform and regulatory means for the diversified production of P(3HB-co-LA). The strategy applied in this invention is the "metabolic transistor" strategy. The "metabolic transistor" strategy is based on the network topology of biosynthetic pathways. This strategy can control certain biosynthetic pathways by adjusting the flow distribution at some newly introduced nodes. This invention uses this strategy to regulate the synthesis of coenzyme Q8. Coenzyme Q8, as a substance that transfers reducing equivalents between electron donors (such as NADH dehydrogenase, succinate dehydrogenase) and the final electron acceptors (such as cytochrome oxidase or reductase), is an important component of the respiratory chain. This invention utilizes the lepgt gene to dynamically control the synthesis of coenzyme Q8, thereby effectively controlling the level of the cellular respiratory chain. Through the "metabolic transistor" strategy based on *E. coli*, more lactate can be produced under aerobic conditions. It provides a genetic means to control the activity of the electron transfer chain in vivo, control the utilization rate of intracellular NADH, and control the production of reduction products. Based on this strategy, the lactate composition in polymers can be regulated. Knocking out cytochrome oxidases can effectively reduce the respiratory chain level in cells. Therefore, by using Red recombination technology to knock out the three cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) located in the electron transport chain of strain WJ01, the strain's ability to synthesize lactic acid can be significantly enhanced, expanding the regulatory range of the lepgt gene on lactic acid components. Furthermore, the regulatory method of this invention flexibly regulates the lactic acid component in the polymer, rather than simply increasing the lactic acid ratio, allowing for precise regulation of the polymer's lactic acid composition within a specific range. Moreover, applying this method to polymer-producing strains with different backgrounds enables precise regulation of the lactic acid component in the polymer.

[0009] Therefore, the first objective of this invention is to provide a genetically engineered bacterium capable of controlling the lactic acid component content in polyhydroxyalkanoates. The second objective of this invention is to provide a method for constructing the aforementioned genetically engineered bacterium. The third objective of this invention is to provide an application of the above-described genetically engineered bacterium in the production of P(3HB-co-LA) with different lactic acid components.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] As a first aspect of the present invention, a genetically engineered bacterium capable of regulating the lactic acid component content in polyhydroxybutyrate esters is comprising the pct gene encoding a key enzyme in the metabolic pathway of pyruvate-to-polyhydroxybutyrate lactate synthesis controlled by the m2 promoter. cpThe recombinant strain WJ01 was constructed by integrating into the genome of Escherichia coli. Then, using the recombinant strain WJ01 as the starting strain, the production strain was obtained by calcium transformation into the pTrc99aABC plasmid and the pBAD-PTrc-lepgt plasmid.

[0012] Furthermore, using recombinant strain WJ01 as the starting strain, the two genes cyoABCD and cbdAB in strain WJ01 were knocked out using Red recombination technology to obtain strains WJ01-CYO and WJ01-CBD.

[0013] According to the present invention, the key enzyme in the metabolic pathway of polyhydroxybutyrate lactate synthesis from pyruvate is a PHA synthase mutant.

[0014] Furthermore, the gene pct cp The nucleotide sequence is shown in SEQ ID NO.12.

[0015] As a second aspect of the present invention, a method for constructing genetically engineered bacteria with adjustable lactic acid content in polyhydroxyalkanoates includes the following steps:

[0016] Step 1: PCT of the gene controlled by the m2 promoter cp The recombinant strain WJ01 was constructed by integrating it into the genome of Escherichia coli.

[0017] Step 2: Using recombinant strain WJ01 as the starting strain, the pTrc99aABC plasmid and pBAD-PTrc-lepgt plasmid were transformed into the strain through calcium transformation to construct the strain.

[0018] According to the present invention, the gene pct in step one cp The steps of genome integration are as follows:

[0019] A. Using Escherichia coli MG1655 as a template, PCR amplification was performed using primers Pct-1-F and Pct-1-R, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, to obtain fragment Pct-1;

[0020] B. Using pct with the gene cp Using the plasmid as a template, PCR amplification was performed using primers Pct-2-F and Pct-2-R, whose nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, to obtain fragment Pct-2;

[0021] C. Using Escherichia coli MG1655 as a template, PCR amplification was performed using primers Pct-3-F and Pct-3-R, whose nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively, to obtain fragment Pct-3;

[0022] D. Using fragments Pct-1, Pct-2, and Pct-3 as templates, PCR amplification was performed using primers Pct-1-F and Pct-3-R, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.6, respectively, to obtain pct-1 fragments that have homologous arms to the insertion site of the E. coli genome. cp Excerpt.

[0023] According to the present invention, the gene pct cp The nucleotide sequence is shown in SEQ ID NO.12.

[0024] As a third aspect of the present invention, a method for constructing genetically engineered bacteria with adjustable lactic acid content in polyhydroxyalkanoates includes the following steps:

[0025] Step 1: PCT of the gene controlled by the m2 promoter cp The recombinant strain WJ01 was constructed by integrating it into the genome of Escherichia coli. The recombinant strains WJ01-CYO and WJ01-CBD were obtained by deleting the cyoABCD and cbdAB genes in the WJ01 strain using Red recombination technology.

[0026] Step 2: Using recombinant strains WJ01-CYO or WJ01-CBD as the starting strain, the pTrc99aABC plasmid and pBAD-PTrc-lepgt plasmid are transformed through calcium conversion to obtain the desired structure.

[0027] According to the present invention, the gene pct cp The nucleotide sequence is shown in SEQ ID NO.12.

[0028] As a fourth aspect of the present invention, the use of the above-described genetically engineered bacteria in the production of P(3HB-co-LA) with different lactic acid components.

[0029] According to the present invention, the application of the genetically engineered bacteria in the production of P(3HB-co-LA) with different lactic acid components is to control the expression of the lepgt gene in the genetically engineered bacteria by adjusting the concentration of the inducer IPTG, thereby regulating the lactic acid component content in the polyhydroxyalkanoate.

[0030] The advantages of this invention are:

[0031] 1. This invention, through analysis of metabolic pathways and regulation, utilizes genetic engineering techniques to modify Escherichia coli, resulting in a genetically engineered Escherichia coli strain that can regulate the lactic acid component in P(3HB-co-LA).

[0032] 2. This invention allows for the control of lepgt gene expression by varying the concentration of different inducers, thereby gradient control of the intensity of the E. coli respiratory chain and flexibly regulating the lactic acid content in polyhydroxyalkanoates produced by recombinant strains, with minimal impact on cell growth. Through analysis and regulation of metabolic pathways, and by modifying E. coli using genetic engineering techniques, polyhydroxyalkanoates with varying lactic acid content can be obtained.

[0033] 3. This invention can dynamically control the content of polyhydroxyalkanoates with different lactic acid components to obtain polymers. The control method is simple; different lactic acid components can be produced simply by changing the inducing agent. Moreover, the inducing agent and the lactic acid component have a certain linear relationship, so the content of different lactic acid components can be quantitatively controlled. Attached Figure Description

[0034] Figure 1 Metabolic diagram of Escherichia coli producing P(3HB-co-LA) using glucose or xylose.

[0035] Figure 2 This explains the mechanism by which the lepgt gene regulates the respiratory chain in E. coli.

[0036] in, Figure 1 and Figure 2 The English annotations are as follows:

[0037] Glucose, D-xylose, Pyruvate, Acetyl-CoA, glyceraldehyde-3-P, Acetoacetyl-CoA, LA-CoA, 3HB-CoA, P(3HB-co-LA), ubiX (3-octenyl-4-hydroxybenzoic acid carboxylase), phaA (β-ketothiolase), phaB (NADPH-dependent acetyl-CoA reductase), pct cp (Propionyl-CoA transferase mutant), phaCm (polyhydroxyalkanoate synthase mutant), Cytochrome oxidase, lepgt (4-hydroxybenzoic acid geranyl transferase), PPP (pentose phosphate pathway), glycolysis, TCA cycle (tricarboxylic acid cycle). Detailed Implementation

[0038] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0039] 1. The strains and plasmids of this invention are derived from the following sources:

[0040] (1) pRedCaS9 plasmid, pGRB-sgRNA plasmid, see "Metabolic engineering of Escherichia coli using CRISPR–Cas9meditated genome editing" for details, Yifan Li, ZhenquanLin, Can Huang, Yan Zhang, Zhiwen Wang, Ya-jie Tang, Tao Chen, Xueming Zhao, Metabolic Engineering, 2015, 31: 13-21.

[0041] (2) PKD4 plasmid, PKD46 plasmid, and MG1655 strain, commercially available.

[0042] (3) Gene pct cp Its sequence is shown in SEQ ID NO.12.

[0043] (4) pTrc99aABC plasmid, pBAD33-trc-pct cp For details on the plasmid, see “The effect of short-chain thioesterase deletion on the synthesis of polyhydroxybutyrate lactate in Escherichia coli”, Wei Xiangju, Wu Ju, Guo Pengye, et al., Chinese Journal of Biotechnology, 2021, 37(1):11.

[0044] (5) pBAD-PTrc-lepgt plasmid, see "Metabolic transistor strategy for controlling electron transfer chain activity in Escherichia coli" for details, Wu H, TuliL, Bennett GN, et al.. Metabolic Engineering, 2015, 28: 159-168.

[0045] 2. The pyruvate-to-polyhydroxybutyrate (PHA) lactate synthesis pathway involves four enzymes: β-ketothiolysis, acetyl-CoA reductase, propionate-CoA transferase mutant, and PHA synthase mutant. In *Ralstonia eutropha*, the genes encoding β-ketothiolysis and acetyl-CoA reductase are phaA and phaB, respectively. In *Pseudomonas fluorescens* strain 2P24, the gene encoding PHA synthase is phaC, which mutates to phaCm. In *Clostridium propionicum*, the gene encoding propionate-CoA transferase is pct, which mutates to pct. cp .

[0046] 3. The sequence of the present invention is shown in Table 1 and Table 2.

[0047] Table 1. Primers and m2 promoter sequences for Donor DNA construction

[0048]

[0049] Table 2 NCBI gene numbers of cytochrome oxidase subunits

[0050]

[0051]

[0052] Example 1. Gene pct cp Genome integration

[0053] First, CRISPR gene editing technology is used to edit the pct gene controlled by the m2 promoter (a constitutive strong promoter). cp The recombinant strain WJ01 was constructed by integrating it into the genome of Escherichia coli.

[0054] The specific steps of gene integration are as follows:

[0055] For gene pct cp For genome integration, primers were first designed (primer sequences are shown in Table 1). First, using primers Pct-1-F and Pct-1-R (containing the m2 promoter sequence) as a template, *E. coli* MG1655 was used to amplify the Pct-1 fragment by PCR. Then, primers Pct-2-F (containing the m2 promoter sequence) and Pct-2-R were used to amplify the fragment containing the pct gene. cp The plasmid (in this example, the plasmid is pBAD33-trc-pct) cpUsing *E. coli* MG1655 as a template, PCR amplification yielded fragment Pct-2. Using primers Pct-3-F and Pct-3-R, using *E. coli* MG1655 as a template, PCR amplification yielded fragment Pct-3. Then, primers pct-1-F and pct-3-R were added to perform PCR amplification using the three fragments Pct-1, Pct-2, and Pct-3 as templates, obtaining pct-3 fragments with homologous arms to the *E. coli* genome insertion site. cp Fragment (Dorner DNA). Prepare E. coli strain MG1655 electroporation competent cells, electroporate pRedCaS9 plasmid (100 μg / ml zirconia-resistant, cultured at 30℃ for approximately 10 h). After single colonies grow on the plate, pick a single colony and transfer it to 3 ml of LB liquid medium. Activate in a shaker at 30℃ for 8 h. Take 1 ml of the bacterial culture and add it to 50 ml of LB liquid medium (100 μg / ml zirconia, 0.1 mM IPTG). Incubate in a shaker at 30℃ until OD600 = 0.4-0.6. Pre-chill on ice for 20 min, centrifuge at 5500 rpm for 5 min, discard the supernatant, resuspend in pre-chilled 10% glycerol (Sigma), and centrifuge at 5500 rpm for 5 min. Repeat once. Discard the supernatant, add 300-500 μl of pre-chilled 10% glycerol, aliquot 50 μl into sterile EP tubes, and store at -80℃. Then, 100 ng of pGRB-sgRNA (which guides the CaS9 protein to cleave the yghx pseudogene) and 100 ng of Dorner fragment were added to the prepared electroporation competent cells containing the pRedCaS9 plasmid. Electroporation was performed using a Bio-Rad electroporator. Electroporation was performed in bacterial mode 1 (1.8 kV, 5 ms). After electroporation, 950 μl of LB was quickly added to the electroporation cuvette, and the cells were incubated at 30°C for 2 h. 100 μl of the bacterial culture was then plated onto plates containing 100 μg / ml zirconia-100 μg / ml ampicillin. The plates were incubated at 30°C for approximately 12 h. Single colonies were selected, and colony PCR was performed using identification primers to screen for successfully knocked-in strains, which were named WJ01.

[0056] Example 2. Knockout of the cytochrome oxidase gene

[0057] Starting with WJ01, strains WJ01-CYO and WJ01-CBD were obtained by knocking out the cyoABCD and cbdAB genes in strain WJ01 using Red recombination technology.

[0058] The specific steps for gene knockout are as follows:

[0059] (1) Obtaining the homologous arm of the knockout gene carrying the Kan fragment

[0060] For the knockout of the cytochrome oxidase gene, primers were first designed (primer sequences are shown in Table 1). Using primers cyoABCD-F and cyoABCD-R as a template, the PKD4 plasmid was used to amplify the cyoABCD-Kan fragment by PCR; using primers cbdAB-F and cbdAB-R as a template, the PKD4 plasmid was used to amplify the cbdAB-Kan fragment by PCR.

[0061] (2) Preparation of electrocompetent cells with PKD46 plasmid

[0062] First, calcium transformation of the PKD46 plasmid was performed into calcium-competent cells of strain WJ01. The calcium transformation steps were as follows: First, the calcium-competent cells were removed from the cryogenic freezer and thawed on ice. 5 μL of the PKD46 plasmid was mixed with the calcium-competent cells, incubated on ice for 20 min, heat-shocked at 42℃ for 90 s, then immediately incubated on ice for 2 min. 900 μL of LB liquid was added for recovery for 1 h, followed by incubation at 37℃ and 220 rpm for 1 h. Then, after centrifugation at 5500 rpm for 3 min, 50 μL was plated onto plates with the appropriate antibiotic resistance. After the strain was constructed, it was stored in glycerol (25% v / v).

[0063] Then, electrocompetent states are prepared, and the specific method is as follows:

[0064] 1) Incubate recombinant E. coli strain WJ01 with pKD46 overnight at 30°C and 220 rpm.

[0065] 2) Take 1 mL of the overnight culture of recombinant Escherichia coli strain WJ01 with pKD46 and inoculate it into 50 mL of liquid LB. Incubate for about 1 hour and measure OD600.

[0066] 3) When OD600 reaches 0.2-0.3, add arabinose to a final concentration of 10mM for induction, and continue culturing until OD600 reaches 0.4-0.6.

[0067] 4) After ice bath for 20 min, centrifuge at 5500 rpm for 5 min at 4℃ and discard the supernatant.

[0068] 5) Resuspend the bacterial cells in 5 mL of pre-cooled 10% glycerol under sterile conditions, centrifuge at 5500 rpm for 5 min at 4°C, remove the supernatant, and repeat this process 3 times.

[0069] 6) After removing the supernatant, add 1 mL of pre-cooled 10% glycerol, gently resuspend, and dispense into 50 μL tubes.

[0070] 7) Store at -80℃.

[0071] (3) Electroconversion

[0072] Thaw the prepared WJ01 electrocompetent cells on ice. Take 2-6 μL of the PCR fragment recovery solution (cydAB-Kan, cyoABCD-Kan, cbdAB-Kan) and mix it with the electrocompetent cells (do not vigorously). Transfer to a pre-chilled electroporation cuvette and electroporate at 2.0-2.5 kV. After electroporation, quickly add 900 μL of fresh LB broth to suspend the cells in the cuvette, then transfer to an EP tube. Incubate at 37℃ and 220 rpm for 2-3 hours, then centrifuge at 5500 rpm for 3 minutes. Discard most of the supernatant, reserving approximately 100 μL. Spread 50 μL onto plates with the appropriate kanamycin resistance. Incubate at 37℃ for approximately 12 hours. Select single colonies and use identification primers to perform colony PCR to screen for successfully knocked-in strains, naming them WJ01-CYO and WJ01-CBD.

[0073] Example 3 describes the production strains that can produce different lactic acid fractions P(3HB-co-LA) through shake-flask fermentation.

[0074] Starting with WJ01, WJ01-CYO, and WJ01-CBD, plasmids pTrc99aABC and pBAD-PTrc-lepgt were introduced via calcium transformation to obtain strains WJ01(pTrc99aABC&pBAD-PTrc-lepgt), WJ01-CYO(pTrc99aABC&pBAD-PTrc-lepgt), and WJ01-CBD(pTrc99aABC&pBAD-PTrc-lepgt). The calcium conversion procedure is as follows: First, the calcium-competent cells were removed from the cryogenic freezer and thawed on ice. 5 μL each of the pTrc99aABC and pBAD-PTrc-lepgt plasmids were mixed with the calcium-competent cells, incubated on ice for 20 min, followed by a heat shock at 42℃ for 90 s, and then immediately incubated on ice for 2 min. 900 μL of LB liquid was added for recovery for 1 h, followed by incubation at 37℃ and 220 rpm for 1 h. After centrifugation at 5500 rpm for 3 min, 50 μL was plated onto plates with the appropriate antibiotic resistance. After the strain was constructed, it was stored in glycerol (25% v / v).

[0075] Shake-flask fermentation procedure: Seed culture stored in glycerol tubes was inoculated into test tubes containing 3 mL LB medium and cultured overnight. Then, it was transferred to Erlenmeyer flasks containing 50 mL M9 medium + 2 g / L yeast extract + 5 g / L xylose medium, with an inoculum size of 1%. The culture conditions were 37°C and 220 rpm. After 10 h of secondary seed culture, the culture was transferred to shake-flask fermentation medium (M9 medium). M9 medium was supplemented with 10 g / L xylose and 2 g / L yeast extract. The inoculum size was 1%, and IPTG inducer was added to final concentrations of 0.1 mM, 0.3 mM, and 0.6 mM. The culture conditions were 30°C and 220 rpm.

[0076] The composition of M9 medium (per liter) is: Na2HPO4·12H2O 15.12g, KH2PO4 3g, NaCl 0.5g, MgSO4·7H2O 0.5g, CaCl2 0.011g, NH4Cl 1g, and 1% vitamin B1 0.2mL.

[0077] Extraction and analysis methods for P(3HB-co-LA):

[0078] (1) Preparation of freeze-dried samples

[0079] After fermentation, take 20 mL of the bacterial culture, centrifuge at 8000 rpm for 10 min, and discard the supernatant. Wash the bacterial cells twice with 40 mL of deionized water, then resuspend in 1.5 mL of deionized water. Quick-freeze with liquid nitrogen and then freeze-dry under vacuum for two days.

[0080] (2) Preparation of esterification solution

[0081] Take 170 mL of methanol, add 1 g / L of benzoic acid, and then slowly add 30 mL of 98 wt% concentrated sulfuric acid (stirring with a glass rod while adding).

[0082] (3) Polymer extraction: Approximately 15 mg of lyophilized sample was placed in an esterification tube, and 1.5 mL of analytical grade chloroform and 1.5 mL of esterification solution were added. Sodium 3-hydroxybutyrate standard was prepared into a 100 g / L aqueous solution, and 45, 75, 105, 135, 165, and 195 μL were placed in esterification tubes, respectively, and treated using the same method as the sample, corresponding to final concentrations of 3, 5, 7, 9, 11, and 13 g / L. Sodium D-lactic acid standard was prepared into a 25 g / L aqueous solution, and 30, 60, 90, 120, and 150 μL were placed in esterification tubes, respectively, and treated using the same method as the sample, corresponding to final concentrations of 0.5, 1, 1.5, 2, and 2.5 g / L. Both the sample and the standard were esterified at a constant temperature of 100 °C for 4 h in a metal bath. After esterification, the sample was cooled in an ice bath. 750 mL of deionized water was added, and the mixture was vortexed for 2 min. Low-speed centrifugation separates the aqueous and organic phases, and the chloroform layer (lower layer) is taken for GC analysis.

[0083] (4) GC analysis

[0084] The monomer composition of polymers in cells was determined using a GC-2014 gas chromatograph (Shimadzu, Japan). The chromatographic column was an RX-5 capillary column, 30 m long and 0.25 mm inner diameter. A flame ionization detector was used. High-purity nitrogen was used as the carrier gas, hydrogen as the fuel gas, and air as the combustion-supporting gas. An AOC-20S automated sampler was used, with ethanol as the cleaning agent. The GC analysis program was as follows: initially held at 54 °C for 4 min, then increased to 80 °C at a rate of 5 °C / min, followed by increases to 125 °C at a rate of 10 °C / min, then to 180 °C at a rate of 30 °C / min, and finally increased to 220 °C at a rate of 20 °C / min and held for 220 min. Biomass was determined by spectrophotometry at 600 nm.

[0085] The results of shake-flask fermentation using xylose as the carbon source are shown in Tables 3, 4 and 5.

[0086] Table 3. P(3HB-co-LA) yield and LA component content of genetically engineered strain WJ01

[0087]

[0088] Table 4. P(3HB-co-LA) yield and LA component content of engineered bacteria with cytochrome oxidase gene deletion.

[0089]

[0090] Table 5. P(3HB-co-LA) yield and LA component content of genetically engineered bacterium JX04

[0091]

[0092]

[0093] The results in Tables 3 and 4 show that, using xylose as a substrate, fermentation experiments were conducted, and by controlling the expression of the lepgt gene with different IPTG concentrations, polymers with different lactic acid content (10.6-27.7 mol% LA) were obtained in the WJ01 recombinant strain. At the same time, polymers with different lactic acid content (29.3-41.8 mol% LA) were also obtained in the cytochrome oxidase gene-deficient strain. This demonstrates the effectiveness of dynamically regulating the lactic acid content in polymers using the "metabolic transistor strategy".

[0094] The results in Table 5 show that this strategy has less impact on cell growth and is more effective in regulating lactate components.

[0095] In summary, this invention uses wild-type *Escherichia coli* MG1655 as the starting strain to construct a heterologous P(3HB-co-LA) production pathway. Lactic acid, as an important precursor in P(3HB-co-LA) production, affects the lactic acid content in P(3HB-co-LA) due to its intracellular accumulation. Therefore, utilizing the *lepgt* gene as a "metabolic transistor strategy" allows for dynamic control of coenzyme Q8 synthesis, thereby effectively controlling the cellular respiratory chain. Through this *E. coli*-based "metabolic transistor" strategy, the activity of the electron transfer chain in vivo can be controlled, as can the utilization rate of intracellular NADH and the production of reduction products. Based on this strategy, the lactic acid composition in polymers can be regulated.

[0096] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> East China University of Science and Technology <120> A genetically engineered bacterium capable of controlling the lactic acid content in polyhydroxyalkanoates, its construction method, and its application. <130> 221032 <141> 2022-02-15 <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 76 <212> DNA <213> Artificial Sequence <400> 1 tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgttaacggt taacaatttc 60 acacaggaaa cagacc 76 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <400> 2 cagccttggt agagataatc agttc 25 <210> 3 <211> 101 <212> DNA <213> Artificial Sequence <400> 3 ggtctgtttc ctgtgtgaaa ttgttaaccg ttaacaattc cacacattat acgagccgga 60 tgattaattg tcaacatcca gcaactcttg tgggaaatct t 101 <210> 4 <211> 101 <212> DNA <213> Artificial Sequence <400> 4 tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgttaacggt taacaatttc 60 acacaggaaa cagaccatga gaaaggttcc cattattacc g 101 <210> 5 <211> 50 <212> DNA <213> Artificial Sequence <400> 5 aagacgtaat cccgtaagag ataaatcagg acttcatttc cttcagaccc 50 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 tttatctctt acgggattac gtctt 25 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <400> 7 tggttatcga tatcatgata ctgcgg 26 <210> 8 <211> 80 <212> DNA <213> Artificial Sequence <400> 8 atgagactca ggaaatacaa taaaagtttg ggatggttgt cattatttgc aggcactgta 60 cgtcttgagc gattgtgtag 80 <210> 9 <211> 80 <212> DNA <213> Artificial Sequence <400> 9 ttagtgcatc atcatgttgt agttgaggtt ccacataatc cagatggagc ctacaaccag 60 gatgtaacgc actgagaagc 80 <210> 10 <211> 80 <212> DNA <213> Artificial Sequence <400> 10 atgtgggatg tcattgattt atcgcgctgg cagtttgctc tgaccgcgct gtatcacttt 60 cgtcttgagc gattgtgtag 80 <210> 11 <211> 80 <212> DNA <213> Artificial Sequence <400> 11 ttagtacaac tcgttttcgt tacggcggag agtttctgtt gtcatgcgcc cccacatttt 60 gatgtaacgc actgagaagc 80 <210> 12 <211> 1575 <212> DNA <213> Artificial Sequence <400> 12 atgagaaagg ttcccattat taccgcagat gaggctgcaa agcttattaa agacggtgat 60 acagttacaa caagtggctt cgttggaaat gcaatccctg aggctcttga tagagctgta 120 gaaaaaagat tcttagaaac aggcgaaccc aaaaacatta catatgttta ttgtggttct 180 caaggtaaca gagacggaag aggtgctgag cactttgctc atgaaggcct tttaaaacgt 240 tacatcgctg gtcactgggc tacagttcct gctttgggta aaatggctat ggaaaataaa 300 atggaagcat ataatgtatc tcagggtgca ttgtgtcatt tgttccgtga tatagcttct 360 cataagccag gcgtatttac aaaggtaggt atcggtactt tcattgaccc cagaaatggc 420 ggcggtaaag taaatgatat taccaaagaa gatattgttg aattggtaga gattaagggt 480<00​​gctgatgaaa gcggaaatat cacatttgag aaagaagctg ctcctctgga aggaacttca 600 gtatgccagg ctgttaaaaa cagtggcggt atcgttgtag ttcaggttga aagagtagta 660 aaagctggca ctcttgaccc tcgtcatgta aaagttccag gaatttatgt tgactatgtt 720 gttgttgctg acccagaaga tcatcagcaa tctttagatt gtgaatatga tcctgcatta 780 tcaggcgagc atagaagacc tgaagttgtt ggagaaccac ttcctttgag tgcaaagaaa 840 gttattggtc gtcgtggtgc cattgaatta gaaaaagatg ttgctgtaaa tttaggtgtt 900 ggtgcgcctg aatatgtagc aagtgttgct gatgaagaag gtatcgttga ttttatgact 960 ttaactgctg aaagtggtgc tattggtggt gttcctgctg gtggcgttcg ctttggtgct 1020 tcttataatg cggatgcatt gatcgatcaa ggttatcaat tcgattacta tgatggcggc 1080 ggcttagacc tttgctattt aggcttagct gaatgcgatg aaaagggcaa tatcaacgtt 1140 tcaagatttg gccctcgcat cgctggttgt ggtggtttca tcaacattac acagaataca 1200 cctaaggtat tcttctgtgg tactttcaca gcaggtggct taaaggttaa aattgaagat 1260 ggcaaggtta ttatgttca agaaggcaag cagaaaaaat tcttgaaagc tgttgagcag 1320 attacattca atggtgacgt tgcacttgct aataagcaac aagtaactta tattacagaa 1380 agatgcgtat tccttttgaa ggaagatggt ttgcacttat ctgaaattgc acctggtatt 1440 gatttgcaga cacagattct tgacgttatg gattttgcac ctattattga cagagatgca 1500 aacggccaaa tcaaattgat ggacgctgct ttgtttgcag aaggcttaat gggtctgaag 1560 gaaatgaagt cctga 1575

Claims

1. A genetically engineered bacterium capable of regulating the content of lactic acid component in polyhydroxyalkanoate, characterized in that, It is a gene controlled by m2 promoter pct cp The recombinant strain WJ01 was constructed by integrating the gene into the genome of E. coli, then the WJ01 strain was used as the starting strain, and the Red recombination technology was used to knockout the two genes in the WJ01 strain to obtain the strains WJ01-CYO and WJ01-CBD, and the production strain was obtained by transforming the pTrc99aABC plasmid and the pBAD-PTrc- cyoABCD cbdAB plasmid through calcium transformation; the nucleotide sequence of the m2 promoter is shown as SEQ ID NO. 1; the nucleotide sequence of the gene lepgt pct cp is shown as SEQ ID NO. 12.​​ 2. A method for constructing a genetically engineered bacterium with adjustable lactic acid content in polyhydroxyalkanoates as described in claim 1, characterized in that, comprising the following steps: Step 1: Genes controlled by the m2 promoter pct cp A recombinant strain, WJ01, was constructed by integrating it into the genome of *E. coli*. The recombinant strain WJ01 was then constructed using Red recombination technology. cyoABCD , cbdAB Two genes were deleted to obtain recombinant strains WJ01-CYO and WJ01-CBD; Step two, using recombinant strain WJ01-CYO or WJ01-CBD as the starting strain, pTrc99aABC plasmid and pBAD-PTrc- lepgt plasmid were transformed by calcium transformation to construct and obtain.

3. The construction method of claim 2, wherein, The gene of step one pct cp The steps for genomic integration of the gene of step one are as follows: A. PCR amplification was performed with the template of E. coli MG1655, the primers Pct-1-F and Pct-1-R with the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3 respectively, to obtain the fragment Pct-1; B. plasmid with gene pct cp PCR amplification was performed with the primers Pct-2-F and Pct-2-R having nucleotide sequences as shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively, using the plasmid with gene C. PCR amplification was performed with the template of E. coli MG1655, the primers Pct-3-F and Pct-3-R with the nucleotide sequences shown in SEQ ID NO. 6 and SEQ ID NO. 7 respectively, to obtain the fragment Pct-3; D. PCR was performed using the primers Pct-1-F and Pct-3-R with the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 7, respectively, as templates and the fragments Pct-1, Pct-2, and Pct-3, to obtain the fragments having homologous arms at the insertion site in the genome of E. coli pct cp fragments.

4. The use of the genetically engineered bacteria according to claim 1 in the production of P(3HB-co-LA) with different lactic acid component contents.

5. The genetically engineered bacteria of claim 4, wherein the genetically engineered bacteria are used in the production of P(3HB-co-LA) with different compositions of lactic acid. It is by adjusting the concentration of inducer IPTG to control the expression of genes in genetically engineered bacteria lepgt The expression of genes, thereby regulating the content of lactic acid component in polyhydroxyalkanoate.

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