Method for constructing cupriavidus necator strain capable of producing methylmalate using organic and inorganic carbon sources and the strain

Abstract

This invention relates to the field of agricultural biotechnology, specifically to a method and strain for constructing an engineered strain of *Hookworm Copper-Gropping Bacteria* capable of producing methylmalic acid using both organic and inorganic carbon sources. This application involves knocking out the restriction endonuclease encoding gene of the type I restriction modification system in the genome of wild-type *Hookworm Copper-Gropping Bacteria* H16. 、 Promoters adapted for methylmalate synthase expression in *Hypotriculus clavatum* were screened, and methylmalate synthases with high catalytic activity in *Hypotriculus clavatum* were obtained, capable of effectively catalyzing methylmalate synthesis in *Hypotriculus clavatum*. A methylmalate transporter protein that promotes methylmalate excretion was also screened, as well as a gene encoding the *Hypotriculus clavatum* endogenous pyruvate dehydrogenase complex subunit that promotes methylmalate synthesis when sodium gluconate is used as a carbon source.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method and strain for constructing an engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources. Background Technology

[0002] Carbon recovery technology based on microbial cell factories has application potential. Recent reports indicate that Hookworm copper-eating bacteria H16, modified using metabolic engineering techniques, can directly convert CO2 into high-value chemicals.

[0003] Methylmalic acid is a C5 precursor in the L-isoleucine synthesis pathway in microorganisms such as *Leptospira questionmark* and *Methanococcus japonicus*. This compound is produced by a one-step condensation of pyruvate and acetyl-CoA via methylmalic acid synthase (CimA, EC2.3.3.21). Therefore, the biosynthesis of methylmalic acid can be further used as a functional probe to study and regulate the carbon flux distribution at the pyruvate-acetyl-CoA node in *Leptospira questionmark*. Furthermore, methylmalic acid is a key precursor for the production of polymethyl methacrylate (PMMA), a polymer with applications in healthcare and food processing. To date, microbial production of methylmalic acid has primarily relied on heterotrophic processes using carbohydrates as carbon sources and photosynthetic bacteria. The direct conversion of CO2 to methylmalic acid using chemoautotrophic platforms independent of light energy has not been reported.

[0004] As a facultative chemoautotrophic bacterium of the β-Proteobacteria phylum, hookworm copper-loving bacteria ( Cupriavidus necatorH16 exhibits metabolic diversity. It can not only undergo heterotrophic growth on multiple carbon sources, including fructose and gluconate, but also autotrophically grow using CO2 through a complete Calvin cycle. Another significant characteristic of *Hookworm Copper-Growing Bacteria* is its ability to synthesize polyhydroxybutyrate (PHB) using acetyl-CoA as a precursor. This is a biodegradable polymer compound that constitutes more than 80% of the cell's dry weight. The three genes involved in this pathway—β-ketothiolysis, acetyl-CoA reductase, and PHB synthase—form a single phaCAB operon, which is constitutively expressed under various growth conditions. Previous studies have shown that *Hookworm Copper-Growing Bacteria* can produce 61 g / L of PHB within 40 hours under autotrophic conditions with CO2 as the sole carbon source. Based on this characteristic, carbon flux has been diverted from PHB synthesis to the production of value-added products by designing pathways starting from acetyl-CoA or its upstream precursor pyruvate, thereby enabling the biosynthesis of compounds such as isobutanol, (R)-1,3-butanediol, and resveratrol. The supply of precursors is a key factor determining the yield of products in microbial cell factories; therefore, increasing precursor supply is an important strategy for enhancing microbial production performance. However, compared to model organisms such as *Escherichia coli*, little is currently known about the physiology and carbon flux regulation at key metabolic branches of *Hookworm* *Copper-Lowering Bacteria*. Summary of the Invention

[0005] To achieve the production of methylmalic acid using sodium gluconate and the greenhouse gas carbon dioxide as carbon sources, the present invention aims to provide a method for constructing an engineered strain of hookworm copper-loving bacteria that can produce methylmalic acid using both organic and inorganic carbon sources.

[0006] Another object of the present invention is to provide an engineered strain of hookworm copper-loving bacteria that can produce methylmalic acid using both organic and inorganic carbon sources.

[0007] Another object of the present invention is to provide a method for producing methylmalic acid by fermentation.

[0008] The method for constructing an engineered hookworm copper-boring bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, according to this application, includes the following steps: Knock out the restriction endonuclease encoding gene of type I restriction modification system in the genome of wild-type hookworm copper-eating bacteria, wherein the amino acid sequence of the restriction endonuclease of type I restriction modification system is shown in SEQ ID NO: 1; The gene encoding methylmalate synthase was overexpressed in the wild-type strain of *Hookworm Copper-Loving Fungus*, wherein the amino acid sequence of the methylmalate synthase is shown in SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 7; and The gene encoding the methylmalate transporter was overexpressed in the wild-type strain of *Hookworm Copper-Loving Bacterium*, and the amino acid sequence of the methylmalate transporter is shown in SEQ ID NO: 9.

[0009] The method for constructing an engineered hookworm copper-boring bacterium capable of producing methyl malic acid using both organic and inorganic carbon sources, according to this application, further comprises the following steps: Using arabinose-induced P BAD The promoter regulates the expression of the gene encoding the methylmalate synthase, wherein the arabinose-inducible P BAD The nucleotide sequence of the promoter is shown in SEQ ID NO: 4.

[0010] The method for constructing an engineered hookworm copper-boring bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, according to this application, further comprises the following steps: When constructing an engineered strain of *Hookworm Copper-Gropping Bacterium* that produces methylmalic acid using an organic carbon source, the encoding gene of an endogenous pyruvate dehydrogenase complex subunit is overexpressed in the wild-type strain of *Hookworm Copper-Gropping Bacterium*. The endogenous pyruvate dehydrogenase complex subunit is pyruvate dehydrogenase complex subunit PdhA1, and the amino acid sequence of pyruvate dehydrogenase complex subunit PdhA1 is shown in SEQ ID NO: 11. When constructing an engineered strain of *Hookworm Copper-Gropping Bacterium* that produces methylmalic acid using an inorganic carbon source, the encoding gene of an endogenous pyruvate dehydrogenase complex subunit is overexpressed in the wild-type strain of *Hookworm Copper-Gropping Bacterium*. The endogenous pyruvate dehydrogenase complex subunit is pyruvate dehydrogenase complex subunit PdhA2, and the amino acid sequence of pyruvate dehydrogenase complex subunit PdhA2 is shown in SEQ ID NO: 13.

[0011] According to the method for constructing an engineered hookworm copper-loving bacterium that can produce methylmalic acid using both organic and inorganic carbon sources, the nucleotide sequence of the gene encoding the methylmalic acid synthase is shown in SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8.

[0012] The method for constructing an engineered hookworm copper-eating bacterium that can produce methylmalic acid using both organic and inorganic carbon sources, according to this application, is characterized in that the nucleotide sequence of the methylmalic acid transporter protein encoding gene is shown in SEQ ID NO: 10.

[0013] The engineered hookworm copper-producing bacterium according to this application, capable of producing methylmalic acid using both organic and inorganic carbon sources, is a wild-type hookworm copper-producing bacterium with the following genomic characteristics: The restriction endonuclease encoding gene of the type I restriction modification system is knocked out, wherein the amino acid sequence of the restriction endonuclease of the type I restriction modification system is shown in SEQ ID NO: 1; Overexpression of the gene encoding methylmalate synthase, wherein the amino acid sequence of said methylmalate synthase is as shown in SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 7; and The gene encoding the methylmalate transporter was overexpressed, and the amino acid sequence of the methylmalate transporter is shown in SEQ ID NO: 9.

[0014] The application of the hookworm copper-boring bacteria engineered by this application, which can utilize both organic and inorganic carbon sources to produce methylmalic acid, for the production of methylmalic acid.

[0015] The method for producing methylmalic acid according to this application is characterized in that the method comprises the following steps: The engineered hookworm copper-loving bacteria, which can produce methyl malic acid using both organic and inorganic carbon sources, was cultured by fermentation using organic or inorganic carbon sources.

[0016] The method for producing methylmalic acid according to this application is characterized in that, during fermentation using an organic or inorganic carbon source, the yield of methylmalic acid is increased by adjusting the concentration of the nitrogen source in the culture medium.

[0017] Advantages of the technical solution in this application: 1. This application improves the transformation efficiency of exogenous plasmids in *Hypophthalmia ulmoides* by knocking out the restriction endonuclease encoding gene of the type I restriction modification system in the wild-type *Hypophthalmia ulmoides* H16 genome. Based on this, an engineered strain of *Hypophthalmia ulmoides* capable of synthesizing methylmalic acid using sodium gluconate and carbon dioxide as carbon sources was constructed.

[0018] The advantages of this application in using the engineered strain H16 of *Hookworm Copper-Greeding Bacterium* for the synthesis of methylmalic acid lie in its broad substrate spectrum. This bacterium can utilize organic matter such as sodium gluconate as a carbon source for product synthesis, while also possessing highly efficient carbon dioxide fixation capabilities, meaning it can directly utilize carbon dioxide for product synthesis without the aid of physical or chemical methods. Furthermore, its heterologous fermentation using carbon dioxide as a carbon source is light-independent, can be carried out under conventional fermentation conditions, and exhibits better robustness than commonly used carbon dioxide-fixing bacteria (such as cyanobacteria), demonstrating greater industrialization advantages and potential. Therefore, based on this technology, not only can the green biosynthesis of methylmalic acid, a key precursor of polymethyl methacrylate (PMMA), be achieved, which can be used in multiple fields such as healthcare and food processing, but it also represents an efficient method for eliminating greenhouse gases from the atmosphere.

[0019] 2. Promoters adapted for methylmalate synthase expression in hookworm *Copper-loving* bacteria were screened. To evaluate the optimal overexpression effect of different promoters on the key enzyme, Methanococcus jannaschii The source of the methylmalate synthase encoding gene Mj The heterologous expression of CimA3.7 in hookworm copper-eating bacteria was achieved by P phaC1 (The natural constitutive promoter regulating the expression of the phaCAB operon in hookworm copper-loving bacteria) or a P operon that can be induced by L-arabinose BAD Promoter control. Results showed that L-arabinose-induced promoter P... BAD Controlling the expression of methylmalate synthase is more conducive to the synthesis of methylmalate in hookworm copper-loving bacteria.

[0020] 3. A methylmalate synthase with high catalytic activity in *Hookworm* was screened and obtained. It was expressed in *Hookworm*. Methanococcus jannaschii ( Mj CimA3.7) Geobacter sulfurreducens ( Gs CimA), Lutibacter profundi ( Lp CimA), Leptospira interrogans ( Li CimA), Methanocaldococcus vulcanius ( Mv CimA) and Methanotorris igneus ( Mi The gene encoding methylmalate synthase derived from CimA was used to synthesize methylmalate using sodium gluconate (10 g / L) as the sole carbon source. The results showed that... Mj CimA3.7 Lp CimA and Mv CimA can effectively catalyze the synthesis of methylmalic acid in hookworm *Amycosis pituitary*. Overexpression of CimA... Mj The hookworm *CimA3.7* strain yielded the highest methylmalic acid production.

[0021] 4. Methylmalate transporters that promote methylmalate secretion were screened and obtained. These transporters were expressed in *Hookworm* and *Copper-loving Bacteroides*. Schizosaccharomyces pombe and Aspergillus oryzae Methylmalate transporter from Sp Mae1 and Ao Mae synthesized methylmalic acid using sodium gluconate (10 g / L) as the sole carbon source. The results showed that... Ao Mae can effectively increase the production of methyl malic acid by Hookworm Copper-Loving Bacterium.

[0022] 5. A gene encoding the endogenous pyruvate dehydrogenase complex subunit in *Hookworm* strain *Copper scavenger* that can promote methylmalate synthesis when sodium gluconate is used as the carbon source was screened and obtained. The overexpression of this gene in *Hookworm* strain *Copper scavenger* was investigated. aceE , pdhA1 , pdhA2 , acoC , pdhL , odhL and H16_B1098 The effect of overexpression on product yield when sodium gluconate is used as the carbon source was investigated. The results showed that overexpression... pdhA1 The methylmalic acid yield of hookworm copper-loving bacteria reached 3.4 g / L.

[0023] 6. A gene encoding the endogenous pyruvate dehydrogenase complex subunit in *Hookworm* *Copper-loving Bacterium* that can promote methylmalate synthesis using carbon dioxide as a carbon source was screened and obtained. The overexpression of this gene in *Hookworm* *Copper-loving Bacterium* was investigated. aceE , pdhA1 , pdhA2 , acoC , pdhL , odhL and H16_B1098 The effect of overexpression on product yield when carbon dioxide is used as the carbon source was investigated. The results showed that overexpression... pdhA2 The highest production of methylmalic acid was found in hookworm copper-loving bacteria. Attached Figure Description

[0024] Figure 1 This demonstrates the impact of knocking out key restriction endonuclease-encoding genes in the Hookworm Copper-Loving Bacterium genome on the electroconversion efficiency of exogenous plasmids; Figure 2 This shows the utilization of sodium gluconate by hookworm copper-loving bacteria expressing methylmalate synthase genes controlled by different promoters. Figure 3 The cell growth of hookworm copper-loving bacteria expressing methylmalate synthase genes controlled by different promoters is shown. Figure 4 This shows the product synthesis of hookworm copper-loving bacteria expressing methylmalate synthase genes controlled by different promoters; Figure 5 This shows the utilization of sodium gluconate by hookworm copper-loving bacteria expressing methylmalate synthase genes from different sources; Figure 6 The cell growth of hookworm copper-loving bacteria expressing methylmalate synthase genes from different sources is shown; Figure 7 This shows the product synthesis of hookworm copper-loving bacteria expressing methylmalate synthase genes from different sources; Figure 8 This study demonstrates the effects of different ammonium sulfate concentrations in the culture medium on sodium gluconate utilization, cell growth, and product synthesis of Hookworm Copper-Loving Bacteria. Figure 9 The effect of overexpression of the methylmalate transporter gene on sodium gluconate utilization by hookworm copper-loving bacteria was shown. Figure 10 The effect of overexpression of the methylmalate transporter gene on the cell growth of hookworm copper-loving bacteria was shown; Figure 11 This study demonstrates the effect of overexpression of the methylmalate transporter gene on product synthesis in hookworm copper-loving bacteria. Figure 12 The effects of overexpression of the gene encoding the pyruvate dehydrogenase complex subunit on cell growth and product yield of Hookworm Copper-loving Bacterium. Figure 13 This study demonstrates the effect of different ammonium sulfate concentrations in the culture medium on cell growth during autotrophic fermentation of Hookworm Copper-Loving Bacterium; Figure 14 This study shows the effect of different ammonium sulfate concentrations in the culture medium on the production of methylmalic acid during autotrophic fermentation of Hookworm Copper-Loving Bacterium; Figure 15 This study demonstrates the effect of overexpression of the gene encoding the pyruvate dehydrogenase complex subunit on cell growth of Hookworm Copper-Loving Bacteria during autotrophic fermentation. Figure 16 This study demonstrates the effect of overexpression of the gene encoding the pyruvate dehydrogenase complex subunit on product synthesis in autotrophic fermentation of Hookworm Copper-Loving Bacterium. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0026] This invention aims to construct an engineered strain of *Hookworm Copper-Gnaphalium* capable of efficiently producing methylmalic acid from sodium gluconate and carbon dioxide. Based on the results of this invention, not only can the synthesis of methylmalic acid, a precursor for the high-value polymer polymethyl methacrylate, be achieved, but it will also provide an effective strategy for carbon flow redirection in microbial cell factories based on *Hookworm Copper-Gnaphalium*, helping *Hookworm Copper-Gnaphalium* become a highly efficient synthetic platform for products using pyruvate and acetyl-CoA as precursors.

[0027] According to the technical solution of this application, the organic carbon source refers to an organic carbon source that can be directly utilized by *Hookworm Copper-Gropping Bacteria*, including sodium gluconate, fructose, glycerol, formic acid, acetic acid, etc., and the inorganic carbon source refers to CO2 that can be directly utilized by *Hookworm Copper-Gropping Bacteria*. The modification process of *Hookworm Copper-Gropping Bacteria* in this application is shown in Table 1 below: Table 1 .

[0028] In the following examples, the concentration of methylmalic acid in the culture medium was determined using HPLC. For the HPLC determination, a differential refractive index detector and a Bio-Rad Aminex HPX-87H analytical column were used. The column temperature was maintained at 35°C, and 5 mM sulfuric acid was used as the mobile phase at a flow rate of 0.5 mL / min. OD of the bacterial culture was... 600 Measurement: The absorbance of the bacterial solution at a wavelength of 600 nm was measured using a visible light spectrophotometer.

[0029] In the following embodiments, the hookworm copper-eating bacteria used can be purchased from microbial culture collection centers. For example, the hookworm copper-eating bacteria H16 used in this application was purchased from the China General Microbiological Culture Collection Center (CGMCC) with the number CGMCC 1.7092; and the German Microbial Culture Collection Center (DSM) with the number DSM 428.

[0030] In the following examples, a fermentation medium with sodium gluconate as the sole carbon source was prepared: 3.5 g Na2HPO4, 1.5 g KH2PO4, 1.0 g (NH4)2SO4, 80 mg MgSO4⋅7H2O, 1 mg CaSO4⋅2H2O, 0.56 mg NiSO4⋅7H2O, 0.4 mg ferric citrate, 200 mg NaHCO3, and 10 g sodium gluconate were accurately weighed, and the volume was adjusted to 1 L with water as the solvent. The medium was then autoclaved at 115°C for 30 minutes to obtain the fermentation medium with sodium gluconate as the sole carbon source.

[0031] When studying the effects of different ammonium sulfate concentrations in the culture medium on sodium gluconate utilization, cell growth, and product synthesis of hookworm copper-loving bacteria, the amount of (NH4)2SO4 in the fermentation medium with sodium gluconate as the sole carbon source was set to 0.1, 0.2, 0.5, 1, and 2 g, respectively.

[0032] In the following examples, a fermentation medium using CO2 as the sole carbon source was prepared: 3.5 g Na2HPO4, 1.5 g KH2PO4, 0.5 g (NH4)2SO4, 80 mg MgSO4⋅7H2O, 1 mg CaSO4⋅2H2O, 0.56 mg NiSO4⋅7H2O, 0.4 mg ferric citrate, 200 mg NaHCO3, 10 g sodium gluconate, and different concentrations of (NH4)2SO4 (0.1, 0.2, 0.5, 1, and 2 g / L) were accurately weighed and diluted to 1 L with water as the solvent. The medium was then autoclaved at 115°C for 30 minutes. During fermentation, a mixture of H2:O2:CO2 ≈ 8:1:1 was introduced as the carbon source at a flow rate of 10 mL / min.

[0033] When studying the effects of different ammonium sulfate concentrations in the culture medium on cell growth and methyl malic acid production during autotrophic fermentation of hookworm copper-loving bacteria, the amount of (NH4)2SO4 in the above-mentioned fermentation culture medium with CO2 as the sole carbon source was set to 0.1, 0.2, 0.5, 1 and 2 g, respectively.

[0034] In the following embodiments, the sequences involved include: SEQ ID NO: 1: H16_A0006 Encoding amino acid sequence; SEQ ID NO: 2: Methylmalate synthase Mj CimA3.7 amino acid sequence; SEQ ID NO: 3: Methylmalate synthase Mj The nucleotide sequence of the gene encoding CimA3.7; SEQ ID NO: 4: Promoter P BAD Nucleotide sequence; SEQ ID NO: 5: L . profundi The amino acid sequence of the methylmalate synthase from which it originated; SEQ ID NO: 6: L . profundi The nucleotide sequence of the methylmalate synthase gene obtained by codon optimization; SEQ ID NO: 7: M . vulcanius The amino acid sequence of the methylmalate synthase from which it originated; SEQ ID NO: 8: M . vulcanius The nucleotide sequence of the methylmalate synthase gene obtained by codon optimization; SEQ ID NO: 9: Methylmalate transporter protein from Aspergillus oryzae Ao Mae amino acid sequence; SEQ ID NO: 10: Gene encoding methylmalate transporter from Aspergillus oryzae Aomae Codon-optimized nucleotide sequences; SEQ ID NO: 11: Amino acid sequence of PdhA1, a subunit of the endogenous pyruvate dehydrogenase complex; SEQ ID NO: 12: Gene encoding the endogenous pyruvate dehydrogenase complex subunit pdhA1 Nucleotide sequence; SEQ ID NO: 13: Amino acid sequence of PdhA2, a subunit of the endogenous pyruvate dehydrogenase complex; SEQ ID NO: 14: Gene encoding the endogenous pyruvate dehydrogenase complex subunit pdhA2 Nucleotide sequence; SEQ ID NO: 15: Amino acid sequence of AceE, a subunit of the endogenous pyruvate dehydrogenase complex; SEQ ID NO: 16: Amino acid sequence of PdhL subunit of endogenous pyruvate dehydrogenase complex; SEQ ID NO: 17: Amino acid sequence of OdhL subunit of endogenous pyruvate dehydrogenase complex.

[0035] Example 1: Construction of a hookworm strain of *Bacillus thuringiensis* lacking key restriction endonuclease genes

[0036] One of the main challenges hindering gene editing and exogenous protein expression using plasmid vectors in *Hookworm Copper-eating Bacteroides* is its low electroporation efficiency. Based on this, this application addresses the issue of the low electroporation efficiency of the restriction endonuclease (SEQ ID NO: 1) encoding the type I restriction modification system gene in the *Hookworm Copper-eating Bacteroides* genome. H16_A0006 Knockout was performed to construct the H16 strain of *Hypophthalmia ulmoides* with improved transformation efficiency using exogenous plasmids. The restriction endonuclease encoding genes of the type I restriction modification system in the *Hypophthalmia ulmoides* genome were also investigated. H16_A0006 The specific method for knocking out is as follows: 1.1 Build with H16_A0006 Knockout plasmid pK18 for upstream and downstream homologous arms of gene sequence - A06 according to H16_A0006 Primers were designed and synthesized based on the gene sequence. Using the H16 genome of *Hookworm* var. *coccidioidomyces* as a template, the designed primers were used to amplify the gene. H16_A0006 The upstream and downstream homologous arms of the gene, with a fragment size of approximately 500 bp, were obtained. The obtained fragment was ligated to the pK18mobSacB plasmid using Gibson Assembly. EcoR I / Sma I site. The plasmid was transformed into E. coli cloning hosts, plated on LB agar plates (kanamycin 50 µg / mL), and incubated overnight at 37°C. Positive clones were then screened, and the target fragment of 1000 bp was named pK18. - A06.

[0037] 1.2 Constructing pK18 - Hookworm Copper-Loving Bacterium strain with A06 plasmid integrated into its genome plasmid pK18 -The A06 plasmid was electroporated into *E. coli* S17-1 host. *E. coli* S17-1 strain carrying the pK18-A06 plasmid and *H. coli* H16 strain were inoculated into LB liquid medium and cultured overnight, with appropriate antibiotics added (kanamycin 50 µg / mL or gentamicin 10 µg / mL). The cells were collected by centrifugation at 4600 rpm for 8 min and washed three times with LB medium. A mixture of *E. coli* S17-1 strain carrying the pK18-A06 plasmid and *H. coli* H16 strain carrying the pK18-A06 plasmid was then spotted onto LB plates and incubated overnight at 30°C. The overnight mixed cells were washed off with LB medium and spread onto LB solid plates (kanamycin 200 µg / mL and gentamicin 10 µg / mL). After incubation at 30°C for 48 hours, positive clones were screened for the kanamycin resistance gene fragment, 500 bp in length. Positive clones were cultured overnight at 30°C in 1.5 mL EP tubes containing LB liquid medium.

[0038] 1.3 Construction H16_A0006 Hookworm copper-loving bacterium strain H16 successfully knocked out Dilute the bacterial culture to 10. -2 The culture was plated on LB agar plates supplemented with 100 g / L sucrose and incubated at 30°C for 48 hours. Colony PCR was performed to detect the target fragment size (1000 bp), and clones with successfully knocked-out target genes were screened. The successfully constructed strain was inoculated into LB liquid medium and named CnΔRe.

[0039] Wild-type strain H16 (CnH16) and strain CnΔRe of hookworm copper-eating bacteria were streaked on antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and cultured in 50 mL LB agar at 30°C and 200 rpm until OD (dose retardation). 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. Add 400 ng pBBR1-MCS2 plasmid to 100 μL of competent cells and transfer to a 2 mm Bio-Rad electroporation cuvette for electroporation at 2.5 kV. Add 200 μL LB resuspending of the cells and incubate at 30°C and 200 rpm for 2 h on a shaker before plating onto LB agar plates (200 µg / mL kanamycin and 10 µg / mL gentamicin). After incubating at 30°C for 48 hours, count the clones and calculate the transformation efficiency. The results show that knocking out the wild-type H16 strain of *Hypophthalmia ulmoides*... H16_A0006The genetically engineered CnΔRe strain showed a transformation efficiency 2583 times higher than that of the wild-type strain. Figure 1 ).

[0040] Example 2: Construction of a methylmalic acid biosynthetic pathway adapted to Hookworm Copper-Loving Bacterium

[0041] The expression of methylmalate synthase in *Hookworm* is a key step in the synthesis of methylmalate from *Hookworm*, and the promoter regulating gene expression is particularly important. This application first describes the expression of methylmalate synthase using the endogenous constitutive promoter (P...). phaC1 ) and arabinose-induced (P BAD Promoter regulation MjcimA3.7 The methylmalate production of engineered strains of *Hookworm* was compared to determine the most suitable promoter. Based on this, genes annotated as methylmalate synthases were obtained from the UniProt database, and methylmalate synthase genes with low amino acid sequence uniformity were selected and heterologously expressed in *Hookworm*. Methylmalate synthases with high catalytic activity were screened based on methylmalate production. The specific methods are as follows:

[0042] 2.1 Construction of hookworm copper-loving bacteria strains with different promoters controlling the expression of methylmalate synthase

[0043] 2.1.1 Construction of the methylmalate synthase gene expression vector P phaC1 -MjcimA3.7-pBBR1 and P BAD -MjcimA3.7-pBBR1 According to the P of hookworm copper-loving bacteria phaC1 Primers were designed and synthesized based on the promoter sequence, and the genome of *Hookworm* *Copper-Loving Bacteria* was used as a template to amplify P... phaC1 Promoter segment. According to P BAD Promoter sequence design and primer synthesis to carry P BAD The promoter's plasmid vector serves as a template for amplifying P BAD Promoter segment.

[0044] Methanococcus japonicus ( Methanococcus jannaschii Methylmalate synthase mutant gene from () MjcimA3.7 (SEQ ID NO: 2) After codon optimization, the entire genome (SEQ ID NO: 3) was synthesized. Primers were designed based on the gene sequence, and using the synthesized full-gene sequence as a template, the methylmalate synthase mutant gene from *Methanococcus japonicus* was amplified using the designed primers. MjcimA3.7 The obtained promoter fragment and gene were ligated to the pBBR1-MCS2 vector plasmid using Gibson Assembly. EcoR I / SmaI site, obtain P phaC1 -MjcimA3.7-pBBR1 and P BAD -MjcimA3.7-pBBR1 expression vector.

[0045] 2.1.2 Construction of recombinant CnCt1 and CnCt2 strains The CnΔRe strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 50 mL LB agar plates and incubated at 30°C and 200 rpm until OD (digestion occurred). 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. 400 ng P... phaC1 -MjcimA3.7-pBBR1 and P BAD The -MjcimA3.7-pBBR1 plasmid was added to 100 μL of competent cells and transferred to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at a voltage of 2.5 kV. The cells were then resuspended in 200 μL of LB broth and incubated at 30°C and 200 rpm for 2 h on a shaker before being plated on LB agar plates (200 µg / mL kanamycin and 10 µg / mL gentamicin). Single colonies were picked to obtain CnCt1 and CnCt2 strains.

[0046] 2.1.3 Shake-flask fermentation of hookworm copper-loving bacteria to produce methylmalic acid The CnCt1 and CnCt2 strains obtained above were inoculated into 40 mL of LB medium at an inoculation rate of 1‰ (200 µg / mL kanamycin and 10 µg / mL gentamicin) and cultured overnight at 30°C and 200 rpm to activate the strains.

[0047] Fermentation medium with sodium gluconate as the sole carbon source was used. Activated CnCt1 and CnCt2 strains were transferred to 50 mL of fermentation medium supplemented with 200 mg / L kanamycin at an inoculation rate of 1‰, with CnΔRe strain (CnCtc) carrying pBBR1-MCS2 as a control.

[0048] CnCt2 strain OD 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentrations of sodium gluconate and methylmalic acid in the culture medium, as well as the OD of the bacterial culture, were measured. 600 .

[0049] like Figure 2As shown, sodium gluconate in the culture medium of CnCtc and CnCt1 strains was completely consumed after 36 hours, while CnCt2 strain required 48 hours to utilize the same amount of carbon source. Corresponding to the carbon source consumption, the maximum OD of CnCtc and CnCt1... 600 After 36 hours of cultivation, the values ​​reached 5.3 and 5.0 respectively, while the maximum OD of CnCt2 was... 600 After 48 hours of cultivation, it reached 3.3 ( Figure 3 ).like Figure 4 As shown, except for CnCtc, both CnCt1 and CnCt2 achieved the production of methylmalic acid. CnCt1 began producing methylmalic acid immediately after inoculation, with the maximum yield reaching 0.5 g / L after 36 hours. Under the induction of L-arabinose, strain CnCt2 began synthesizing methylmalic acid, with the product concentration in the culture medium reaching a peak of 2.1 g / L after 48 hours. Figure 4 The efficiency was approximately four times higher than that of CnCt1. These results indicate that strain CnCt2, when treated with arabinose-induced (P...)... BAD The promoter (SEQ ID NO: 4) regulates the expression of methylmalate synthase, which is more conducive to the synthesis of methylmalate in hookworm copper-eating bacteria. Since the CnCtc strain does not have the methylmalate synthase gene, it does not synthesize methylmalate although it consumes the carbon source sodium gluconate.

[0050] 2.2 Construction of hookworm copper-loving bacteria strains expressing methylmalate synthase genes from different microbial sources

[0051] 2.2.1 Construction of expression vectors for methylmalate synthase genes from different microbial sources Genes annotated as methylmalate synthase in the UniProt database were collected and a phylogenetic tree was constructed. Five sequences with low amino acid sequence identity were codon-optimized before full-gene synthesis. To eliminate the feedback inhibition of enzyme activity by L-isoleucine, the L-isoleucine-binding domain coding region was deleted from the methylmalate synthase gene. These five genes include C-terminal 136-amino acid truncated sequences from... Geobacter sulfurreducens of Gs CimA, truncated from the C-terminus of 116 amino acids Lutibacter profundi of Lp CimA (SEQ ID NO: 6), truncated at the C-terminus of 128 amino acids Leptospira interrogans of LiCimA 117 amino acids truncated at the C-terminus Methanocaldococcus vulcanius of Mv CimA (SEQ ID NO: 8), truncated at the C-terminus of 119 amino acids Methanotorris igneus of Mi CimA. L . profundi The amino acid sequence of methylmalate synthase is shown in SEQ ID NO: 5. M . vulcanius The amino acid sequence of methylmalate synthase is shown in SEQ ID NO: 7.

[0052] according to Gs CimA, Lp CimA, Li CimA Mv CimA and Mi Primers were designed based on the coding gene sequence of CimA. The synthesized gene was used as a template for PCR, and the obtained fragment was ligated into the pBBR1-MCS2 vector plasmid using Gibson Assembly. EcoR I / Sma I site. The plasmid was chemically transformed into *E. coli* Trans1 T1 host, plated on LB agar plates (200 mg / L kanamycin), and incubated overnight at 37°C. Positive clones were screened by colony PCR and sequenced for verification. The correctly constructed plasmid was named P. BAD -GscimA-pBBR1、P BAD -LpcimA-pBBR1、P BAD -LicimA-pBBR1、P BAD -MvcimA-pBBR1 and P BAD -MicimA-pBBR1.

[0053] 2.2.2 Construction of CnCt3, CnCt4, CnCt5, CnCt6 and CnCt7 engineered strains The CnΔRe strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 50 mL LB agar plates and incubated at 30°C and 200 rpm until OD (digestion occurred). 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. 400 ng P... BAD -GscimA-pBBR1、P BAD -LpcimA-pBBR1、P BAD -LicimA-pBBR1、P BAD -MvcimA-pBBR1 and P BADThe MicimA-pBBR1 plasmid was added to 100 μL of competent cells and transferred to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at a voltage of 2.5 kV. The cells were then resuspended in 200 μL of LB broth and incubated at 30°C and 200 rpm for 2 h on an LB agar plate (200 µg / mL kanamycin and 10 µg / mL gentamicin). Single colonies were picked to obtain engineered strains CnCt3, CnCt4, CnCt5, CnCt6, and CnCt7.

[0054] 2.2.3 Shake-flask fermentation of engineered strains CnCt2, CnCt3, CnCt4, CnCt5, CnCt6 and CnCt7 The engineered strains CnCt2, CnCt3, CnCt4, CnCt5, CnCt6, and CnCt7 were inoculated at an inoculum size of 1‰ into 40 mL of LB medium (200 µg / mL kanamycin and 10 µg / mL gentamicin) and cultured overnight at 30°C and 200 rpm to activate the strains.

[0055] Fermentation medium with sodium gluconate as the sole carbon source was used. The activated CnCt2, CnCt3, CnCt4, CnCt5, CnCt6 and CnCt7 engineered strains were transferred to 50 mL of fermentation medium supplemented with 200 mg / L kanamycin at an inoculation rate of 1‰.

[0056] When the strain OD 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentrations of sodium gluconate and methylmalic acid in the culture medium, as well as the OD of the bacterial culture, were measured. 600 .

[0057] like Figure 5 As shown, the sodium gluconate consumption of CnCt2, CnCt3, CnCt4, CnCt5, CnCt6, and CnCt7 after 36 hours of culture was 8.2, 9.2, 9.3, 8.7, 7.8, and 10.0 g / L, respectively, and the carbon source in the culture medium was completely depleted after 60 hours of culture. After 36 hours of culture, the OD values ​​of CnCt3, CnCt4, CnCt5, and CnCt7 were... 600 The values ​​reached 4.6, 4.4, 4.4, and 4.8 respectively. CnCt2 and CnCt6 reached their maximum OD after 48 hours of incubation. 600 The figures are 2.9 and 4.2 respectively. Figure 6 After induction with the inducer, only CnCt2, CnCt4, and CnCt6 successfully synthesized methylmalic acid, while no product synthesis was detected in the culture medium of strains CnCt3, CnCt5, and CnCt7. Figure 7This result indicates that... Mj CimA3.7 (CnCt2) Lp CimA (CnCt4) and Mv CimA (CnCt6) can effectively catalyze the biosynthesis of methylmalic acid in hookworm *Amycosis pituitary*. CnCt2, CnCt4, and CnCt6 exhibit different OD values. 600 The highest methylmalic acid yields were achieved at peak values ​​of 2.0, 1.8, and 1.1 g / L, respectively. Although CnCt4 had a faster methylmalic acid production rate, its yield was 10% lower than that of CnCt2. Figure 7 Overall, except Mj In addition to CimA3.7, from L . profundi (SEQ ID NO: 5) and M . vulcanius Two methylmalate synthases (SEQ ID NO: 6) were first demonstrated to be active in hookworm *Copper-loving*. However, consistent with other cell factories that produce methylmalate, the most widely used currently... Mj CimA3.7 still exhibits superior biosynthetic capacity for methylmalate. In summary, through P... BAD overexpression under promoter regulation Mj The cimA3.7 method successfully constructed a methylmalic acid biosynthetic pathway in hookworm copper-loving bacteria.

[0058] Example 3: Effects of nitrogen source concentration and transport protein overexpression on methylmalic acid production

[0059] Based on the previously constructed CnCt2 strain capable of synthesizing methylmalic acid using sodium gluconate as a carbon source, this application further investigates the effects of nitrogen source concentration and product transport proteins on methylmalic acid yield. First, the effect of different nitrogen source concentrations in the culture medium on the methylmalic acid yield of *Hypophthalmia ulmoides* was explored. Then, methylmalic acid transport proteins were selected from the literature and heterologously expressed in *Hypophthalmia ulmoides*. Based on the methylmalic acid yield, transport proteins that promote product synthesis in *Hypophthalmia ulmoides* were screened. The specific methods are as follows:

[0060] 3.1 Effects of different nitrogen source concentrations on methylmalic acid synthesis by Hookworm Copper-Loving Bacterium

[0061] The CnCt2 engineered strain was inoculated into 40 mL of LB medium at an inoculation rate of 1‰ (200 µg / mL kanamycin and 10 µg / mL gentamicin) and cultured overnight at 30°C and 200 rpm to activate the strain.

[0062] Fermentation media with sodium gluconate as the sole carbon source were prepared using different concentrations of (NH4)2SO4 (0.1, 0.2, 0.5, 1, and 2 g). The activated CnCt2 engineered strain was transferred at an inoculum rate of 1‰ to 50 mL of fermentation medium containing different nitrogen sources, supplemented with 200 mg / L kanamycin. When the strain OD... 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentrations of sodium gluconate and methylmalic acid in the culture medium, as well as the OD of the bacterial culture, were measured. 600 .

[0063] like Figure 8 As shown, the carbon source consumption and OD of all strains 600 The yields of both methylmalic acid and nitrogen source increased with increasing nitrogen source concentration. When the (NH₄)₂SO₄ concentration in the culture medium was 0.1, 0.2, and 0.5 g / L, the carbon source consumption of the strains was 2.7, 3.0, and 7.0 g / L, respectively. The OD₂ of the corresponding strains... 600 The concentrations were 0.4, 0.6, and 1.5, respectively, and the yields of methylmalic acid were 0.6, 0.7, and 1.7 g / L, respectively. When the (NH4)2SO4 concentration reached 1 g / L, the carbon source was completely depleted, and the OD... 600 At a concentration of 2.4, the highest concentration of methylmalic acid (2.1 g / L) was achieved. When the (NH₄)₂SO₄ concentration was 2 g / L, biomass accumulation increased, and OD₂O₃ levels also improved. 600 The concentration reached 2.7. In contrast, the yield of methylmalic acid decreased to 1.9 g / L, a 9.5% reduction compared to the strain in a medium containing 1 g / L (NH4)2SO4. Figure 8 This comparison highlights the strong dependence of methylmalic acid production in *Hookworm Copper-Gropping Bacterium* on nitrogen source supply and identifies an initial (NH4)2SO4 concentration of 1 g / L as the optimal level. This is likely because sodium gluconate is not depleted under low initial nitrogen source concentrations, and carbon source depletion is the main factor affecting methylmalic acid production. However, although carbon source depletion is complete at an initial (NH4)2SO4 concentration of 2 g / L, the strain tends to increase biomass (…). Figure 8 Based on the above results, regulating the initial nitrogen source concentration is an effective strategy for reprogramming metabolic flux and thereby increasing the supply of methylmalic acid synthesis precursors in Hookworm Copper-Loving Bacterium.

[0064] 3.2 Construction of hookworm copper-loving bacteria strains expressing methylmalate transporter genes from different microbial sources

[0065] 3.2.1 Constructing expression vectors for genes encoding methylmalate transporters from different microbial sources The source of Aspergillus oryzae ( Aspergillus oryzae ) and Saccharomyces cerevisiae ( Schizosaccharomyces pombe ) methylmalate transporter Ao Mae (SEQ ID NO: 9) and Sp The coding sequence of Mae1 was codon-optimized before whole-genome synthesis, including the methylmalate transporter protein. Ao The codon-optimized sequence of the Mae-encoded sequence is SEQ ID NO: 10. According to... Aomae and Spmae1 Primers were designed based on the coding gene sequence, and the RBS upstream of the phaC1 gene coding sequence was added to the primers. PCR was performed using the synthesized gene as a template, and the obtained fragments were ligated to P using GibsonAssembly. BAD -MjcimA3.7-pBBR1 Spe Site I. The recombinant plasmid was chemically transformed into *E. coli* Trans1 T1 host, plated on LB agar plates (200 mg / L kanamycin), and incubated overnight at 37°C. Positive clones were screened by colony PCR and sequenced for verification. The correctly constructed plasmid was named P. BAD -MjcimA-Aomae1-pBBR1 and P BAD -MjcimA3.7-Spmae1-pBBR1.

[0066] 3.2.2 Construction of CnCt8 and CnCt9 engineered strains The CnΔRe strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 50 mL LB agar plates and incubated at 30°C and 200 rpm until OD (digestion occurred). 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. 400 ng P... BAD -MjcimA-Aomae1-pBBR1 and P BAD The plasmid -MjcimA3.7-Spmae1-pBBR1 was added to 100 μL of competent cells and transferred to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at a voltage of 2.5 kV. The cells were then resuspended in 200 μL of LB broth and incubated at 30°C and 200 rpm for 2 h on a shaker before being plated on LB agar plates (200 µg / mL kanamycin and 10 µg / mL gentamicin). Single clones were picked to obtain the CnCt8 and CnCt9 engineered strains.

[0067] 3.2.3 Shake-flask fermentation of engineered strains CnCt2, CnCt8, and CnCt9 The engineered strains CnCt2, CnCt8, and CnCt9 were inoculated at an inoculum rate of 1‰ into 40 mL of LB medium (200 µg / mL kanamycin and 10 µg / mL gentamicin) and cultured overnight at 30°C and 200 rpm to activate the strains.

[0068] A fermentation medium was prepared using sodium gluconate as the sole carbon source. The activated CnCt2, CnCt8, and CnCt9 engineered strains were transferred at an inoculation rate of 1‰ to 50 mL of fermentation medium supplemented with 200 mg / L kanamycin.

[0069] When the strain OD 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentrations of sodium gluconate and methylmalic acid in the culture medium, as well as the OD of the bacterial culture, were measured. 600 .

[0070] like Figure 9 As shown, after 48 hours of fermentation, the sodium gluconate in the culture medium was almost completely depleted by the strain. After 24 hours of fermentation, the maximum OD of CnCt2... 600 The value is 2.2, which is 1.2 times and 1.1 times that of CnCt8 and CnCt9 (1.9 and 2.0), respectively. Figure 10 This may be attributed to the growth inhibition of the strain caused by excessive insertion of transmembrane proteins. The methylmalic acid yield of CnCt2 was 2.1 g / L, while that of CnCt8 was 2.6 g / L, representing a 23.8% increase. Figure 11 This indicates that overexpression of a highly efficient efflux protein... Ao Mae increased the production of methylmalic acid by *Amycium clavatum*. This is consistent with previous literature reports. Spmae1 Overexpression helps increase methylmalic acid production in Saccharomyces cerevisiae. Conversely, CnCt2 showed a methylmalic acid production of 2.1 g / L and a yield of Y per unit substrate. P / C The yield per unit biomass is 0.21 g / g, and the yield per unit biomass is Y. P / B It is 0.84 g / (L·OD) 600 Compared with strain CnCt2, CnCt9 produced methylmalic acid (1.8 g / L) and yielded Y per unit substrate. P / C (0.18 g / g) and yield per unit biomass Y P / B (0.75 g / (L·OD) 600 These figures represent decreases of 14.3%, 14.3%, and 10.7%, respectively. (Compared to...) Ao Compared to Mae, SpMae1 exhibits a stronger ability to efflux citric acid, malic acid, fumaric acid, and succinic acid, which are key nodes in the tricarboxylic acid cycle, thus negatively impacting the cell growth and methylmalic acid production of CnCt9.

[0071] Example 4: Enhancing Acetyl-CoA Supply to Increase Methylmalic Acid Production by Hookworm Copper-Gropping Bacteria

[0072] In the synthesis of methylmalic acid, the supply of acetyl-CoA is crucial for the synthesis of the product. Theoretically, overexpression of the gene encoding the pyruvate dehydrogenase complex subunit can promote the synthesis of acetyl-CoA, which uses pyruvate as a precursor. This application involves overexpressing the gene encoding the pyruvate dehydrogenase complex subunit in *Hookworm Copper-Gnawing Bacterium*, and screening for the optimal overexpressed subunit by measuring the product yield of strains expressing different subunits, thereby increasing the yield of methylmalic acid. The specific method is as follows:

[0073] 4.1 Construction of an overexpression vector for the gene encoding the endogenous pyruvate dehydrogenase complex subunit

[0074] Primers were designed and synthesized based on the gene sequence encoding the endogenous pyruvate dehydrogenase complex subunit of hookworm *Hookworm* *Copper-loving Bacterium*, and... phaA RBS upstream of the gene coding sequence was added to the primers. Using the H16 genome of *Hypoxanthus pumilum* as a template, the gene encoding the endogenous pyruvate dehydrogenase complex subunit of *Hypoxanthus pumilum* was amplified using the primers designed above. The obtained gene was ligated to P using Gibson Assembly. BAD -MjcimA3.7-Aomae1-pBBR1 vector plasmid Spe I site, obtain P BAD -MjcimA3.7-Aomae1-aceE-pBBR1,P BAD -MjcimA3.7-Aomae1-pdhA1-pBBR1,P BAD -MjcimA3.7-Aomae1-pdhA2-pBBR1,P BAD -MjcimA3.7-Aomae1-acoC-pBBR1,P BAD -MjcimA3.7-Aomae1-pdhL-pBBR1,P BAD -MjcimA3.7-Aomae1-odhL-pBBR1 and P BAD -MjcimA3.7-Aomae1-B1098-pBBR1 expression vector.

[0075] 4.2 Construction of strains overexpressing the gene encoding the endogenous pyruvate dehydrogenase complex subunit

[0076] The CnΔRe strain was streaked onto antibiotic-free LB agar plates and incubated at 30°C for 48 h. Single colonies were picked and placed in 50 mL LB agar plates and incubated at 30°C and 200 rpm until OD (digestion occurred). 600 ≈0.3-0.5. Place on ice for 20 min. Wash the cells three times with pre-chilled 10% glycerol. Then resuspend the cells in 1.8 mL of 10% glycerol and aliquot into 1.5 mL EP tubes (100 μL / tube), flash freeze in liquid nitrogen, and store at -80°C. 400 ng P... BAD -MjcimA3.7-Aomae1-aceE-pBBR1,P BAD -MjcimA3.7-Aomae1-pdhA1-pBBR1,P BAD -MjcimA3.7-Aomae1-pdhA2-pBBR1,P BAD -MjcimA3.7-Aomae1-acoC-pBBR1,P BAD -MjcimA3.7-Aomae1-pdhL-pBBR1,P BAD -MjcimA3.7-Aomae1-odhL-pBBR1 and P BAD The plasmid -MjcimA3.7-Aomae1-B1098-pBBR1 was added to 100 μL of competent cells and transferred to a 2 mm Bio-Rad electroporation cuvette for electroporation transformation at a voltage of 2.5 kV. The cells were then resuspended in 200 μL of LB broth and incubated at 30°C and 200 rpm for 2 h on a shaker before being plated on LB agar plates (200 µg / mL kanamycin and 10 µg / mL gentamicin). Single colonies were picked to obtain CnCt10, CnCt11, CnCt12, CnCt13, CnCt14, CnCt15, and CnCt16.

[0077] 4.3 Shake-flask fermentation of engineered hookworm copper-loving bacteria to produce methylmalic acid

[0078] CnCt8, CnCt10, CnCt11, CnCt12, CnCt13, CnCt14, CnCt15, and CnCt16 were inoculated at an inoculum size of 1‰ into 40 mL of LB medium (200 µg / mL kanamycin and 10 µg / mL gentamicin) and cultured overnight at 30°C and 200 rpm to activate the strains.

[0079] Fermentation medium with sodium gluconate as the sole carbon source was used. Activated CnCt8 and CnCt10, CnCt11, CnCt12, CnCt13, CnCt14, CnCt15, and CnCt16 strains were transferred at an inoculum rate of 1‰ to 50 mL of fermentation medium supplemented with 200 mg / L kanamycin. The OD of the strains was then measured. 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentrations of sodium gluconate and methylmalic acid in the culture medium, as well as the OD of the bacterial culture, were measured. 600 .

[0080] like Figure 12 As shown, overexpression acoC (CnCt13) pdhL (CnCt14) and B1098 (CnCt16) promoted strain proliferation. The maximum OD of CnCt8 was... 600 The maximum OD of CnCt13, CnCt14, and CnCt16 is 2.2. 600 These are 3.0, 2.8, and 2.5 respectively, which are 1.4, 1.3, and 1.1 times that of CnCt8. aceE and pdhA1 Overexpression negatively impacted strain proliferation, affecting the OD values ​​of strains CnCt10 and CnCt11. 600 All were 2.0. Furthermore, overexpression of most subunits significantly increased methylmalic acid production. Only CnCt13 and CnCt16 showed lower methylmalic acid titers (2.3 and 2.4 g / L, respectively), representing reductions of 11.5% and 7.7% compared to CnCt8 (2.6 g / L). Overexpression pdhA1 The strain CnCt11 had the highest methylmalic acid yield, reaching 3.4 g / L, which is 1.3 times that of CnCt8. Figure 12 Furthermore, the yields of CnCt10, CnCt12, CnCt14, and CnCt15 were significantly higher than those of CnCt8, at 3.3, 3.3, 3.2, and 3.3 g / L, respectively. The acetyl-CoA content in CnCt11 was significantly increased, reaching 3.02 μmol / gDCW, a 19.4% increase compared to CnCt8 (2.53 μmol / gDCW). These studies indicate that overexpression of the gene encoding the endogenous pyruvate dehydrogenase complex subunit can enhance acetyl-CoA production, thereby increasing methylmalic acid production in *Hookworm Copper-Gropping Bacterium*.

[0081] Example 5: Production of methylmalic acid by fermentation using an engineered strain of hookworm copper-loving bacteria with CO2 as a carbon source.

[0082] Based on the previously constructed CnCt8 strain capable of synthesizing methylmalic acid using sodium gluconate as a carbon source, this application further investigated the effect of nitrogen source concentration on the yield of methylmalic acid synthesized by the engineered strain of *Hookworm Copper-Gnawing* using CO2 as a carbon source. Subsequently, this application overexpressed the gene encoding the pyruvate dehydrogenase complex subunit in *Hookworm Copper-Gnawing*, and screened the optimal overexpressed subunit by measuring the yield of methylmalic acid produced by strains expressing different subunits using CO2 as a carbon source, thereby increasing the yield of methylmalic acid. The specific methods are as follows:

[0083] 5.1 Effects of different nitrogen source concentrations on the fermentation production of methylmalic acid by hookworm copper-loving bacteria using CO2 as a carbon source

[0084] The CnCt8 engineered strain was inoculated at an inoculum rate of 1‰ into 40 mL of LB medium (200 µg / mL kanamycin and 10 µg / mL gentamicin), and cultured overnight at 30°C and 200 rpm in a shaker to activate the strain.

[0085] Fermentation media with different concentrations of (NH4)2SO4 (0.1, 0.2, 0.5, 1, and 2 g / L) and CO2 as the sole carbon source were used. During fermentation, a mixed gas of H2:O2:CO2 ≈ 8:1:1 was introduced as the carbon source at a flow rate of 10 mL / min. The activated CnCt8 engineered strain was transferred at an inoculum rate of 1‰ to 50 mL of fermentation medium containing different concentrations of nitrogen source, supplemented with 200 mg / L kanamycin. When the strain OD... 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentration of methylmalic acid in the culture medium and the OD of the bacterial culture were measured. 600 .

[0086] like Figure 13 As shown, higher nitrogen source concentrations correspond to higher OD values ​​in engineered strains. 600 The range is from 0.5 to 2.4. When the OD of the strain... 600When the nitrogen source concentration reached 0.5 g / L, 1 g / L L-arabinose was added to initiate methylmalic acid synthesis. Methylmalic acid was detectable on day 5 of fermentation in media containing 0.5, 1, and 2 g / L (NH4)2SO4, while it was only detectable on day 7 in media containing 0.1 and 0.2 g / L (NH4)2SO4. At the end of the 11-day culture period, the strain reached its highest methylmalic acid yield of 129.2 mg / L when the medium contained 0.5 g / L (NH4)2SO4. However, while increasing the nitrogen source concentration promoted strain proliferation, it was detrimental to methylmalic acid biosynthesis, indicating a balance between growth and production under autotrophic conditions. When the (NH4)2SO4 concentration in the medium reached 1 g / L and 2 g / L, the methylmalic acid titers were 85.6 mg / L and 56.1 mg / L, respectively, representing decreases of 33.7% and 56.6% compared to the medium containing 0.5 g / L (NH4)2SO4. Figure 14 These results indicate that, consistent with the results under heterotrophic fermentation conditions, autotrophic methylmalic acid production is also affected by nitrogen source concentration, with the highest yield observed at a concentration of 0.5 g / L (NH4)2SO4.

[0087] 5.2 Increasing acetyl-CoA supply enhances the production of methylmalic acid by hookworm *Amycosis pilosa* using CO2 as a carbon source.

[0088] CnCt8, CnCt10, CnCt11, CnCt12, CnCt13, CnCt14, CnCt15, and CnCt16 were inoculated at an inoculum size of 1‰ into 40 mL of LB medium (200 µg / mL kanamycin and 10 µg / mL gentamicin) and cultured overnight at 30°C and 200 rpm to activate the strains.

[0089] A fermentation medium with CO2 as the sole carbon source was used, and a mixed gas of H2:O2:CO2 ≈ 8:1:1 was introduced at a flow rate of 10 mL / min as the carbon source. Activated engineered strains CnCt8, CnCt10, CnCt11, CnCt12, CnCt13, CnCt14, CnCt15, and CnCt16 were transferred at an inoculum rate of 1‰ to 50 mL of fermentation medium supplemented with 200 mg / L kanamycin, using CO2 as the sole carbon source. When the OD of the strains... 600 When the concentration of L-arabinose was approximately 0.5, a final concentration of 1 g / L was added for induction. The concentration of methylmalic acid in the culture medium and the OD of the bacterial culture were measured. 600 .

[0090] like Figure 15 As shown, even in the absence of the inducer L-arabinose, except pdhA2 Besides (CnCt12), leakage expression of the subunit-encoding gene severely inhibited the growth of the strain. Only the OD of CnCt12... 600 With increasing fermentation time, it reached a peak value of 1.0, which was 28.6% lower than that of CnCt8 (1.4). The OD values ​​of CnCt10, CnCt11, CnCt13, CnCt14, CnCt15, and CnCt16 were also observed. 600 The concentration was kept below 0.3 until fermentation was complete. Figure 15 This is likely due to excessive carbon loss and NADH generation caused by pyruvate decarboxylation, which creates an inefficient cycle with the CBB cycle and exacerbates the redox imbalance under autotrophic conditions. Furthermore, pyruvate is a major metabolic branching point in bacteria, serving not only as a precursor to acetyl-CoA but also as a substrate for the anastomosis reaction, amino acid biosynthesis, and overflow or fermentation pathways. The significant redirection of pyruvate to acetyl-CoA limits the precursors available for biomass accumulation. In autotrophic culture, CnCt12 produced 77.7 mg / L of methylmalate within 7 days, a level comparable to the control strain CnCt8 (63.3 mg / L). Interestingly, methylmalate accumulation was faster in CnCt12, despite slower cell proliferation relative to CnCt8. At the end of fermentation, CnCt12 reached a final titer of 174.7 mg / L, a 1.4-fold increase compared to CnCt8 (129.2 mg / L). Figure 16 ), yield per unit biomass Y P / B It is 174.7 mg / (L·OD) 600 ), which is the control group strain CnCt8 (92.3 mg / (L·OD) 600 1.9 times that of )). That is, only pdhA2 Overexpression under autotrophic conditions promotes the production of methylmalate using CO2 as the sole carbon source. These results highlight that the endogenous pyruvate dehydrogenase complex subunit encoding genes with positive functions screened under heterotrophic conditions cannot be directly applied to autotrophic metabolism, emphasizing the necessity of condition-specific enzyme selection.

[0091] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for constructing an engineered strain of *Hookworm* *Copper-Greeding Bacterium* capable of producing methylmalic acid using both organic and inorganic carbon sources, characterized in that, The method includes the following steps in manipulating the genome of a wild-type strain of hookworm copper-boring fungus: Knock out the restriction endonuclease encoding gene of the type I restriction modification system, wherein the amino acid sequence of the restriction endonuclease of the type I restriction modification system is shown in SEQ ID NO: 1; Overexpression of the gene encoding methylmalate synthase, wherein the amino acid sequence of said methylmalate synthase is as shown in SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 7; and Overexpression of the gene encoding methylmalate transporter, wherein the amino acid sequence of the methylmalate transporter is shown in SEQ ID NO:

9.

2. The method for constructing an engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, as described in claim 1, is characterized in that... The method further includes the following steps: The expression of the gene encoding methylmalate synthase is regulated using an arabinose-inducible promoter, wherein the nucleotide sequence of the arabinose-inducible promoter is shown in SEQ ID NO:

4.

3. The method for constructing an engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, as described in claim 1, is characterized in that... The method further includes the following steps: When constructing an engineered strain of *Hookworm Copper-Gropping Bacterium* that produces methylmalic acid using an organic carbon source, the encoding gene of an endogenous pyruvate dehydrogenase complex subunit is overexpressed in the wild-type strain of *Hookworm Copper-Gropping Bacterium*. The endogenous pyruvate dehydrogenase complex subunit is pyruvate dehydrogenase complex subunit PdhA1, and the amino acid sequence of pyruvate dehydrogenase complex subunit PdhA1 is shown in SEQ ID NO:

11. When constructing an engineered strain of *Hookworm Copper-Gropping Bacterium* that produces methylmalic acid using an inorganic carbon source, the encoding gene of an endogenous pyruvate dehydrogenase complex subunit is overexpressed in the wild-type strain of *Hookworm Copper-Gropping Bacterium*. The endogenous pyruvate dehydrogenase complex subunit is pyruvate dehydrogenase complex subunit PdhA2, and the amino acid sequence of pyruvate dehydrogenase complex subunit PdhA2 is shown in SEQ ID NO:

13.

4. The method for constructing an engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, as described in claim 3, is characterized in that... The nucleotide sequence of the gene encoding the pyruvate dehydrogenase complex subunit PdhA1 is shown in SEQ ID NO: 12, and the nucleotide sequence of the gene encoding the pyruvate dehydrogenase complex subunit PdhA2 is shown in SEQ ID NO:

14.

5. The method for constructing an engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, as described in claim 1, is characterized in that... The nucleotide sequence of the gene encoding the methylmalate synthase is shown in SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO:

8.

6. The method for constructing an engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, as described in claim 1, is characterized in that... The nucleotide sequence of the gene encoding the methylmalate transporter is shown in SEQ ID NO:

10.

7. An engineered hookworm bacterium capable of producing methylmalic acid using both organic and inorganic carbon sources, characterized in that, The genome of the engineered hookworm copper-producing bacterium that utilizes both organic and inorganic carbon sources to produce methylmalic acid is the genome of the wild-type hookworm copper-producing bacterium with the following modified characteristics: The restriction endonuclease encoding gene of the type I restriction modification system is knocked out, wherein the amino acid sequence of the restriction endonuclease of the type I restriction modification system is shown in SEQ ID NO: 1; Overexpression of the gene encoding methylmalate synthase, wherein the amino acid sequence of said methylmalate synthase is as shown in SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 7; and The gene encoding the methylmalate transporter was overexpressed, and the amino acid sequence of the methylmalate transporter is shown in SEQ ID NO:

9.

8. The application of the hookworm copper-boring bacteria engineered by claim 7, which can utilize both organic and inorganic carbon sources to produce methylmalic acid, in the production of methylmalic acid.

9. A method for producing methylmalic acid, characterized in that, The method includes the following steps: The hookworm copper-loving bacteria of claim 7, which can produce methyl malic acid using both organic and inorganic carbon sources, is cultured by fermentation using organic or inorganic carbon sources.

10. The method for producing methylmalic acid according to claim 9, characterized in that, When fermenting the engineered hookworm copper-loving bacteria that can produce methylmalic acid using both organic and inorganic carbon sources, the yield of methylmalic acid can be increased by adjusting the concentration of nitrogen source in the culture medium.

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