Recombinant escherichia coli, construction method and application thereof, and method for producing L-leucine
By overexpressing cstA, ptsG, and pfkA in E. coli and knocking out fbp, a synergistic metabolic network was constructed, solving the problems of low L-leucine production and numerous byproducts, and achieving efficient and stable L-leucine production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- COFCO NUTRITION AND HEALTH RESEARCH INSTITUTE CO LTD
- Filing Date
- 2026-01-12
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies for constructing microbial cell factories suffer from problems such as low L-leucine yield, slow cell growth, reduced conversion rate, and increased byproducts, making it difficult to balance high yield with cell physiological stability.
By constructing recombinant Escherichia coli, overexpressing pyruvate transporter CSTA, glucose transporter PTSG, and 6-phosphofructokinase PFKA, and knocking out fructose-1,6-bisphosphatase FBP, a synergistic metabolic network is formed, which improves glucose utilization and L-leucine production and inhibits byproduct formation.
It significantly improved the yield of L-leucine and the glucose conversion rate, reduced the generation of by-products, and achieved efficient biosynthesis with cost savings.
Smart Images
Figure CN121950651A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, specifically to a recombinant Escherichia coli, its construction method and application, and a method for producing L-leucine. Background Technology
[0002] L-Leucine serves as a raw material for protein synthesis, participating in the direct synthesis of proteins. Its catabolites provide the carbon source needed for molecules involved in maintaining cell structure and function, and supply nitrogen for the de novo synthesis of nucleotides and amino acids. Furthermore, L-leucine metabolism influences the epigenome by altering cofactor levels, ultimately regulating gene expression. Due to its important physiological functions in promoting muscle metabolism and energy supply, regulating the immune system, and affecting glucose and lipid metabolism, it has been widely applied in animal nutrition, food, and pharmaceutical industries.
[0003] The main methods for producing branched-chain amino acids (BCAAs) include chemical synthesis, enzymatic catalysis, hydrolysis extraction, and microbial fermentation. Chemical synthesis methods for amino acids include the Strecker process, α-haloacid ammonolysis, malonic acid ester synthesis, and phase transfer catalysis. Among these, microbial fermentation, with its advantages of being environmentally friendly, low-cost, and having high conversion rates, has become the primary method for BCAA synthesis. Microbial fermentation for BCAA synthesis involves constructing microbial cell factories using synthetic biology techniques such as genetic engineering and metabolic engineering, enabling them to efficiently synthesize the target products from carbon sources such as glucose through metabolic pathways.
[0004] Traditional random mutation construction of microbial cell factories leads to random changes in cell physiology. Even if the production of the target product amino acids increases, it is accompanied by negative effects such as slow cell growth, reduced conversion rate, and increased byproducts.
[0005] Therefore, there is an urgent need to provide a high-yield recombinant strain of L-leucine that balances high L-leucine production with cellular physiological stability, while also improving raw material conversion rate and inhibiting byproduct formation. Summary of the Invention
[0006] The purpose of this invention is to overcome the above-mentioned problems existing in the prior art and to provide a recombinant Escherichia coli, its construction method and application, and a method for producing L-leucine.
[0007] To achieve the above objectives, the first aspect of the present invention provides a recombinant Escherichia coli that simultaneously contains an overexpressed gene encoding a pyruvate transporter. cstA The gene encoding the overexpressed glucose transporter ptsG and the gene encoding overexpressed 6-phosphofructokinase pfkA It does not contain the gene encoding fructose-1,6-bisphosphatase. fbp .
[0008] A second aspect of the present invention provides a method for constructing recombinant Escherichia coli with increased L-leucine production, the method comprising: causing the starting strain to simultaneously overexpress a gene encoding a pyruvate transporter. cstA Genes encoding glucose transporters ptsG and the gene encoding 6-phosphofructokinase pfkA And knock out the gene encoding fructose-1,6-bisphosphatase in the starting strain. fbp .
[0009] The third aspect of this invention provides the application of the recombinant Escherichia coli described in the first aspect, or the recombinant Escherichia coli constructed by the method described in the second aspect, in increasing L-leucine production.
[0010] The fourth aspect of the present invention provides a method for producing L-leucine, the method comprising inoculating the recombinant Escherichia coli described in the first aspect, or the recombinant Escherichia coli constructed by the method described in the second aspect, into a fermentation medium for fermentation culture.
[0011] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The recombinant Escherichia coli provided by this invention can significantly improve the utilization rate of raw glucose, increase L-leucine production, and inhibit the formation of byproducts. In a preferred embodiment of this invention, when the recombinant Escherichia coli provided by this invention is fermented in a 5L fermenter for 20 hours, its L-leucine production reaches 70 g / L, the sugar-acid conversion rate reaches 36%, the daily output is significantly increased, and production costs are greatly reduced. Attached Figure Description
[0012] Figure 1 This is a schematic diagram of the modification mechanism of recombinant Escherichia coli provided by the present invention. Detailed Implementation
[0013] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0014] The inventors of this invention conducted a series of studies on the substrate conversion rate of recombinant Escherichia coli used in fermentation engineering, including exploring different pyruvate and glucose-related genes, such as glucose transporter genes (…). ptsG ), 6-phosphofructokinase gene ( pfkA ), pyruvate kinase gene ( pykFand pykA ), pyruvate transporter ( cstA and yhjX The invention investigated the effects of genes related to L-leucine substrate conversion. However, the inventors found that not all genes involved in pyruvate and glucose metabolism significantly improved L-leucine substrate conversion. Crossover experiments with several genes demonstrated that the gene performing best in single-factor experiments did not necessarily achieve the best results in crossover experiments. Synergistic effects existed among the genes, and only precise regulation through the merging of genes with positive synergistic effects could maximize substrate conversion.
[0015] Based on the above findings, the first aspect of the present invention provides a recombinant Escherichia coli, which simultaneously contains the gene encoding an overexpressed pyruvate transporter. cstA The gene encoding the overexpressed glucose transporter ptsG and the gene encoding overexpressed 6-phosphofructokinase pfkA It does not contain the gene encoding fructose-1,6-bisphosphatase. fbp .
[0016] Numerous genes are involved in the utilization and transformation of pyruvate and glucose, and changes in gene expression in other pathways may potentially interfere with the utilization and transformation of pyruvate and glucose. Limited single-factor or combination screening experiments cannot cover all possible gene combinations. Based on an in-depth analysis of the synergistic mechanism between the pyruvate-glucose metabolic network and the L-leucine synthesis pathway, the inventors of this invention have obtained the optimal gene combination through targeted design and systematic verification. Specifically, this invention significantly improves raw material utilization by integrating the metabolic pathways of pyruvate and glucose, thereby achieving efficient biosynthesis of L-leucine. Among these, overexpression of… cstA It can promote the reuse of pyruvate, thereby increasing the concentration of pyruvate, a precursor to L-leucine synthesis; overexpression ptsG and pfkA The conversion of glucose to leucine can be promoted by increasing the glucose transport rate and utilization efficiency; furthermore, by knocking out... fbp It can promote the directed inflow of glucose into the glycolysis pathway, thereby improving glucose utilization efficiency. The above four genes, through multi-target synergistic regulation, form a linked metabolic network of "glucose transport enhancement - glycolysis flux increase - pyruvate precursor enrichment", thereby achieving a synergistic improvement in L-leucine synthesis efficiency and substrate conversion rate.
[0017] In this invention, preferably, the recombinant Escherichia coli further contains an overexpressed gene encoding phosphotransketase. fpkt Genes encoding acetylphosphotransferase pta and the gene encoding overexpressed pyruvate kinasepykA At least one of them.
[0018] The recombinant E. coli overexpressing the above-mentioned gene can interact with the overexpressed gene. cstA , ptsG and pfkA It exerts a synergistic promoting effect by increasing the concentration of L-leucine synthesis precursors acetyl-CoA and pyruvate to further improve the synthesis efficiency of L-leucine.
[0019] More preferably, the fpkt It is derived from at least one of Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium breve, with Bifidobacterium adolescentis being more preferred.
[0020] More preferably, the fpkt Nucleotide sequences including those with optional codon optimization, including the nucleotide sequence shown in GenBank accession number PQ149030.1.
[0021] According to a preferred embodiment of the present invention, the fpkt This includes the nucleotide sequence shown in GenBank accession number PQ149030.1, which is a codon-optimized nucleotide sequence based on the codon preference of E. coli.
[0022] It should be noted that, in this invention, the aforementioned exogenous... fpkt The gene can be either its naturally derived wild-type gene sequence or a codon-optimized gene sequence. Because the exogenous gene used is not from *E. coli*, its expression efficiency in *E. coli* may be limited; therefore, codon optimization of the exogenous gene is necessary. The codon optimization method is a conventional technique known to those skilled in the art, and the optimized gene only adjusts the nucleotide sequence, without changing the amino acid sequence of the encoded enzyme or its corresponding biological activity; therefore, this will not be elaborated further.
[0023] According to a preferred embodiment of the present invention, and more preferably, before performing optional codon optimization, the... fpkt The nucleotide sequence selected is one that has at least 90% identity with the nucleotide sequence shown in GenBank accession number PQ149030.1. According to some specific embodiments of the present invention, the... fpkt Nucleotide sequences selected that have 90-92% identity with the nucleotide sequence shown in GenBank accession number PQ149030.1.
[0024] Those skilled in the art can use the gene encoding phosphotransketase fpktKnowing the amino acid sequence obtained from transcription and translation allows us to deduce the nucleotide sequence of the encoding gene from that sequence. Due to the existence of degenerate bases, the same amino acid sequence can correspond to multiple different nucleotide sequences. Specifically, of the 20 amino acids that make up proteins, except for methionine and tryptophan which correspond to only one codon, the remaining amino acids correspond to two or more synonymous codons. This codon degeneracy makes reverse deduction possible. fpkt When analyzing the nucleotide sequence of a gene, the original nucleotide sequence of the gene cannot be uniquely determined solely by the amino acid sequence of the phosphate transketolase it encodes; furthermore, different designs based on degenerate bases... fpkt Homologous genes, as long as they encode the same amino acid sequence or their core functional domains are conserved, can encode phosphotransketases with the same or similar catalytic activities.
[0025] In this invention, "identity" refers to fpkt Similarity to the above sequences. For example, based on sequence A with a length of 100 bp, sequence B obtained by substituting and mutating 3 nucleotides has 97% identity with sequence A; sequence C obtained by deleting 20 bp consecutively from its 5' end has 100% identity with sequence A, and sequence A has 80% identity with sequence C.
[0026] Preferably, the present invention pfkA The identity with the sequences listed above can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 100%, or it can be a range consisting of any two of the above values, or any intermediate value in that range.
[0027] More preferably, the fpkt , pta, pykA , cstA , ptsG and pfkA Overexpression is achieved by each using a strong promoter independently.
[0028] More preferably, the strong promoter is selected from at least one of Ptrc, P102, P119, Tac and PrrnB.
[0029] More preferably, the fpkt Overexpression is achieved by using P102.
[0030] More preferably, the pykA Overexpression is achieved using Ptrc and / or P119.
[0031] More preferably, the pta, cstA , ptsG andpfkA The overexpressions are implemented independently using Ptrc.
[0032] When the gene provided by this invention is overexpressed using the aforementioned strong promoter, its expression level enables the key enzyme activities and metabolic fluxes of each pathway to be matched and matched, forming a coordinated and unified metabolic flux regulation network. This network regulates key steps such as glucose uptake, glycolysis, pyruvate precursor enrichment, and L-leucine synthesis, ultimately significantly improving L-leucine production and substrate conversion.
[0033] According to a preferred embodiment of the present invention, preferably, the recombinant Escherichia coli further contains a gene encoding a weakly expressed α-ketoglutarate dehydrogenase. sucAB .
[0034] More preferably, the sucAB Weak expression is achieved using weak promoters.
[0035] More preferably, the weak promoter is selected from at least one of PflgC, PfliC and PfliA, and is preferably PflgC.
[0036] According to a preferred embodiment of the present invention, preferably, the recombinant Escherichia coli does not contain the gene encoding lactate dehydrogenase. ldhA.
[0037] According to a preferred embodiment of the present invention, preferably, the recombinant Escherichia coli does not contain the gene encoding pyruvate oxidase. poxB.
[0038] According to some specific embodiments of the present invention, cstA , ptsG , pfkA, fpkt , pta, pykA and sucAB It is present in the genome of the recombinant *E. coli*. This invention is for making... cstA , ptsG , pfkA, fpkt , pta, pykA and sucAB There are no particular limitations on the specific methods and techniques used to achieve this purpose in the genome of the recombinant E. coli; any method in the art that can achieve this purpose can be used. For example, gene editing technologies (such as CRISPR-Cas9 technology) can be employed.
[0039] According to some specific embodiments of the present invention, the recombinant Escherichia coli contains an overexpressed gene encoding acetylhydroxyl synthase that enables normal expression of acetylhydroxyl synthase. ilvH fbr(2) The genes encoding the synthase, dehydrogenase, and isomerase of α-isopropylmalate that enable normal expression of these enzymes. leuABCD And (3) the gene encoding a branched-chain amino acid transporter that promotes the expulsion of L-leucine from the cell. brnFE.
[0040] According to some specific embodiments of the present invention, in order to further promote the excretion of L-leucine from cells, recombinant Escherichia coli does not contain the gene encoding the carrier protein of the branched-chain amino acid transport system. brnQ .
[0041] In a preferred embodiment of the present invention, to further improve glucose utilization efficiency, the recombinant Escherichia coli also contains an overexpressed gene encoding a phosphoglycerate mutase. gpmA and the gene encoding the overexpressed enolase eno .
[0042] In a preferred embodiment of the present invention, the overexpression of the gpmA as well as eno The method can be the same as or similar to the overexpression of other genes defined above. For example, it is preferable to use the strong promoter Ptrc. gpmA as well as eno Overexpression.
[0043] According to a preferred embodiment of the present invention, in order to further improve glucose utilization efficiency, the recombinant *Escherichia coli* does not contain the gene encoding phosphoenolpyruvate synthase. ppsA .
[0044] The modification mechanisms of each gene in some preferred recombinant Escherichia coli provided in this invention can be found by referring to... Figure 1 In the diagram, red arrows represent overexpression, black arrows with red crosses represent knockout, and gray arrows represent weak expression; Leu represents L-leucine, Lac represents lactate, Ace represents acetic acid, PYR represents pyruvate, PEP represents phosphoenolpyruvate, Glu represents glucose, Acetyl-CoA represents acetyl-CoA, AcP represents acetyl phosphate, and X5P represents xylose-5-phosphate. ackA The gene encodes acetate kinase. In a preferred embodiment of the present invention, by constructing and establishing a highly efficient end-to-end system of "glucose uptake - glycolysis propulsion - pyruvate precursor enrichment - L-leucine synthesis", a significant increase in L-leucine production is ultimately achieved.
[0045] According to some specific embodiments of the present invention, the overexpression, knockout, or weak expression of all genes is carried out on the genome of Escherichia coli.
[0046] This invention utilizes a strategy for constructing synergistically regulated metabolic pathways through overexpression, attenuation, or knockout of specific genes. This strategy is applicable to all *E. coli* strains in the field that possess or have the potential to produce L-leucine, including wild-type strains, strains with L-leucine production potential, and engineered strains related to L-leucine synthesis that have undergone preliminary modification, such as *E. coli* MG1655, wild-type *E. coli* K12, and wild-type *E. coli* W3110. According to some specific embodiments of this invention, the starting strain is wild-type *E. coli* W3110.
[0047] A second aspect of the present invention provides a method for constructing recombinant Escherichia coli with increased L-leucine production, the method comprising: causing the starting strain to simultaneously overexpress a gene encoding a pyruvate transporter. cstA Genes encoding glucose transporters ptsG and the gene encoding 6-phosphofructokinase pfkA And knock out the gene encoding fructose-1,6-bisphosphatase in the starting strain. fbp .
[0048] Preferably, the recombinant Escherichia coli also overexpresses the gene encoding phosphotransketase. fpkt Genes encoding acetylphosphotransferase pta, The gene encoding pyruvate kinase pykA At least one of them.
[0049] Preferably, the recombinant Escherichia coli also weakly expresses the gene encoding α-ketoglutarate dehydrogenase. sucAB .
[0050] Preferably, the recombinant Escherichia coli has the gene encoding lactate dehydrogenase knocked out. ldhA and / or the gene encoding pyruvate oxidase poxB.
[0051] According to some specific embodiments of the present invention, cstA , ptsG , pfkA, fpkt , pta, pykA and sucAB Overexpression, weak expression, and knockout operations were all performed within the genome of the recombinant Escherichia coli.
[0052] According to some specific embodiments of the present invention, the recombinant Escherichia coli also overexpresses (1) the gene encoding acetylhydroxyl synthase, which enables normal expression of acetylhydroxyl synthase. ilvH fbr (2) The genes encoding the synthase, dehydrogenase, and isomerase of α-isopropylmalate that enable normal expression of these enzymes. leuABCDAnd (3) the gene encoding a branched-chain amino acid transporter that promotes the expulsion of L-leucine from the cell. brnFE.
[0053] According to some specific embodiments of the present invention, in order to further promote the expulsion of L-leucine from the cell, the gene encoding the branched-chain amino acid transport system carrier protein was knocked out in recombinant Escherichia coli. brnQ .
[0054] According to a preferred embodiment of the present invention, in order to further improve glucose utilization efficiency, the recombinant Escherichia coli also overexpresses the gene encoding phosphoglycerate mutase. gpmA and the gene encoding enolase eno .
[0055] According to a preferred embodiment of the present invention, the overexpression of the gpmA as well as eno The method can be the same as or similar to the overexpression of other genes defined above. For example, it is preferable to use the strong promoter Ptrc. gpmA as well as eno Overexpression.
[0056] According to a preferred embodiment of the present invention, in order to further improve glucose utilization efficiency, the recombinant Escherichia coli also has phosphoenolpyruvate synthase knocked out. ppsA .
[0057] According to some specific embodiments of the present invention, the starting strain is wild-type Escherichia coli W3110.
[0058] Preferably, the starting strain overexpresses the gene encoding the pyruvate transporter. cstA Genes encoding glucose transporters ptsG and the gene encoding 6-phosphofructokinase pfkA The methods include gene editing.
[0059] According to some specific embodiments of the present invention, the overexpression and weak expression of the coding gene can be achieved by replacing the promoter. The methods for replacing the promoter are well known in the art, such as promoter replacement technology based on homologous recombination and CRISPR-Cas9-mediated promoter replacement, which will not be described in detail here.
[0060] According to some specific embodiments of the present invention, overexpression or weak expression of the coding gene can be achieved by replacing the promoter, and the replaced promoter (strong promoter or weak promoter) has the same definition as the definition.
[0061] According to some specific embodiments of the present invention, the knockout of the coding gene is achieved by replacing the target gene with the upper and lower homologous arms of a nonfunctional target gene whose nucleotide sequence length is adapted to the target gene (the upper and lower homologous arms are abbreviated as gene abbreviations). ko )accomplish, ko The length of the gene is adaptively adjusted according to the length of the target gene that has been knocked out. The gene replacement method is well known in the field and will not be described in detail here.
[0062] The third aspect of this invention provides the application of the recombinant Escherichia coli described in the first aspect, or the recombinant Escherichia coli constructed by the method described in the second aspect, in increasing L-leucine production.
[0063] The present invention further provides the application of the recombinant Escherichia coli described in the first aspect, or the recombinant Escherichia coli constructed by the method described in the second aspect, in improving glucose conversion rate.
[0064] The fourth aspect of the present invention provides a method for producing L-leucine, the method comprising inoculating the recombinant Escherichia coli described in the first aspect, or the recombinant Escherichia coli constructed by the method described in the second aspect, into a fermentation medium for fermentation culture.
[0065] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.
[0066] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from legitimate chemical or biological reagent / material suppliers, and all reagents are chemically pure.
[0067] In the following embodiments, unless otherwise specified, the primer sequences used for gene amplification can be easily obtained by those skilled in the art through conventional deduction methods based on known gene template sequences or publicly available information on the E. coli genome.
[0068] In the following embodiments, unless otherwise specified, the “operation sites” in Table 1 are known to those skilled in the art, for example, the methods described in Wang, Xiaolei, et al. "Integration site library for efficient construction of plasmid-free microbial cell factories in Escherichia coli." Journal of Agricultural and Food Chemistry 72.44 (2024): 24687-24696.
[0069] Example 1 I. Construction Method 1. Achieving gene overexpression or attenuation by replacing the promoter: (1.1) Plasmid construction Using the pTargetF plasmid as a template, a linear vector with a 20bp specific cleavage site (original or pseudogenomic site) was obtained by PCR amplification using primers. After digestion with DpnI restriction endonuclease (DMT) at 37°C for 1 hour, the vector was chemically transformed into *E. coli* DH5α competent cells and plated on streptomycin-resistant plates containing 40 μg / mL for verification. Single colonies carrying the corresponding pTargetF plasmid were screened. After overnight incubation in LB medium at 37°C, the plasmid was extracted using a plasmid extraction kit.
[0070] (1.2) Construction of recombinant gene fragments Constructing the recombinant gene fragment: upstream homologous arm - promoter - target gene - downstream homologous arm. The nucleotide sequences of both the upstream and downstream homologous arms were obtained by PCR amplification using primers according to the operating sites shown in Table 1. Those skilled in the art can clearly obtain the specific nucleotide sequences of the upstream and downstream homologous arms described in this invention based on the site information disclosed in Table 1.
[0071] (1.3) Integration site of recombinant gene fragments The constructed plasmid and recombinant gene fragment were electroporated into competent cells of a strain containing the pCas9 plasmid. The cells were plated on antibiotic-resistant plates containing 40 μg / mL streptomycin and 50 μg / mL kanamycin and incubated at 30°C for 24 h. Positive transformants were obtained by routine colony PCR screening. Recombinant strains with correctly sequenced positive transformants underwent IPTG treatment and incubation at 42°C, resulting in plasmid loss. After integration, the "original gene" at the manipulation site was replaced with the "promoter gene-target gene," for example, in operation sequence 3, the gene encoding the branched-chain amino acid transport system carrier protein was knocked out. brnQ And the Ptrc promoter is used to control the coding gene of branched-chain amino acid transporters. brnFE Overexpression (about to) brnQ Replace with overexpression brnFE ).
[0072] 2. Gene knockout The plasmids are prepared according to the overexpression or attenuation method, the recombinant gene fragments are constructed, and the recombinant gene fragments are integrated into the gene locus. The difference is that the target gene is replaced with the upstream homologous arm-downstream homologous arm, and no promoter is used.
[0073] II. Using wild-type Escherichia coli W3110 as the starting strain, strain A1 was obtained by overexpressing, knocking out, or weakening its expression according to the order in Table 1. Except for... fpkt Apart from (GenBank: PQ149030.1), all genes involved in this embodiment are derived from the starting strain W3110 (genetic information can be found in NC_000913.3). The names of each target gene and its location on NC_000913.3 are shown in Table 1. In Table 1, " / "This indicates that gene editing is performed at the target gene location." — "" indicates that no promoter was used. The nucleotide sequences of the promoters used are shown in Table 2.
[0074] Table 1
[0075] Table 2
[0076] Example 2 Following the method in Example 1, using strain A1 as the starting strain, the strains listed in Table 3 were introduced into it. gpmA Genes were obtained from strain A2; using strain A2 as the starting strain, the strains listed in Table 3 were introduced into it. eno Strains A3 were obtained; using A3 as the starting strain, the strains listed in Table 3 were knocked out. ppsA Strains A4 were obtained.
[0077] Table 3
[0078] Comparative Example 1 Recombinant Escherichia coli was constructed according to the method in Example 1, except that it was not knocked out. fbp strain B1 was obtained.
[0079] Test Example 1 Seed culture medium: glucose 20 g / L, peptone 5 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 4 g / L, ammonium sulfate 5 g / L, sodium citrate 2 g / L, magnesium sulfate heptahydrate 2 g / L, ferrous sulfate heptahydrate 0.04 g / L, manganese sulfate tetrahydrate 0.03 g / L, anhydrous disodium hydrogen phosphate 6 g / L.
[0080] Fermentation medium: glucose 20g / L, yeast extract 2g / L, citric acid 2g / L, K2HPO4 7g / L, (NH4)2SO4 3g / L, MgSO4·7H2O 1g / L, L-methionine (methionine) 1g / L, biotin 0.2mg / L, complex vitamin B 20mg / L, trace elements 97.93mg / L, methionine 0.5g / L, monosodium glutamate 0.5g / L, isoleucine 0.05g / L, threonine 0.05g / L, lysine 0.05g / L, aspartic acid 0.05g / L.
[0081] The strain was inoculated into a 5L mechanically stirred fermenter containing seed culture medium and cultured at 37℃. During cultivation, the pH was maintained at 7.2 by automatically adding 20% ammonia solution. The fermentation time was 12-16 hours until OD (Organic Demand) was reached. 600 When the value reaches 10, seed liquid is obtained.
[0082] The seed culture was inoculated at a ratio of 10 vol% into a 5L mechanically stirred fermenter containing fermentation medium and cultured at 35°C. During the fermentation, the pH was maintained at 7.2 by automatically adding 20% ammonia solution, the dissolved oxygen value was maintained at 30% by adjusting the stirring speed or ventilation, and the glucose concentration in the tank was maintained at 0.2 g / L by adding 80% glucose solution. Fermentation was carried out for 24 hours to obtain the fermentation broth.
[0083] The total glucose consumption during fermentation (addition amount - remaining amount) was calculated. The residual glucose and L-leucine content in the fermentation broth were measured using a high-performance liquid chromatograph (Thermo Fisher Scientific) and an amino acid analyzer (Secam). The OD value in the fermentation broth was measured using a UV spectrophotometer (Shimadzu). 600 The results are shown in Table 3. The sugar-acid conversion rate is calculated as follows: (L-leucine yield (g / L) × fermentation broth volume (L)) / total glucose consumed (g) × 100%. The results show that compared to the starting strain or existing engineered strains, the recombinant *E. coli* strain provided by this invention achieves a significant increase in both L-leucine yield and substrate glucose conversion rate.
[0084] In addition, to comprehensively evaluate the product specificity and raw material utilization efficiency of the fermentation system, amino acid byproducts were further measured to clarify the ratio between the target product L-leucine and the byproducts, thus demonstrating the superiority of the fermentation process of this invention. The types and contents of the measured byproducts are shown in Table 4. In Table 4, "ND" indicates not detected. The results show that the byproduct content of strain A4 provided by this invention is low after fermentation. Among them, the contents of aspartic acid, serine, alanine, valine, isoleucine, and proline are below the detection limit and therefore not detected. This indicates that this invention, through targeted regulation of key genes, effectively blocks the diversion of carbon source to other bypass amino acids, significantly reduces the generation of byproducts, and significantly improves the synthesis specificity of the target product L-leucine.
[0085] Table 3
[0086] Table 4
[0087] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A recombinant Escherichia coli, characterized in that, This recombinant E. coli also contains the gene encoding an overexpressed pyruvate transporter. cstA The gene encoding the overexpressed glucose transporter ptsG and the gene encoding overexpressed 6-phosphofructokinase pfkA It does not contain the gene encoding fructose-1,6-bisphosphatase. fbp .
2. The recombinant Escherichia coli according to claim 1, wherein, The recombinant E. coli also contains an overexpressed gene encoding phosphotransferase. fpkt The gene encoding overexpressed acetylphosphotransferase pta and the gene encoding overexpressed pyruvate kinase pykA At least one of them; And / or, the recombinant Escherichia coli does not contain the gene encoding lactate dehydrogenase. ldhA ; And / or, the recombinant Escherichia coli does not contain the gene encoding pyruvate oxidase. poxB; And / or, the recombinant Escherichia coli also contains a gene encoding a weakly expressed α-ketoglutarate dehydrogenase. sucAB .
3. The recombinant Escherichia coli according to claim 1 or 2, wherein, The fpkt It comes from at least one of Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium breve; Preferably, the fpkt From Bifidobacterium adolescentis; More preferably, the fpkt It has at least 90% identity with the nucleotide sequence shown in GenBank accession number PQ149030.1; More preferably, the fpkt Nucleotide sequences that include the nucleotide sequence shown in GenBank accession number PQ149030.1, with optional codon optimization.
4. The recombinant Escherichia coli according to any one of claims 1-3, wherein, The fpkt , pta, pykA , cstA , ptsG and pfkA Overexpression is achieved independently using strong promoters; Preferably, the strong promoter is selected from at least one of Ptrc, P102, P119, Tac, and PrrnB; More preferably, the fpkt Overexpression was achieved using P102; More preferably, the pykA Overexpression is achieved by using Ptrc and / or P119; More preferably, the pta, cstA , ptsG and pfkA The overexpressions are implemented independently using Ptrc.
5. The recombinant Escherichia coli according to any one of claims 1-4, wherein, The sucAB Weak expression is achieved by using a weak promoter; Preferably, the weak promoter is selected from at least one of PflgC, PfliC, and PfliA; More preferably, the weak promoter is PflgC.
6. The recombinant *Escherichia coli* according to any one of claims 1-5, wherein, cstA , ptsG , pfkA、fpkt , pta, pykA and sucAB It is present in the genome of the recombinant Escherichia coli.
7. A method for constructing recombinant Escherichia coli with increased L-leucine production, characterized in that, The method includes: causing the starting strain to simultaneously overexpress the gene encoding the pyruvate transporter. cstA Genes encoding glucose transporters ptsG and the gene encoding 6-phosphofructokinase pfkA And knock out the gene encoding fructose-1,6-bisphosphatase in the starting strain. fbp .
8. The method according to claim 7, wherein, The starting strain was wild-type Escherichia coli W3110; Preferably, the starting strain overexpresses the gene encoding the pyruvate transporter. cstA Genes encoding glucose transporters ptsG and the gene encoding 6-phosphofructokinase pfkA The methods include gene editing.
9. The recombinant Escherichia coli according to any one of claims 1-6, or the recombinant Escherichia coli constructed according to the method of claim 7 or 8, in increasing L-leucine production.
10. A method for producing L-leucine, characterized in that, The method includes inoculating the recombinant Escherichia coli according to any one of claims 1-6, or the recombinant Escherichia coli constructed according to the method of claim 7 or 8, into a fermentation medium for fermentation culture.