Genetically engineered bacteria for producing alpha-ketoglutarate and application thereof
By genetically engineering Corynebacterium glutamicum and optimizing its metabolic network, the problems of insufficient precursor supply, consumption, and efflux in the production of α-ketoglutarate were solved, and efficient and stable production of α-ketoglutarate was achieved.
Patent Information
- Application Number
- CN202610814271.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-08
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies make it difficult to achieve efficient, controllable, and large-scale production of α-ketoglutarate within microbial cells, mainly due to problems such as insufficient supply of precursor substances, easy consumption of target products, carbon diversion, and efflux of intermediates. Furthermore, hydrogen peroxide produced during the L-glutamate oxidase catalysis process causes oxidative damage to cells.
By genetically engineering Corynebacterium glutamicum, including weakening or knocking out the expression of genes in multiple key metabolic pathways, while enhancing the expression and regulation of key enzymes, genetically engineered bacteria were constructed to optimize the metabolic network, thereby enhancing the ability to synthesize α-ketoglutarate and inhibiting competitive consumption.
It achieves efficient accumulation and stable production of α-ketoglutarate, significantly improves conversion efficiency, reduces by-product generation, and is suitable for large-scale production.
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Figure CN122326504A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering and metabolic engineering, specifically relating to a genetically engineered bacterium that produces α-ketoglutarate and its applications. Background Technology
[0002] α-Ketoglutarate (α-KG) is a key intermediate metabolite in the tricarboxylic acid cycle (TCA cycle), playing a pivotal role in cellular carbon and nitrogen metabolism. It not only participates in the synthesis and conversion of amino acids but also plays important functions in energy metabolism, reducing power balance, and various physiological regulatory processes. Furthermore, as an important functional small molecule, α-ketoglutarate has wide applications in medicine, health foods, sports nutrition, and fine chemicals. For example, it can be used to regulate nitrogen metabolism, promote protein synthesis, improve fatigue, and shows promising application prospects in clinical nutritional support and adjunctive treatment of chronic diseases. Therefore, developing efficient, green, and scalable α-ketoglutarate production technologies is of great significance.
[0003] Currently, the main methods for preparing α-ketoglutaric acid include chemical synthesis, bio-fermentation, and enzyme-catalyzed biotransformation. Chemical synthesis typically requires high temperatures, high pressures, or strong acid / base conditions, resulting in high energy consumption and environmental pollution. While bio-fermentation offers advantages such as a wide range of raw material sources and a green and environmentally friendly process, the complex microbial metabolic network often leads to the accumulation of byproducts such as pyruvate, lactic acid, and other organic acids, limiting yield and conversion efficiency. Enzyme-catalyzed biotransformation offers advantages such as mild reaction conditions, high selectivity, and fewer side reactions, but its efficiency largely depends on the catalytic activity of key enzymes and substrate supply capacity.
[0004] Within microbial cells, the synthesis and consumption of α-ketoglutarate are dynamically regulated by multiple metabolic pathways. From a metabolic network perspective, its accumulation is limited by several factors: First, insufficient supply of precursor substances, such as the limited supply of key intermediates like pyruvate and oxaloacetate, directly affects the efficiency of α-ketoglutarate production. Second, α-ketoglutarate is easily further metabolized within the cell, for example, entering the downstream TCA cycle via the α-ketoglutarate dehydrogenase complex or being converted into amino acids through the glutamate synthesis pathway, leading to the loss of the target product. Third, some carbon flux is diverted through byproduct pathways such as lactate, reducing overall carbon utilization efficiency. In addition, some intracellular metabolic intermediates may be exported extracellularly via transport protein systems, further reducing their effective intracellular accumulation concentration.
[0005] In recent years, metabolic engineering strategies have been widely applied in the biosynthesis of α-ketoglutaric acid, including enhancing precursor supply, knocking out competing pathways, and optimizing the expression of key enzymes. However, existing studies mostly focus on the regulation of single or a few metabolic nodes, lacking systematic optimization of the entire metabolic network. This makes it difficult to simultaneously achieve multiple objectives such as precursor enhancement, branching inhibition, and intermediate retention in the same system (Chen X, et al. Pathway engineering of Escherichia coli for α-ketoglutaric acid production. Biotechnol Bioeng. 2020,117:2791-2801; Li Y, et al. Efficient production of α-ketoglutarate in thegdh deleted Corynebacterium glutamicum by novel double-phase pH and biotin control strategy. Bioprocess Biosyst Eng. 2016, 39:967-76.). Furthermore, in biotransformation systems using L-glutamate as a substrate, hydrogen peroxide produced during the catalysis of L-glutamate oxidase may cause oxidative damage to cells, thereby affecting enzyme activity and system stability. This problem has not been adequately solved in the existing technology.
[0006] Therefore, there is an urgent need to develop an engineered bacteria construction strategy that can synergistically regulate multiple metabolic pathways, enhance the synthesis capacity of α-ketoglutarate, effectively reduce competitive consumption and improve system stability, thereby achieving its efficient, controllable and large-scale production. Summary of the Invention
[0007] To address the problems existing in the prior art, this invention provides a strategy for constructing engineered bacteria based on metabolic flux redistribution. By directionally regulating key metabolic nodes, the synthesis capacity of the target product is enhanced while its competitive consumption is suppressed, thereby achieving efficient accumulation of α-ketoglutarate.
[0008] On one hand, the present invention provides a genetically engineered bacterium for producing α-ketoglutarate, which is obtained by modifying the central metabolic pathway of Corynebacterium glutamicum by performing gene modification at any one or more of the following sites, based on the starting bacterium: *Corynebacterium glutamicum*; and / or *odhana* (α-ketoglutarate dehydrogenase gene odhA);
[0009] Attenuate the expression of the glutamate transporter gene cg1434; and / or
[0010] Reduce the expression of the key subunit gene gltB of glutamate synthase; and / or
[0011] Reduce the expression of the lactate dehydrogenase gene ldh; and / or
[0012] Reduce the expression of the glutamate dehydrogenase gene gdh; and / or
[0013] Attenuate the expression of the serine / threonine protein kinase gene pknG; and / or
[0014] Enhance the expression of the pyruvate carboxylase gene pyc; and / or
[0015] Introduce and express the glutamate oxidase gene IGOX at the lactate dehydrogenase ldh gene locus; and / or
[0016] Enhance the expression of the catalase gene katA.
[0017] Specifically, "weakening" or "reducing" refers to lowering the expression or activity of one or more genes in the genetically engineered bacteria. In a preferred embodiment, "weakening" or "reducing" means that the expression or activity of the weakened gene is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the expression or activity of the unweakened gene; preferably, the gene is completely inactivated. In a preferred embodiment, the "weakened gene expression or activity" can be achieved, for example, by adding an inhibitor to the culture medium of the genetically engineered bacteria or by knocking out the gene in the genetically engineered bacteria.
[0018] Preferably, "enhancement" refers to increasing the expression or activity of endogenous genes present in the genetically engineered bacteria; or exogenously introducing genes not present in the genetically engineered bacteria, causing the exogenously introduced genes to be expressed or function in the genetically engineered bacteria. Specifically, the enhancement of gene expression or activity can be achieved by using a strong promoter to express endogenous or heterologous genes, and / or increasing the copy number of the gene, and / or modifying the promoter of the gene to enhance transcription initiation speed, and / or modifying the translation regulatory region or rare codons of the messenger RNA carrying the gene to enhance translation intensity, and / or modifying the gene itself to enhance mRNA stability and protein stability. Preferably, the promoter is a strong promoter, such as Ptuf, Psod, or an artificially constructed strong promoter to achieve enhanced expression of the target gene.
[0019] Specifically, the modification is achieved through homologous recombination.
[0020] Specifically, the GenBank accession number corresponding to the amino acid sequence encoded by odhA is BAB98522.1; the GenBank accession number corresponding to the amino acid sequence encoded by cg1434 is BAB98663.1; the GenBank accession number corresponding to the amino acid sequence encoded by gltB is BAB97577.1; the GenBank accession number corresponding to the amino acid sequence encoded by ldh is BAC00305.1; the GenBank accession number corresponding to the amino acid sequence encoded by gdh is BAB99472.1; the GenBank accession number corresponding to the amino acid sequence encoded by pknG is BAC00145.1; the GenBank accession number corresponding to the amino acid sequence encoded by pyc is BAB98082.1; the GenBank accession number corresponding to the amino acid sequence encoded by lgox is KY315918.1; and the GenBank accession number corresponding to the amino acid sequence encoded by katA is BAB97648.1.
[0021] Specifically, the glutamate oxidase gene lgox is derived from Streptomyces mobara.
[0022] Specifically, the originating strain of Corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032 (which can be purchased for a fee from the American Type Culture Collection Center ATCC or obtained from the China Industrial Microbial Culture Collection Center, and is a commercially available standard strain).
[0023] On the one hand, the present invention provides the application of the genetically engineered bacteria in the production of α-ketoglutaric acid.
[0024] On the other hand, the present invention provides a method for producing α-ketoglutarate, which utilizes the genetically engineered bacteria described above for fermentation culture to produce α-ketoglutarate. Preferably, the inorganic ammonium salt content in the culture medium is less than 1 g / L, constituting an ammonia-limited culture condition, thereby limiting the excessive allocation of carbon sources to the amino acid synthesis pathway by the cells, which is conducive to promoting the accumulation of carbon flow towards α-ketoglutarate and increasing the yield of the target product.
[0025] Specifically, the fermentation conditions are a temperature of 30℃~35℃, a rotation speed of 150r / min~250r / min, a culture time of 60h~72h, and the pH of the culture system is adjusted by calcium carbonate to make the pH=6.0~8.0.
[0026] Specifically, it also includes the step of separating or purifying the resulting α-ketoglutarate.
[0027] Compared with the prior art, the present invention has the following beneficial effects: The present invention achieves effective regulation of carbon flow distribution by systematically optimizing the metabolic network: On the one hand, by strengthening the precursor supply pathway and key conversion reactions, the production capacity of α-ketoglutarate is improved; on the other hand, by weakening competitive branching pathways and reducing intermediate efflux losses, the consumption of target products is significantly reduced, thereby improving the overall conversion efficiency.
[0028] The genetically engineered bacteria constructed in this invention can maintain a stable metabolic state even under high substrate concentrations, achieving effective accumulation of α-ketoglutarate while generating fewer byproducts, which is beneficial for subsequent separation and purification processes. In the preferred embodiment, the genetically engineered bacteria of this invention, after 48 hours of shake-flask fermentation, achieved an α-ketoglutarate content of 18.1 ± 0.5 g / L. The method for producing α-ketoglutarate using this genetically engineered bacteria has the advantages of clear control strategy, strong engineering operability, and suitability for large-scale production, showing promising application prospects in the biomanufacturing of α-ketoglutarate and its related derivatives. Attached Figure Description
[0029] Figure 1 This is a schematic diagram of the biosynthesis and metabolism of α-ketoglutarate. Detailed Implementation
[0030] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.
[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0033] Example 1: Construction of knockout / integration plasmids for α-ketoglutarate metabolism modification
[0034] A schematic diagram of α-ketoglutarate biosynthesis and metabolism is shown below. Figure 1 As shown, the synthesis and consumption of α-ketoglutarate in microbial cells are dynamically regulated by multiple metabolic pathways. Enhancing the expression of the pyruvate carboxylase gene pyc (GenBank accession number BAB98082.1) improves the conversion efficiency of pyruvate to oxaloacetate, thereby strengthening the supply capacity of upstream carbon sources in the tricarboxylic acid cycle. Enhancing the expression of the L-glutamate oxidase gene lgox (GenBank accession number KY315918.1) promotes the efficient conversion of L-glutamate to α-ketoglutarate, increasing the rate of target product formation. Simultaneously, enhancing the expression of the catalase gene katA (GenBank accession number BAB97648.1) promptly removes hydrogen peroxide generated during L-glutamate oxidation, mitigating the inhibitory effect of oxidative stress on cell metabolism and enzyme activity, thereby maintaining the stability of the cell system. In terms of reducing competitive metabolism, knocking out or weakening the α-ketoglutarate dehydrogenase gene odhA (GenBank accession number BAB98522.1) reduces the consumption of α-ketoglutarate further entering the downstream of the tricarboxylic acid cycle; regulating the glutamate synthase gene gltB (GenBank accession number BAB97577.1) and the glutamate dehydrogenase gene gdh (GenBank accession number BAB99472.1) reduces the diversion of α-ketoglutarate to the amino acid synthesis pathway; weakening the lactate dehydrogenase gene ldh (GenBank accession number BAC00305.1) reduces the conversion of carbon sources to byproducts such as lactate, improving carbon utilization efficiency; and regulating the serine / threonine protein kinase gene pknG (GenBank accession number BAC00145.1) optimizes the intracellular metabolic regulatory network, thereby further promoting the accumulation of target products. Furthermore, this invention reduces the extracellular transport of the key metabolic intermediate glutamate by knocking out the transporter protein system-related gene cg1434 (whose encoded amino acid sequence corresponds to the GenBank accession number BAB98663.1), thereby increasing the effective accumulation of α-ketoglutarate in cells.
[0035] In this embodiment, the pK18mobsacB shuttle plasmid is used as the vector backbone to achieve the knockout or integration of the target gene at a specific site in the Corynebacterium glutamicum genome. This vector contains a sacB anti-selection marker, which can be used to achieve double-crossover homologous recombination, thereby improving gene editing efficiency.
[0036] Using Corynebacterium glutamicum genomic DNA or existing plasmids as templates, the target gene fragment and its upstream and downstream homologous arm sequences were obtained by PCR amplification. After purification, the amplified product was ligated into the linearized pK18mobsacB vector backbone. In the specific construction process, a one-step seamless cloning technique (ClonExpress® II One Step Cloning Kit, Vazyme Biotech, China) was used to directionally splice each DNA fragment, ensuring that the target gene fragment and its homologous arms were inserted into the vector in a predetermined order, thereby constructing recombinant plasmids for gene knockout or gene integration. These recombinant plasmids, after transformation, screening, and sequencing verification, were used for the subsequent construction of Corynebacterium glutamicum metabolically engineered strains. Primers used for constructing the recombinant plasmids were all synthesized by commercial companies.
[0037] (1) Knockout of α-ketoglutarate dehydrogenase odhA gene
[0038] In the production process targeting α-ketoglutarate, the presence of the odhA gene leads to the continuous consumption of the target product, thus limiting its accumulation. Therefore, knocking out or weakening the odhA gene can help block this metabolic branch and increase the proportion of carbon allocated to α-ketoglutarate.
[0039] To knock out the odhA gene on the chromosome, a recombinant plasmid PK-odhA for gene knockout needs to be constructed. The specific steps are as follows: ① Using Corynebacterium glutamicum genomic DNA as a template, primers are designed according to the target gene sequence to amplify the upstream and downstream homologous arm sequences of the odhA gene, respectively. The homologous arm length is 1000 bp. The amplification primers introduce a nucleotide sequence homologous to the multiple cloning site (MCS) region of the plasmid vector at their 5' end. The homologous sequence length is approximately 20 bp, so as to achieve directional recombination ligation between the amplified fragment and the vector backbone during subsequent seamless cloning. ② The pK18mobsacB shuttle plasmid is linearized after digestion with BamHI and XbaI restriction endonucleases to obtain the vector backbone. ③ Using the Novizan one-step high-efficiency seamless cloning kit, the plasmid backbone and upstream and downstream homologous arm fragments obtained from amplification were ligated to construct the recombinant plasmid PK-odhA for odhA gene knockout; ④ The obtained knock-in plasmid PK-odhA was transformed into Corynebacterium glutamicum, and single-crossover integrated strains were obtained through kanamycin resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thus achieving odhA gene knockout. The α-ketoglutarate dehydrogenase gene knockout strain was obtained using a two-step homologous recombination method, and its genotype is represented as ΔodhA.
[0040] (2) Knockout of the cg1434 gene of glutamate transporter
[0041] In the production process targeting α-ketoglutarate, glutamate, as an important metabolic intermediate, is effluxed, which reduces the efficient utilization of intracellular carbon flux, thus indirectly affecting the accumulation of α-ketoglutarate. Therefore, knocking out or inhibiting the expression of the glutamate transporter gene cg1434 (the corresponding GenBank accession number for its encoded amino acid sequence is BAB98663.1) helps reduce the extracellular transport of glutamate, increases the retention level of intracellular metabolic intermediates, and thus promotes the allocation of carbon flux to α-ketoglutarate.
[0042] The recombinant plasmid PK-cg1434 for gene knockout was constructed using the following steps: ① Using Corynebacterium glutamicum genomic DNA as a template, primers were designed based on the target gene sequence to amplify the upstream and downstream homologous arm sequences of the cg1434 gene, with each homologous arm being 1000 bp in length. A nucleotide sequence homologous to the multiple cloning site region of the plasmid vector was introduced at the 5' end of each primer, with a homologous sequence length of 20 bp, to facilitate directional recombination between the amplified fragment and the vector backbone during subsequent seamless cloning. ② The pK18mobsacB shuttle plasmid was linearized after digestion with BamHI and XbaI restriction endonucleases to obtain the vector backbone. ③ Using the Novizan one-step high-efficiency seamless cloning kit, the plasmid backbone and the amplified upstream and downstream homologous arm fragments were ligated to construct the recombinant plasmid PK-cg1434 for cg1434 gene knockout. ④ The obtained knock-in plasmid PK-cg1434 was transformed into Corynebacterium glutamicum, and single-exchange integrated strains were obtained through resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thus achieving the knockout of the cg1434 gene. A glutamate transporter gene knockout strain was obtained using a two-step homologous recombination approach, and its genotype is represented as Δcg1434.
[0043] (3) Glutamate synthase gltB gene knockout
[0044] In production systems targeting α-ketoglutarate, expression of the gltBCD gene, an enzyme that synthases glutamate, leads to the diversion of α-ketoglutarate to the glutamate synthesis pathway, thereby reducing its intracellular accumulation. Therefore, knocking out the gltB gene (whose encoded amino acid sequence corresponds to the GenBank accession number BAB97577.1) helps disrupt the function of the glutamate synthase complex, reducing the consumption of α-ketoglutarate by this branch pathway and increasing the accumulation of the target product.
[0045] The recombinant plasmid PK-gltB for gene knockout was constructed using the following steps: ① Using Corynebacterium glutamicum genomic DNA as a template, primers were designed based on the target gene sequence to amplify the upstream and downstream homologous arm sequences of the gltB gene, with each homologous arm being 1000 bp in length. A nucleotide sequence homologous to the multiple cloning site region of the plasmid vector was introduced at the 5' end of each primer, with a homologous sequence length of 20 bp, to facilitate directional recombination between the amplified fragment and the vector backbone during subsequent seamless cloning. ② The pK18mobsacB shuttle plasmid was linearized after digestion with BamHI and XbaI restriction endonucleases to obtain the vector backbone. ③ Using the Novizan one-step high-efficiency seamless cloning kit, the plasmid backbone and the amplified upstream and downstream homologous arm fragments were ligated to construct the recombinant plasmid PK-gltB for gltB gene knockout. ④ The obtained knock-in plasmid PK-gltB was transformed into Corynebacterium glutamicum, and single-exchange integrated strains were obtained through resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thus achieving the knockout of the gltB gene. The glutamate synthase gene knockout strain was obtained using a two-step homologous recombination method, and its genotype was represented as ΔgltB.
[0046] Following the methods described above, recombinant plasmids PK-ldh, PK-gdh, and PK-pknG were constructed sequentially, knocking out the lactate dehydrogenase gene (ldh), the glutamate dehydrogenase gene (gdh), and the serine / threonine protein kinase gene (pknG). These knock-in plasmids were then transformed into *Corynebacterium glutamicum*, and single-crossover integrated strains were obtained through resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thus achieving the knockout of the ldh, gdh, and pknG genes. A two-step homologous recombination method was used to obtain a lactate dehydrogenase gene knockout strain (genotype Δldh), a glutamate dehydrogenase gene knockout strain (genotype Δgdh), and a serine / threonine protein kinase gene knockout strain (genotype ΔpknG).
[0047] (4) Enhanced expression of the pyc gene encoding pyruvate carboxylase
[0048] In a production system targeting α-ketoglutarate, increasing the expression level of the pyc gene (the GenBank accession number for its encoded amino acid sequence is BAB98082.1) can help enhance the supply of precursors such as oxaloacetate, thereby promoting the entry of carbon streams into the tricarboxylic acid cycle and improving the synthesis capacity of α-ketoglutarate.
[0049] In situ enhanced expression of the pyruvate carboxylase gene pyc was achieved using the strong promoter Psod, specifically by inserting the strong promoter P before the start codon of the pyc gene on the chromosome. sod Constructing the knock-in plasmid PK-P sod -pyc, primers were designed based on the target gene sequence, and Corynebacterium glutamicum genomic DNA was used as a template to amplify the upstream homologous arm and strong promoter P of the pyc gene (its amino acid sequence corresponds to the GenBank accession number BAB98082.1). sod The sequence and downstream homologous arm of the pyc gene were obtained. The homologous arm is 1000 bp in length. A 20 bp homologous sequence was introduced at the 5' end of the amplification primers to facilitate directional recombination ligation between the amplified fragments and the vector backbone during subsequent seamless cloning. The pK18mobsacB shuttle plasmid was linearized after digestion with BamHI and XbaI restriction endonucleases to obtain the vector backbone. Subsequently, the vector backbone was ligated with the amplified fragments using a one-step seamless cloning kit to construct the recombinant plasmid PK-P for enhanced pyc gene expression. sod -pyc. The recombinant plasmid was transformed into *Corynebacterium glutamicum* competent cells, and single-crossover integrated strains were obtained through resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thereby achieving a strong promoter P on the chromosome. sod In situ substitution expression of the pyc gene. A strain with enhanced pyc gene expression was obtained using a two-step homologous recombination approach; its genotype is represented as P. sod -pyc.
[0050] Following the method described above, using the strong promoter P sod To achieve in situ enhanced expression of the catalase gene katA (whose encoded amino acid sequence corresponds to the GenBank accession number BAB97648.1), a strong promoter was inserted before the start codon of the katA gene on the chromosome, and a recombinant plasmid PK-P with enhanced katA gene expression was constructed. sod -katA. The recombinant plasmid was transformed into competent Corynebacterium glutamicum cells, and single-crossover integrated strains were obtained through resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thereby achieving a strong promoter P on the chromosome. sod In situ substitution expression of the katA gene. A strain with enhanced katA gene expression was obtained using a two-step homologous recombination approach; its genotype is represented as P. sod -katA.
[0051] (5) Enhanced expression of the glutamate oxidase encoding gene Igox
[0052] The lgox gene encodes glutamate oxidase, an enzyme belonging to the flavin-dependent oxidoreductase family. This enzyme catalyzes the oxidation of L-glutamate to α-ketoglutarate, along with the production of ammonia and hydrogen peroxide. This type of enzyme exhibits high substrate specificity and can catalyze reactions without the need for exogenous cofactors, thus showing great potential for application in the biotransformation of α-ketoglutarate. In production systems targeting α-ketoglutarate, increasing the expression level of the lgox gene enhances the catalytic activity of this reaction pathway, thereby improving the efficiency of target product formation.
[0053] The expression of the L-glutamate oxidation lgox gene (GenBank accession number KY315918) was enhanced at the locus of the lactate dehydrogenase-encoding gene ldh (GenBank accession number BAC00305.1). Specifically, the lgox gene replaced the ldh gene on the chromosome, and a strong promoter P was introduced upstream of it. tuf Thus, an engineered strain that highly expresses Igox was constructed.
[0054] Construction of recombinant plasmid pK-P for Igox gene expression tuf The specific method for amplifying lgox is as follows: Primers were designed based on the sequences flanking the ldh gene locus to amplify the upstream and downstream homologous arms of the ldh gene using Corynebacterium glutamicum genomic DNA as a template; primers were designed based on the tuf gene promoter sequence to amplify the strong promoter Ptuf sequence using Corynebacterium glutamicum genomic DNA as a template; primers were designed based on the lgox coding gene sequence to amplify the target gene fragment using the glutamate oxidase gene from Streptomyces mobaraensis as a template. The amplification primers described above introduced a 20bp homologous sequence at their 5' ends to facilitate directional recombination between the amplified fragment and the vector backbone during subsequent seamless cloning. The pK18mobsacB shuttle plasmid was linearized after digestion with BamHI and XbaI restriction endonucleases to obtain the vector backbone. Subsequently, the vector backbone was ligated with the amplified fragments using a one-step seamless cloning kit to construct the recombinant plasmid pK-P, which is used to replace lgox at the ldh site and introduces the strong promoter tuf sequence upstream of it. tuf -lgox. The recombinant plasmid was transformed into competent Corynebacterium glutamicum cells, and single-crossover integrated strains were obtained through resistance selection. Subsequently, reverse selection was performed in a sucrose-containing medium to obtain double-crossover mutants that underwent a second homologous recombination, thereby achieving the replacement of the ldh gene with the lgox gene and its integration into the strong promoter P. tufIgX-enhanced expression was achieved through a two-step homologous recombination approach, and its genotype was denoted as P. tuf -lgox.
[0055] Example 2: Construction of genetically engineered bacteria for producing α-ketoglutaric acid
[0056] Using *Corynebacterium glutamicum* ATCC 13032 as the starting strain (obtained from the China Industrial Microbial Culture Collection Center, a commercially available standard strain), the odhA gene was knocked out sequentially via a two-step homologous recombination method according to Example 1 to obtain the α-ketoglutarate dehydrogenase-deficient strain CgAKG1 (ΔodhA) to reduce the metabolic loss of α-ketoglutarate to succinyl-CoA. Subsequently, the cg1434 gene was further knocked out on the ΔodhA strain to obtain the CgAKG2 strain (ΔodhA; Δcg1434) to reduce the efflux loss of glutamate and other related metabolic intermediates. Furthermore, the gltB gene was knocked out on the above strains to obtain the CgAKG3 strain (ΔodhA; Δcg1434; ΔgltB) to reduce the diversion of α-ketoglutarate to the glutamate synthesis pathway. Furthermore, by knocking out the ldh gene from the above strains, the CgAKG4 (ΔodhA; ΔgltB; Δcg1434; Δldh) strains were obtained, reducing the conversion of carbon sources into byproducts such as lactic acid.
[0057] To enhance precursor supply capacity, based on the aforementioned engineered strain CgAKG4, the expression of the pyc gene was enhanced at the chromosomal level through a promoter substitution strategy. Specifically, a strong promoter, Psod, was introduced upstream of the start codon of the pyc gene, resulting in the pyc gene-enhanced strain CgAKG5 (ΔodhA; Δcg1434; ΔgltB; Δldh; Psod-pyc). This modification helps improve the conversion efficiency of pyruvate to oxaloacetate, thereby enhancing the supply of precursors for the tricarboxylic acid cycle. Building upon this, the gdh gene was further knocked out to obtain the CgAKG6 (ΔodhA;Δcg1434;ΔgltB;Δldh;Psod-pyc;Δgdh) strain, in order to reduce the reversible consumption of α-ketoglutarate towards glutamate; and the pknG gene was further knocked out to obtain the CgAKG7 (ΔodhA;Δcg1434;ΔgltB;Δldh;Psod-pyc;Δgdh;ΔpknG) strain, in order to weaken the promoting effect of the relevant global transcriptional regulatory network on the downstream metabolic allocation of α-ketoglutarate.
[0058] Furthermore, to enhance the target response pathway, the Igox gene was introduced at the ldh locus, and a strong promoter P was introduced upstream of it. tufTo achieve efficient expression of the L-glutamate oxidase gene, and simultaneously replace the ldh gene to reduce lactate byproduct formation, CgAKG8 (ΔodhA;Δcg1434;ΔgltB;Δldh::P) was obtained. tuf -logx;P sod The engineered strain CgAKG9 (ΔodhA;Δgdh;ΔpknG) was developed to reduce oxidative stress on cells caused by hydrogen peroxide produced during L-glutamate oxidation. Further, a promoter substitution strategy was used to enhance katA gene expression, resulting in the katA gene-enhanced strain CgAKG9 (ΔodhA;Δcg1434;ΔgltB;Δldh::P). tuf -logx;P sod -pyc;Δgdh;ΔpknG;P sod -katA).
[0059] Through multiple rounds of metabolic engineering modifications, the genetically engineered bacterium CgAKG9 for the production of α-ketoglutarate was finally constructed. This engineered bacterium possesses multiple characteristics, including enhanced precursor supply, weakened competitive branching, reduced efflux of metabolic intermediates, enhanced target catalytic reactions, and reduced oxidative stress. It can effectively increase the proportion of carbon allocated to α-ketoglutarate, laying the foundation for subsequent fermentation production and whole-cell biotransformation. Colony PCR and sequencing were performed to verify the correctness of each target gene modification site. The constructed engineered bacterium was then used for subsequent fermentation performance evaluation and α-ketoglutarate production experiments.
[0060] Example 3: Fermentation culture and detection of genetically engineered bacteria producing α-ketoglutarate
[0061] The constructed genetically engineered bacteria were fermented to produce α-ketoglutaric acid, and their fermentation performance was analyzed. The method includes seed culture, shake-flask fermentation, and product detection. Details are as follows:
[0062] After plate activation, the genetically engineered strains (CgAKG1-CgAKG9) were inoculated into seed culture medium and cultured with shaking at 30–32℃ and 200–220 rpm for 16–20 h to obtain seed culture. Subsequently, the seed culture was transferred to fermentation medium at an inoculation rate of 5%–10% and cultured in shake flasks at 30–35℃ and 200–220 rpm for 48–72 h. During fermentation, calcium carbonate was added to the culture system to adjust the pH; the preferred amount of calcium carbonate added was approximately 20 g / L, thereby maintaining the fermentation system within a suitable pH range.
[0063] The seed culture medium consisted of 40 g / L glucose, 30 g / L corn steep liquor, 4 g / L urea, 1 g / L KH₂PO₄, 0.4 g / L MgSO₄·7H₂O, 10 mg / L FeSO₄·7H₂O, 10 mg / L MnSO₄·5H₂O, 200 μg / L vitamin B1, and 50 μg / L biotin, with the initial pH adjusted to approximately 7.0 using KOH. The fermentation culture medium consisted of 40 g / L glucose, 1 g / L (NH₄)₂SO₄, 6 g / L L-glutamate sodium, 1.3 g / L KH₂PO₄, 0.8 g / L MgSO₄·7H₂O, 600 μg / L vitamin B1, 1 g / L alanine, and 30 g / L corn steep liquor. The inorganic ammonium salt content in the seed culture medium and fermentation culture medium is controlled at a low level (below 1 g / L), which constitutes ammonia-limited culture conditions. This limits the cells from allocating too much carbon source to the amino acid synthesis pathway, which is conducive to promoting the accumulation of carbon flow towards α-ketoglutarate and increasing the yield of the target product.
[0064] After fermentation, the fermentation broth sample was centrifuged, and the supernatant was collected. The α-ketoglutaric acid content was determined using high-performance liquid chromatography (HPLC). For the test, an appropriate amount of supernatant was diluted and injected (10 μL). Separation was performed using an organic acid analytical column under acidic mobile phase (5 mM dilute sulfuric acid) conditions, and detection was performed at a wavelength of 210 nm. Qualitative analysis was performed based on retention time, and the α-ketoglutaric acid content was quantitatively calculated using a standard curve.
[0065] The fermentation results of each genetically engineered strain are shown in Table 1. The results indicate that each metabolic engineering step promoted the accumulation of α-ketoglutarate to varying degrees. Compared with the wild-type strain, the α-ketoglutarate yield of each engineered strain showed a significant upward trend after stepwise metabolic engineering, indicating that weakening competitive metabolic pathways and optimizing carbon flux allocation can effectively promote the synthesis of the target product. In the intermediate modification stage, knocking out the glutamate dehydrogenase gene gdh significantly reduced the reverse conversion of α-ketoglutarate to glutamate, allowing more carbon flux to remain in the target product pathway. Further knocking out the pknG gene, by regulating related signaling pathways, reduced the loss of α-ketoglutarate to downstream metabolism, thereby enhancing the efficiency of carbon flux allocation to α-ketoglutarate from a metabolic regulation perspective, resulting in a significant increase in yield. This shows that gdh and pknG play a key role in regulating carbon flux allocation. Based on the above modifications, the direct conversion of L-glutamate to α-ketoglutarate was significantly enhanced by introducing and efficiently expressing the glutamate oxidase gene lgox at the ldh locus, thus further increasing the synthesis rate of the target product. At the same time, by enhancing the expression of the catalase gene katA, the oxidative stress of hydrogen peroxide produced during the glutamate oxidation reaction on the cells was effectively alleviated, improving cell stability and sustained catalytic capacity, thereby further promoting the accumulation of α-ketoglutarate.
[0066] The engineered strain CgAKG9, finally constructed, exhibited optimal α-ketoglutarate production performance, with a yield of 21.6 ± 0.6 g / L, significantly higher than the starting strain ATCC 13032 (0.7 ± 0.2 g / L) by approximately 30 times, demonstrating excellent product synthesis capability. These results indicate that systematic optimization of the central metabolic pathway, combined with enhancement of key catalytic steps and improved antioxidant capacity, can significantly increase the accumulation level of α-ketoglutarate, validating the effectiveness and superiority of the metabolic engineering strategy described in this invention.
[0067] Table 1. Detection of fermentation yield of α-ketoglutarate-engineered bacteria
[0068] ;
[0069] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A genetically engineered bacterium that produces α-ketoglutaric acid, characterized in that, Based on the original strain Corynebacterium glutamate, the following gene modifications were made to its central metabolic pathway, including: weakening the expression of the α-ketoglutarate dehydrogenase gene odhA; Reduce the expression of the glutamate transporter gene cg1434; Reduce the expression of gltB, a key subunit gene of glutamate synthase; Reduce the expression of the lactate dehydrogenase gene ldh; Reduce the expression of the glutamate dehydrogenase gene gdh; Reduce the expression of the serine / threonine protein kinase gene pknG; Enhance the expression of the pyruvate carboxylase gene pyc; The glutamate oxidase gene lgox was introduced and expressed at the lactate dehydrogenase ldh gene site; and Enhance the expression of the catalase gene katA.
2. The genetically engineered bacterium according to claim 1, characterized in that, The weakening refers to reducing gene expression or activity; the enhancement refers to increasing the expression or activity of endogenous genes and / or introducing heterologous genes; the modification is achieved through homologous recombination.
3. The genetically engineered bacteria according to claim 2, characterized in that, The attenuation is achieved through inhibitors and / or knockout; the enhancement is achieved through promoter substitution to enhance the expression of endogenous or heterologous genes, wherein the promoter is a strong promoter P. sod and / or P tuf。 4. The genetically engineered bacterium according to claim 1, characterized in that, The GenBank accession number corresponding to the amino acid sequence encoded by odhA is BAB98522.1; the GenBank accession number corresponding to the amino acid sequence encoded by cg1434 is BAB98663.1; the GenBank accession number corresponding to the amino acid sequence encoded by gltB is BAB97577.1; the GenBank accession number corresponding to the amino acid sequence encoded by ldh is BAC00305.1; the GenBank accession number corresponding to the amino acid sequence encoded by gdh is BAB99472.1; the GenBank accession number corresponding to the amino acid sequence encoded by pknG is BAC00145.1; the GenBank accession number corresponding to the amino acid sequence encoded by pyc is BAB98082.1; the GenBank accession number corresponding to the amino acid sequence encoded by lgox is KY315918.1; and the GenBank accession number corresponding to the amino acid sequence encoded by katA is BAB97648.
1.
5. The genetically engineered bacterium according to claim 4, characterized in that, The glutamate oxidase gene lgox is derived from Streptomyces mobara.
6. The genetically engineered bacterium according to claim 4, characterized in that, The original strain of Corynebacterium glutamicum was selected from Corynebacterium glutamicum ATCC 13032.
7. The use of the genetically engineered bacteria according to any one of claims 1-6 in the production of α-ketoglutaric acid.
8. A method for producing α-ketoglutaric acid, characterized in that, Fermentation culture using the genetically engineered bacteria according to any one of claims 1-6 to produce α-ketoglutaric acid.
9. The method according to claim 8, characterized in that, The inorganic ammonium salt content in the fermentation culture medium is less than 1 g / L. The fermentation culture conditions are a temperature of 30℃~35℃, a rotation speed of 150 r / min~250 r / min, a culture time of 60 h~72 h, and the pH of the culture system is adjusted by calcium carbonate to make the pH=6.0~8.
0.
10. The method according to any one of claims 8-9, characterized in that, It also includes the step of separating or purifying the resulting α-ketoglutaric acid.