Genetically engineered bacteria for producing 2-oxoglutaric acid, method for producing 2-oxoglutaric acid and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a gene engineering bacterium for producing 2-oxoglutaric acid, a production method of 2-oxoglutaric acid and application thereof. The gene engineering bacterium contains L-glutamic acid oxidase gene and catalase gene in vivo; the gene engineering bacterium is a prokaryotic bacterium. The production method is as follows: inoculating the gene engineering bacterium into a whole cell catalytic fermentation system containing glutamic acid or glutamate, and performing whole cell catalytic fermentation to obtain 2-oxoglutaric acid. The application also relates to application of the gene engineering bacterium and the production method in production of 2-oxoglutaric acid. The application realizes high-efficiency biological preparation of 2-oxoglutaric acid by constructing an arabinose inducible expression system containing L-glutamic acid oxidase gene and catalase gene.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to genetically engineered bacteria that produce 2-oxoglutaric acid, methods for producing 2-oxoglutaric acid, and their applications. Background Technology

[0002] 2-Oxopentanedioic acid (also known as α-ketoglutaric acid, α-KG) is an important intermediate in the tricarboxylic acid cycle and has high application value in pharmaceuticals, food, feed, and fine chemicals. Therefore, developing efficient, green, and low-cost methods for preparing 2-oxopentanedioic acid is of great significance. Among existing technologies, the biocatalytic method for converting glutamic acid or glutamate into 2-oxopentanedioic acid has attracted attention due to its mild reaction conditions, wide availability of substrates, and high selectivity.

[0003] Existing research has yielded several methods for converting glutamate to 2-oxoglutaric acid by expressing glutamate oxidase in *Escherichia coli*. However, these methods still have some limitations. First, the process of glutamate oxidase catalyzing the conversion of glutamate to 2-oxoglutaric acid produces hydrogen peroxide. The accumulation of hydrogen peroxide not only causes oxidative stress to cells but may also inhibit the activity of related enzymes, thus affecting the whole-cell catalytic fermentation efficiency. Therefore, it is necessary to simultaneously introduce catalase (CAT) to decompose hydrogen peroxide into water and oxygen, eliminating its inhibitory effect. However, co-expression of both methods suffers from insufficient soluble expression and poor enzyme activity synergy. Second, conventional *E. coli* expression systems may also have issues such as high endotoxin levels and insufficient biosafety, thus limiting their applicability in subsequent industrial applications. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a genetically engineered bacterium that produces 2-oxoglutaric acid, a method for producing 2-oxoglutaric acid, and its application, so as to solve the problems of low expression efficiency of the target enzyme, high induction cost, and the need to further improve the whole-cell catalytic fermentation efficiency in the bio-preparation process of 2-oxoglutaric acid in the prior art.

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

[0006] The first aspect of the present invention provides a genetically engineered bacterium that produces 2-oxoglutarate, wherein the genetically engineered bacterium contains an L-glutamate oxidase gene and a catalase gene; the genetically engineered bacterium is a prokaryotic bacterium.

[0007] In some embodiments, the lipopolysaccharide (LPS) synthesis-related gene of the prokaryotic bacteria is knocked out. Further, the knocked-out LPS synthesis-related gene is the waaA gene.

[0008] In some embodiments, the genetically engineered bacteria further contain an inducible expression regulatory element; the inducible expression regulatory element is used to regulate the expression of the L-glutamate oxidase gene and the catalase gene in the genetically engineered bacteria in the presence of an inducer. Further, the inducible expression regulatory element is an arabinose-induced expression regulatory element, and the inducer is arabinose.

[0009] In some embodiments, the genetically engineered bacteria further contains a nucleotide sequence encoding an MBP tag, which is fused with L-glutamate oxidase and expressed such that the MBP tag in the resulting fusion protein is located at the N-terminus of the L-glutamate oxidase.

[0010] In some embodiments, the L-glutamate oxidase gene is derived from *Streptomyces mobaraensis*, and the catalase gene is derived from *Micrococcus*. Further, the amino acid sequence encoded by the L-glutamate oxidase gene is shown in SEQ ID No. 48; the amino acid sequence encoded by the catalase gene is shown in SEQ ID No. 49. In some preferred embodiments, the nucleotide sequence of the L-glutamate oxidase gene is shown in SEQ ID No. 2, and the nucleotide sequence of the catalase gene is shown in SEQ ID No. 5.

[0011] In some embodiments, the prokaryotic bacterium is *Escherichia coli* or *Corynebacterium glutamicum*, preferably *Escherichia coli* Nissle1917. Further, both the endogenous plasmid pMUT1 and pMUT2 of the *E. coli* are knocked out. In some preferred embodiments, the prokaryotic bacterium with the waaA gene knocked out is *E. coli* Nissle1917, and both the endogenous plasmids pMUT1 and pMUT2 of *E. coli* Nissle1917 are knocked out.

[0012] In some embodiments, the L-glutamate oxidase gene and catalase gene are carried by a recombinant vector; preferably, the starting plasmid of the recombinant vector is pET22b or pXMJ19.

[0013] A second aspect of the invention provides the use of the genetically engineered bacteria in the preparation of 2-oxoglutaric acid.

[0014] A third aspect of the present invention provides a method for producing 2-oxoglutaric acid, wherein the genetically engineered bacteria are inoculated into a whole-cell catalytic fermentation system containing glutamic acid or glutamate, and whole-cell catalytic fermentation is carried out to obtain 2-oxoglutaric acid.

[0015] In some embodiments, when the genetically engineered bacteria contain arabinose-inducible expression regulatory elements, arabinose is used to induce the expression of L-glutamate oxidase and catalase in the genetically engineered bacteria before inoculating them into the whole-cell catalytic fermentation system.

[0016] In some embodiments, when inducing the expression of L-glutamate oxidase and catalase in the genetically engineered bacteria containing arabinose-inducible expression regulatory elements, arabinose is added to the bacterial culture containing the genetically engineered bacteria for the first time to start the induction culture, and arabinose is added a second time during the induction culture; after the induction culture is completed, the bacterial culture is subjected to solid-liquid separation, and the genetically engineered bacteria are collected.

[0017] In some embodiments, the induction culture has one or more of the following characteristics: the induction culture temperature is 20–28°C; the induction culture duration is 6–10 h; when arabinose is added to the bacterial solution for the first time, the OD600 value of the genetically engineered bacteria in the bacterial solution is 0.6–0.8; after half of the induction time has been reached, arabinose is added to the bacterial solution for the second time; each time arabinose is added to the bacterial solution, the ratio of arabinose mass to bacterial solution volume is 1–5 g / L.

[0018] In some embodiments, the whole-cell catalytic fermentation method has one or more of the following characteristics: the whole-cell catalytic fermentation system is prepared by dispersing glutamic acid or glutamate in water or a buffer solution; the buffer solution is PBS buffer or MES buffer, preferably PBS buffer; the whole-cell catalytic fermentation system contains 80–200 g / L of glutamic acid or glutamate; the pH of the whole-cell catalytic fermentation system is 5–9, preferably 7–9; in the whole-cell catalytic fermentation system, the inoculum size of the induced cultured genetically engineered bacteria is 0.01–0.50 g / mL based on the wet cell mass; the whole-cell catalytic fermentation time is 4–20 h; the whole-cell catalytic fermentation temperature is 28–37 °C; and the whole-cell catalytic fermentation is carried out in a baffle flask.

[0019] A fourth aspect of the invention provides the application of the production method in the production of 2-oxoglutaric acid.

[0020] As described above, the present invention has the following beneficial effects:

[0021] 1. By knocking out the endogenous plasmids pMUT1 and pMUT2 in the host bacteria, interference with the introduction and stable maintenance of exogenous recombinant vectors is reduced, thus providing the prerequisite for the subsequent construction of genetically engineered bacteria that stably express the L-glutamate oxidase gene and catalase gene. Based on this, a recombinant vector containing the L-glutamate oxidase gene, catalase gene, and arabinose-induced expression regulatory element is introduced into the host bacteria, enabling the two key enzymes to be synergistically expressed within the same host system. Furthermore, this invention identifies a superior enzyme combination by screening L-glutamate oxidase and catalase from different sources; and by replacing the IPTG induction system with an arabinose induction system, combined with host bacteria screening, both induction costs and potential toxicity are reduced, while the adaptability of the expression system to industrial applications is improved. Furthermore, by optimizing the concentration of arabinose added during induction culture, the induction culture temperature, the induction culture time, and the strategy of secondary arabinose supplementation, the expression level of the target enzyme in the genetically engineered bacteria was improved. Based on this, by introducing an MBP tag at the N-terminus of L-glutamate oxidase, the soluble expression level of the enzyme was improved, inclusion body formation was reduced, allowing the target enzyme to function more fully. Subsequently, by optimizing the pH, buffer system, mass transfer conditions, inoculum size, substrate concentration, catalytic temperature, and catalytic time during whole-cell catalytic fermentation, the advantages brought about by the aforementioned strain construction and expression optimization were further reflected in the yield and molar conversion rate of 2-oxoglutaric acid. In other words, this invention, by constructing a continuous technical chain of "host bacteria modification → recombinant vector construction → enzyme combination screening → induction system optimization → enhanced soluble expression → catalytic condition optimization," enables the synergistic effect of various technical means to achieve the effects of improving the production efficiency of 2-oxoglutaric acid, improving the biosafety of the microbial fermentation system, and reducing production costs.

[0022] 2. The knockout of the waaA gene in this invention not only improves the biosafety of the engineered bacteria but also brings unexpected performance enhancements. Typically, waaA gene knockout is primarily understood to be associated with reduced endotoxin accumulation and improved biosafety, but not necessarily with a simultaneous improvement in catalytic performance. However, based on the completion of pMUT1 and pMUT2 knockout, optimal enzyme combination screening, establishment of the arabinose induction system, optimization of MBP-induced solubilization expression, and optimization of catalytic conditions, further knocking out the waaA gene in the host bacteria revealed that the yield of 2-oxoglutaric acid could still be further increased. This indicates that waaA gene knockout in this invention is not simply a safety modification, but rather, given the established high-efficiency expression and catalytic system, it further amplifies the effects of various 2-oxoglutaric acid yield optimization measures, thus giving the engineered bacteria of this invention both good catalytic performance and high application value. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the reaction process in which L-glutamate oxidase and catalase catalyze the production of 2-oxoglutarate from sodium glutamate in an embodiment of the present invention.

[0024] Figure 2 This is a graph showing the yield of 2-oxoglutaric acid whole-cell catalytic fermentation during the screening of the optimal enzyme combination in Example 2 of the present invention;

[0025] Figure 3 This is a graph showing the whole-cell catalytic fermentation yield of 2-oxoglutaric acid by different strains after induction with arabinose replacement in Example 3 of the present invention.

[0026] Figure 4 The figure shows the concentration of arabinose, induction culture time, induction culture temperature, and the yield of 2-oxoglutaric acid whole-cell catalytic fermentation after secondary induction in Example 4 of the present invention.

[0027] Figure 5 This is an SDS-PAGE gel electrophoresis image of MBP with added solubilization tag in Example 5 of the present invention. In the figure, 1 is the supernatant without solubilization tag, 2 is the precipitate without solubilization tag, 3 is the supernatant with solubilization tag, and 4 is the precipitate with solubilization tag.

[0028] Figure 6 The graph shows the yield of 2-oxoglutaric acid after optimization under different whole-cell catalytic fermentation conditions in Example 6 of this invention. Detailed Implementation

[0029] To make the inventive purpose, technical solution, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. However, it should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this application. All equivalent substitutions or modifications made by those skilled in the art to the technical solution of this application without departing from the spirit and substance of this application should fall within the scope of protection of this application.

[0030] Before further describing the specific embodiments of this application, it should be understood that the terminology used in this application specification is only used to describe specific embodiments and is not intended to limit this application. Unless otherwise expressly stated, the singular forms "an," "a," "described," etc., also include the plural forms. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the methods, reagents, and instruments used in this application are all conventional methods, conventional reagents, and conventional instruments in the art, and are commercially available.

[0031] In this application, the names of a protein and its encoding gene are sometimes used interchangeably. Those skilled in the art should be able to understand the actual meaning based on the specific context. For example, for LgoX, when describing its enzymatic function, expression product, or catalytic action, it usually refers to the L-glutamate oxidase protein; when describing cloning, amplification, ligation, expression vector construction, or nucleotide sequence, it usually refers to the gene encoding the protein. Similarly, for sgRNA, when describing its targeted recognition and guided cleavage function, it usually refers to the sgRNA molecule itself; when describing its design, cloning, introduction, or expression by plasmids, it may also refer to the DNA sequence encoding the sgRNA. Those skilled in the art can clearly distinguish its meaning based on the context.

[0032] The gene editing tool in this application can be a dual-plasmid editing system consisting of a pCas plasmid and a pTarget plasmid. The pCas plasmid provides the Cas protein expression element required for genome editing, while the pTarget plasmid provides the sgRNA guide sequence targeting the target site, thereby enabling site-specific editing of the host bacterial genome at the target site.

[0033] In this application, the dual plasmid editing system can be used to knock out endogenous plasmids pMUT1, pMUT2, and / or the waaA gene in the host bacteria of *Escherichia coli*. For example, a recombinant pTarget plasmid for simultaneously knocking out pMUT1 and pMUT2 can be constructed by introducing target sequences for pMUT1 and pMUT2 into the pTarget plasmid; alternatively, the waaA gene can be knocked out by designing a target sequence for the waaA gene.

[0034] The gene editing tool described in this application is preferably a dual-plasmid editing system using a pCas plasmid and a recombinant pTarget plasmid. The recombinant pTarget plasmid contains an sgRNA guide sequence designed for the target site, used to recognize and edit the target sequence in the host bacteria. After gene editing, to obtain a host bacterium without the edited plasmid, the edited plasmid (i.e., the pCas plasmid and the recombinant pTarget plasmid) needs to be eliminated. The resulting *E. coli* host bacterium, lacking endogenous plasmids pMUT1 and pMUT2 and with the waaA gene removed, can be further used to introduce a recombinant vector containing the L-glutamate oxidase gene, catalase gene, and arabinose-inducible expression regulatory element, thereby constructing a genetically engineered bacterium producing 2-oxoglutarate.

[0035] The genetically engineered bacteria that produce 2-oxoglutarate described in this application contain L-glutamate oxidase and catalase genes; the genetically engineered bacteria are prokaryotes.

[0036] In some embodiments, the lipopolysaccharide synthesis-related genes of the prokaryotic bacteria are knocked out.

[0037] In other embodiments, the genetically engineered bacteria further contain an inducible expression regulatory element; the inducible expression regulatory element is used to regulate the expression of the L-glutamate oxidase gene and the catalase gene in the genetically engineered bacteria in the presence of an inducer. In some specific embodiments, the inducible expression regulatory element is an arabinose inducible expression regulatory element, and the inducer is arabinose.

[0038] Furthermore, in some embodiments, the genetically engineered bacteria also contains a nucleotide sequence encoding an MBP tag, which is fused with L-glutamate oxidase and expressed such that the MBP tag in the resulting fusion protein is located at the N-terminus of the L-glutamate oxidase.

[0039] The amino acid sequence of the MBP tag is shown in SEQ ID No. 50:

[0040] SEQ ID No. 50:

[0041] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVG VDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT

[0042] Preferably, the nucleotide sequence encoding the MBP tag may be as shown in SEQ ID No. 7:

[0043] MBP (SEQ ID NO.7)

[0044]

[0045] In some embodiments, the sources of the L-glutamate oxidase and catalase are as follows: the nucleotide sequence of L-glutamate oxidase from *Streptomyces mobaraensis* is shown in SEQ ID No. 2; the nucleotide sequence of L-glutamate oxidase from *Streptomyces albus* is shown in SEQ ID No. 3; the nucleotide sequence of catalase from *Escherichia coli* is shown in SEQ ID No. 4; the nucleotide sequence of catalase from *Micrococcus* is shown in SEQ ID No. 5; and the nucleotide sequence of catalase from *Bos taurus* is shown in SEQ ID No. 6. Unless otherwise specified, the corresponding enzyme genes used in the following examples can be obtained by gene synthesis, PCR amplification, or other conventional methods in the art.

[0046] LGOX (SEQ ID No.2)

[0047]

[0048] StLGOX(SEQ ID No.3)

[0049]

[0050] katE(SEQ ID No.4)

[0051]

[0052] MiCat (SEQ ID No. 5)

[0053]

[0054] BoCat(SEQ ID No.6)

[0055]

[0056] For illustrative purposes, the amino acid sequence of L-glutamate oxidase from *Streptomyces mobara* in this application is shown in SEQ ID No. 48.

[0057] SEQ ID No. 48:

[0058] *

[0059] The amino acid sequence of catalase derived from Micrococcus is shown in SEQ ID No. 49.

[0060] SEQ ID No. 49:

[0061] MEHQKTTPHATGSTRQNGAPAVSDRQSLTVGSEGPIVLHDTHLLETHQHFNRMNIPERRPHAKGSGAFGEFEVTEDVSKYTKALVFQPGTKTETLLRFSTVAGELGSPDTWRDVRGFALRFYTEEG NYDLVGNNTPIFFLRDPMKFTHFIRSQKRLPDSGLRDATMQWDFWTNNPESAHQVTYLMGPRGLPRTWREMNGYGSHTYLWVNAQGEKHWVKYHFISQQGVHNLSNDEATKIAGENADFHRQDLFE SIAKGDHPKWDLYIQAIPYEEGKTYRFNPFDLTKTISQKDYPRIKVGTLTLNRNPENHFAQIESAAFSPSNTVPGIGLSPDRMLLGRAFAYHDAQLYRVGAHVNQLPVNRPKNAVHNYAFEGQMWY DHTGDRSTYVPNSNGDSWSDETGPVDDGWEADGTLTREAQALRADDDDFGQAGTLVREVFSDQERDDFVETVAGALKGVRQDVQARAFEYWKNVDATIGQRIEDEVKRHEGDGIPGVEAGGEARM*

[0062] In some embodiments, the L-glutamate oxidase gene is derived from *Streptomyces mobaraensis*, and the catalase gene is derived from *Micrococcus*. Further, in some specific embodiments, the amino acid sequence encoded by the L-glutamate oxidase gene is shown in SEQ ID No. 48, and the amino acid sequence encoded by the catalase gene is shown in SEQ ID No. 49.

[0063] In some embodiments, the prokaryotic bacterium is *Escherichia coli* or *Corynebacterium glutamicum*, preferably *Escherichia coli* Nissle1917. Further, in some embodiments, both the endogenous plasmid pMUT1 and pMUT2 of the *E. coli* are knocked out. In other embodiments, the knocked-out lipopolysaccharide synthesis-related gene is the waaA gene. Further, in some preferred embodiments, the prokaryotic bacterium with the waaA gene knocked out is *Escherichia coli* Nissle1917, and both the endogenous plasmids pMUT1 and pMUT2 of *E. coli* Nissle1917 are knocked out.

[0064] As an illustration, knockout of endogenous plasmids pMUT1 and pMUT2 is beneficial for improving the stable inheritance and expression of exogenous genes after introduction into the host bacteria. Knockout of the waaA gene is generally considered to be associated with impaired lipopolysaccharide synthesis, reduced endotoxin levels, and improved biosafety, but not with improved whole-cell catalytic fermentation efficiency for the production of 2-oxoglutaric acid using glutamate or glutamate as substrates. Furthermore, as a gene related to the host bacterial cell surface structure, knockout of the waaA gene may even adversely affect bacterial growth, material transport, or the enzyme catalytic environment. However, based on the completion of endogenous plasmid removal from the host bacteria, optimal enzyme combination screening, induction system optimization, and enhanced lysing expression in this application, knockout of the waaA gene resulted in an increased yield of 2-oxoglutaric acid.

[0065] In some embodiments, the waaA gene sequence of the *E. coli* genome in this application is shown in SEQ ID No. 1. As further illustrative, SEQ ID No. 1 can be used for the construction of the corresponding knockout vector and for gene editing of the host bacterium.

[0066] waaA(SEQ ID NO:1)

[0067]

[0068] In some embodiments, the L-glutamate oxidase gene and catalase gene are carried by a recombinant vector; preferably, the starting plasmid of the recombinant vector is pET22b or pXMJ19.

[0069] In some embodiments, the recombinant vector may further contain an arabinose-inducible expression regulatory element, thereby regulating the expression of the L-glutamate oxidase gene and the catalase gene in the presence of arabinose. In other embodiments, the recombinant vector may further contain a nucleotide sequence encoding an MBP tag, enabling the MBP tag to be fused with L-glutamate oxidase for expression, thereby improving the soluble expression of L-glutamate oxidase.

[0070] In some embodiments, the reaction process catalyzing the production of 2-oxoglutarate from sodium glutamate by L-glutamate oxidase and catalase is as follows: Figure 1 As shown. To further illustrate, L-glutamate oxidase catalyzes the oxidative deamination of glutamate to produce 2-oxoglutarate, accompanied by the generation of hydrogen peroxide; catalase further decomposes the hydrogen peroxide, thereby helping to reduce the impact of hydrogen peroxide on bacterial cells and enzyme activity.

[0071] The method for producing 2-oxoglutaric acid described in this application includes the following steps: inoculating the genetically engineered bacteria into a whole-cell catalytic fermentation system containing glutamic acid or glutamate, and carrying out whole-cell catalytic fermentation to obtain 2-oxoglutaric acid.

[0072] In some embodiments, when the genetically engineered bacteria contain arabinose-inducible expression regulatory elements, arabinose is used to induce the expression of L-glutamate oxidase and catalase in the genetically engineered bacteria before inoculating them into the whole-cell catalytic fermentation system. Specifically, in some embodiments, when inducing the expression of L-glutamate oxidase and catalase in the genetically engineered bacteria using arabinose, arabinose can be added once to the bacterial culture containing the genetically engineered bacteria, and induction culture can begin after the addition of arabinose; alternatively, arabinose can be added twice to the bacterial culture containing the genetically engineered bacteria, and induction culture can begin after the first addition of arabinose.

[0073] Furthermore, in some embodiments, arabinose is added once to the bacterial culture containing the genetically engineered bacteria, and induction culture is started after the addition of arabinose; the amount of arabinose added is calculated as the arabinose mass / bacterial culture volume ratio, and is 1–10 g / L. In some preferred embodiments, the amount of arabinose added is calculated as the arabinose mass / bacterial culture volume ratio, and is 3 g / L.

[0074] Furthermore, in some embodiments, when inducing the expression of L-glutamate oxidase and catalase by the genetically engineered bacteria containing arabinose-inducible expression regulatory elements, arabinose is added to the bacterial culture containing the genetically engineered bacteria for the first time to start the induction culture, and arabinose is added a second time during the induction culture; after the induction culture is completed, the bacterial culture is separated into solid and liquid, the genetically engineered bacteria are collected, and the collected genetically engineered bacteria after induction culture are inoculated into the whole-cell catalytic fermentation system.

[0075] Further, in some embodiments, each time arabinose is added to the bacterial culture, it is added at a mass-to-volume ratio of arabinose to bacterial culture of 1–5 g / L. In some embodiments of this application, the amount of arabinose added each time can be 1–1.5 g / L, 1.5–2 g / L, 2–2.5 g / L, 2.5–3 g / L, 3.5–4 g / L, or 4.5–5 g / L; in a preferred embodiment of this application, the amount of arabinose added the first time is 3 g / L, and the amount added the second time is 2 g / L.

[0076] Furthermore, in some embodiments, arabinose can be added to the bacterial culture a second time after half of the induction culture time has been reached.

[0077] As an explanation, adding an excessively high concentration of arabinose all at once during the initial induction stage can subject the engineered bacteria to strong induction stress in a short period, leading to an increased metabolic burden on the cells. In contrast, adding arabinose in two stages helps maintain bacterial activity and increases the expression levels of L-glutamate oxidase and catalase.

[0078] Further, in some embodiments, when arabinose is first added to the bacterial solution, the OD600 value of the genetically engineered bacteria in the bacterial solution is 0.6–0.8. In some embodiments of this application, the OD600 of the bacterial solution can be 0.6–0.65, 0.65–0.7, 0.7–0.75, or 0.75–0.8. For illustration, the bacterial solution containing the genetically engineered bacteria is obtained by picking a single clone of the genetically engineered bacteria and inoculating it into LB medium for activation culture. During the activation culture process, the OD600 value of the genetically engineered bacteria in the LB medium gradually increases. When the OD600 value of the genetically engineered bacteria in the LB medium increases to 0.6–0.8, it is considered that the genetically engineered bacteria have reached the logarithmic growth phase.

[0079] Furthermore, in some embodiments, the induction culture temperature is 20–28°C. In some embodiments of this application, the induction culture temperature can be 20–22°C, 22–24°C, 24–26°C, or 26–28°C; in the preferred embodiment of this application, the induction culture temperature is 28°C.

[0080] Furthermore, in some embodiments, the induction culture time is 6–10 h. In some embodiments of this application, the induction culture time can be 6–7 h, 7–8 h, 8–9 h, or 9–10 h; in the preferred embodiment of this application, the induction culture time is 8 h.

[0081] Furthermore, in some embodiments, the whole-cell catalytic fermentation system is prepared by dispersing glutamic acid or glutamate in water or a buffer solution; the buffer solution is PBS buffer or MES buffer, preferably PBS buffer.

[0082] Further, in some embodiments, the whole-cell catalytic fermentation system contains 80–200 g / L of glutamic acid or glutamate. In some embodiments of this application, the concentration of the glutamic acid or glutamate can be 80–100 g / L, 100–120 g / L, 120–140 g / L, 140–160 g / L, 160–180 g / L, or 180–200 g / L; in some embodiments, the glutamic acid or glutamate can be 80 g / L; in other embodiments, the glutamic acid or glutamate can be 200 g / L. In one embodiment of this application, when the substrate is monosodium glutamate, its concentration is 200 g / L.

[0083] In some embodiments of this application, the glutamate salt can be monosodium glutamate, potassium glutamate, ammonium glutamate, or a combination thereof. The glutamate salt can be added as a hydrate or an anhydrous salt. Unless otherwise specified, the concentration of glutamate or glutamate salt is based on the actual mass concentration of the substrate compound added to the whole-cell catalytic fermentation system. Those skilled in the art should know that glutamate salt can dissociate into glutamate-related ions in the whole-cell catalytic fermentation system, and glutamate itself mainly exists in ionic form in the reaction system. Therefore, regardless of whether the initial feed is glutamate or glutamate salt, there is no essential difference in the substrate skeleton actually recognized and catalyzed by L-glutamate oxidase. L-glutamate oxidase catalyzes the oxidative deamination reaction of glutamate to produce 2-oxoglutarate, accompanied by the generation of hydrogen peroxide; catalase further decomposes the hydrogen peroxide. Since the ions that salt precipitate glutamate do not participate in the above enzymatic reaction and do not change the glutamate skeleton itself, both glutamate and glutamate salt can be used as suitable substrates in this application. In some other embodiments of this application, the glutamate can be sodium glutamate monohydrate, and the concentration of sodium glutamate monohydrate can be 80 g / L.

[0084] Furthermore, in some embodiments, the pH of the whole-cell catalytic fermentation system is 5-9. In some embodiments of this application, the pH of the whole-cell catalytic fermentation system can be 5-6, 6-7, 7-8, or 8-9; in a preferred embodiment of this application, the pH of the whole-cell catalytic fermentation system is 7-9; in a more preferred embodiment, the pH of the whole-cell catalytic fermentation system is 8.

[0085] Furthermore, in some embodiments, the inoculum amount of the induced cultured genetically engineered bacteria is 0.01–0.50 g / mL, calculated based on the wet cell mass. In some embodiments of this application, the inoculum size of the induced cultured genetically engineered bacteria can be 0.01–0.05 g / mL, 0.05–0.10 g / mL, 0.10–0.15 g / mL, 0.15–0.20 g / mL, 0.20–0.25 g / mL, 0.25–0.30 g / mL, 0.30–0.35 g / mL, 0.35–0.40 g / mL, or 0.45–0.50 g / mL; it can also be 0.01 g / mL, 0.02 g / mL, 0.03 g / mL, 0.05 g / mL, 0.08 g / mL, 0.13 g / mL, 0.21 g / mL, 0.34 g / mL, or 0.50 g / mL; in a preferred embodiment of this application, the inoculum size of the induced cultured genetically engineered bacteria is 0.03 g / mL.

[0086] Furthermore, in some embodiments, the whole-cell catalytic fermentation time is 4–20 h. In some embodiments of this application, the whole-cell catalytic fermentation time can be 4–5 h, 5–6 h, 6–7 h, 7–8 h, 8–9 h, 9–10 h, 10–11 h, 11–12 h, 12–13 h, or 13–14 h; in a preferred embodiment of this application, the whole-cell catalytic fermentation time is 12 h.

[0087] Further, in some embodiments, the whole-cell catalytic fermentation temperature is 28–37 °C. In some embodiments of this application, the whole-cell catalytic fermentation temperature can be 28–29 °C, 29–30 °C, 30–31 °C, 31–32 °C, 32–33 °C, 33–34 °C, 34–35 °C, 35–36 °C, or 36–37 °C; in a preferred embodiment of this application, the whole-cell catalytic fermentation temperature is 32 °C.

[0088] As a further explanation of the above production method, in some embodiments, before inoculating the whole-cell catalytic fermentation system, the genetically engineered bacteria can be activated and cultured to obtain a bacterial solution containing the genetically engineered bacteria suitable for induction culture. The activation culture can be carried out in accordance with the conventional activation culture method for preserved bacterial strains, and the temperature and time of the activation culture can be set according to the specific host bacteria.

[0089] Furthermore, in some embodiments, the whole-cell catalytic fermentation is carried out in a baffle flask or an Erlenmeyer flask, preferably a baffle flask. In some embodiments of this application, the baffle flask can be a 250 mL baffle flask. In some specific embodiments, the 250 mL baffle flask contains 50 mL of the whole-cell catalytic fermentation system.

[0090] The culture media used in the following examples are as follows:

[0091] The LB liquid medium formula is: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0092] The LB solid medium formula is: add 20 g / L of agar powder to the above LB liquid medium.

[0093] The preparation method for competent *E. coli* cells is as follows: Dip an inoculating loop in glycerol-preserved bacterial culture and streak it onto an antibiotic-free LB agar plate, then incubate at 37°C for 12 h. Pick a single colony and inoculate it into LB liquid medium, incubating at 37°C and 220 rpm for 8–10 h. Take 500 μL of the above bacterial culture and inoculate it into a 250 mL shake flask containing 50 mL of LB medium, incubating at 37°C and 220 rpm until the OD600 reaches 0.35–0.7. Transfer the bacterial culture to a 50 mL centrifuge tube, incubate on ice for 10–15 min, then centrifuge at 4000 rpm for 5 min, discarding the supernatant. Resuspend the bacterial cells in 5 mL of solution A, centrifuge again, and discard the supernatant. Resuspend the bacterial cells in 5 mL of solution B, then aliquot 100 μL into sterile EP tubes that have been in an ice bath and store at -80°C.

[0094] The method for chemical transformation of *E. coli* is as follows: Competent cells stored at -80℃ are removed, thawed on ice, and plasmids or ligation products are added. The mixture is then pipetted and stirred 3–4 times, followed by an ice bath for 30 min. The cells are then heat-shocked at 42℃ for 1–2 min, followed by an immediate ice bath for 2 min. Next, 1 mL of antibiotic-free LB medium is added, mixed, and incubated at 37℃ and 220 rpm for 40–60 min. The mixture is then centrifuged at 4000 rpm for 3 min, a portion of the supernatant is discarded, and 70–100 μL of the bacterial culture is retained, mixed, and spread onto LB agar plates containing the appropriate antibiotic. The plates are then incubated upside down at 37℃ overnight.

[0095] In embodiments requiring electroporation transformation, *E. coli* electroporation competent cells can be prepared as follows: The strain inoculated with the pCas plasmid is cultured in LB medium containing the appropriate antibiotic; after expansion culture, arabinose is added during the logarithmic growth phase to induce expression of the relevant recombinant system on the pCas plasmid; after culture, the cells are collected by centrifugation at low temperature, washed three times with 10% glycerol, and aliquoted to prepare electroporation competent cells. During electroporation transformation, the plasmid is added to the electroporation competent cells, which are then transferred to electroporation cuvettes for electroporation. Immediately after electroporation, antibiotic-free medium is added for recovery, and the cells are then plated onto solid medium containing the appropriate antibiotic for screening. Specific electroporation conditions, recovery conditions, and antibiotic screening conditions can be routinely adjusted according to the strain, plasmid, and electroporation instrument model used.

[0096] In the following examples, the content of 2-oxoglutaric acid was determined by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: mobile phase 5 mmol / L dilute sulfuric acid, flow rate 0.6 mL / min, column temperature 35℃, injection volume 20 μL, and detection wavelength 210 nm.

[0097] In the following embodiments, all microbial strains used are publicly available. For example, *Escherichia coli* Nissle 1917 can be purchased from the China General Microbiological Culture Collection Center, and other strains can be purchased from commercial collection institutions such as the China Center for Type Culture Collection, thus meeting the requirement that biological materials are publicly available. Those skilled in the art should also understand that any identical microbial strains that can be obtained from other commercial collection institutions can be used to implement the technical solution of this application without affecting the realization of the technical solution of this application.

[0098] Example 1: Knockout of endogenous plasmids pMUT1 and pMUT2

[0099] Because the endogenous plasmids pMUT1 and pMUT2 are present in *E. coli* Nissle1917, their presence can affect the introduction of exogenous recombinant vectors and their stable maintenance within the bacteria. Therefore, the endogenous plasmids pMUT1 and pMUT2 in *E. coli* Nissle1917 were first knocked out. The pTarget / pCas dual-plasmid editing system was used for the knockout. The pTarget and pCas plasmids used have been disclosed in the 2015 paper "Multigene Editing in the *Escherichia coli* Genome via the CRISPR-Cas9 System" (Applied and Environment, Microbiology, 81) by Jiang Y, Chen B, Duan C, Sun B, Yang J, and Yang S.

[0100] (1) Constructing the pTarget-pMUT12 plasmid

[0101] Using pMUT12-F (SEQ ID No. 8) and pMUT12-R (SEQ ID No. 9) as primers and pTarget plasmid as a template, the first 20 bp sequences (i.e., the N20 sequences) of the PAM sites of pMUT1 and pMUT2 were introduced into the pTarget plasmid as guide sequences for the sgRNA. This resulted in the pTarget plasmid containing the N20 sequences of pMUT1 and pMUT2 (as shown in SEQ ID No. 44 and SEQ ID No. 45, respectively; see Table 7 for specific sequences), thus enabling simultaneous recognition of pMUT1 and pMUT2, yielding the dual-target knockout plasmid pTarget-pMUT12 (the specific construction method and restriction enzymes used are fully disclosed in the aforementioned paper by Jiang Y et al.; see the construction method of the pTargetT-ΔmaeAΔmaeB plasmid in the paper). The constructed pTarget-pMUT12 was sent for sequencing verification, and plasmids with correct sequencing results were retained for subsequent operations. The sequences of pMUT12-F and pMUT12-R are shown in Table 7.

[0102] (2) pCas plasmid was introduced into Escherichia coli Nissle1917 and electrocompetent cells were prepared.

[0103] The pCas plasmid was introduced into *E. coli* Nissle1917 (using the chemical transformation method described above). Single colonies were picked and inoculated into LB tubes containing 0.05 mg / L kanamycin and cultured overnight at 37°C. Then, a 1% inoculum was added to a 250 mL Erlenmeyer flask containing 50 mL of LB medium, along with 500 μL of 1 mol / L arabinose. The flask was cultured at 37°C and 220 rpm until the OD600 reached 0.4–0.6, at which point another 500 μL of 1 mol / L arabinose was added. After culturing, the cells were collected by centrifugation at 5000 rpm and 4°C, washed three times with 10% glycerol, and aliquoted to obtain electrotransformed competent cells.

[0104] (3) Electroconversion of pTarget-pMUT12 and screening for positive clones

[0105] Take 500 ng of the constructed pTarget-pMUT12 plasmid, add it to 100 μL of electroporation competent cells, and transfer it into a pre-chilled 1 mm electroporation cuvette. Perform electroporation transformation using an electroporator. Immediately after electroporation, add 1 mL of LB medium, gently pipette to mix, and transfer to a 1.5 mL centrifuge tube. Incubate at 37℃ and 220 rpm for 1.5–2 h. Spread the recovered bacterial culture onto LB agar plates containing 0.05 mg / L kanamycin and streptomycin, and incubate at 37℃ for 16 h. Colonies were picked and colony PCR was performed using muta1-F / muta1-R and muta2-F / muta2-R (sequences shown in SEQ ID No. 10~13, specific sequences are shown in Table 7) as primers, with the non-knockout bacteria as a control. If the non-knockout bacteria control showed a band while the colony PCR in this step did not show a band, it indicates that the picked clone was a positive clone, and the plasmids pMUT1 and pMUT2 in the positive clone had been successfully knocked out.

[0106] (4) Plasmid elimination

[0107] Verified colonies (i.e., the positive clones obtained in the previous step) were transferred to LB medium containing 1 mM rhamnose and 0.05 mg / L kanamycin and cultured overnight to remove the pTarget-pMUT12 plasmid. After culturing, the colonies were streaked overnight on kanamycin-resistant plates. Once colonies had grown, single colonies were picked and streaked on both kanamycin-resistant and streptomycin-resistant plates. Strains that could grow on kanamycin-resistant plates but not on streptomycin-resistant plates were considered strains with pTarget-pMUT12 removed. The strains with pTarget-pMUT12 removed were then transferred to LB medium containing 1% (1 M) sucrose and cultured overnight to eliminate the pCas9 plasmid. After culturing, the cells were streaked overnight on antibiotic-free plates. Once colonies had grown, single clones were picked and streaked on antibiotic-free plates, kanamycin-resistant plates, and streptomycin-resistant plates, respectively. The strains that could grow on antibiotic-free plates but not on kanamycin-resistant or streptomycin-resistant plates were identified as Nissle1917 strains with pCas9 and pTarget plasmids knocked out.

[0108] Example 2 Screening of combinations of highly active L-glutamate oxidase and catalase

[0109] The inventors used databases to screen L-glutamate oxidase and catalase from different sources in order to find a highly efficient combination of L-glutamate oxidase and catalase.

[0110] After screening, the inventors initially selected the following enzymes and combined them to test their catalytic effect on the conversion of glutamate to 2-oxoglutarate: L-glutamate oxidase LgoX from *Streptomyces morihara*, L-glutamate oxidase StLgoX from *Streptomyces blazei*, catalase katE from *Escherichia coli*, catalase MiCat from *Micrococcus*, and catalase BoCat from bovine liver. Among them, *E. coli* catalase katE was obtained by PCR amplification using the *E. coli* genome as a template, while the other genes were synthesized by Qingke Biotechnology. The corresponding nucleotide sequences are shown in SEQ ID No. 2 to SEQ ID No. 6, respectively.

[0111] Using pET22b as the starting plasmid, L-glutamate oxidases from different sources were combined with catalases from different sources to construct plasmids pET22b-BosCat-LgoX, pET22b-MiCat-LgoX, pET22b-katE-LgoX, pET22b-BosCat-StLgoX, pET22b-katE-StLgoX, and pET22b-MiCat-StLgoX.

[0112] Taking the construction of pET22b-MiCat-LgoX as an example: First, the catalase gene MiCat is inserted into the pET22b plasmid. Primers are designed based on the sequence of the MiCat gene (SEQ ID No. 5) to perform PCR on the MiCat gene. Sequences for homologous recombination are added to both ends of the MiCat gene (the complete coding sequence). Then, homologous recombination is used to insert the MiCat gene into the pET22b plasmid to obtain the pET22b-MiCat plasmid. This step uses a commercially available homologous recombination kit (such as the Novizan ClonExpress® kit, catalog number C112-01), and the procedure should be followed according to the kit instructions.

[0113] Using pET22b-MiCat-R (SEQ ID No. 14) and pET22b-LogX-F (SEQ ID No. 15) as primers and pET22b-MiCat plasmid as template, a linearized pET22b-MiCat vector was amplified. Using MiCat-LgoX-F (SEQ ID No. 16) and LgoX-R (SEQ ID No. 17) as primers and LgoX gene (SEQ ID No. 2) as template, an LgoX fragment with homologous sequences was amplified. The LgoX fragment with homologous sequences obtained by PCR was purified using a DNA purification kit. Then, homologous recombination ligation of the LgoX fragment with pET22b-MiCat plasmid was performed using the Novizan ClonExpress® kit (catalog number: C112-01) to obtain the recombinant vector pET22b-MiCat-LgoX (see kit instructions for specific operation methods). The obtained recombinant vector was transformed into Escherichia coli JM109. Colony PCR was performed using LogX-yz-F (SEQ ID No. 18) and pET22b-yz-R (SEQ ID No. 19) as verification primers. Colonies with PCR bands of approximately 500 bp were selected, plasmids were extracted, and sequencing was performed to verify the correct recombinant vector pET22b-MiCat-LgoX.

[0114] The specific sequences of the primers used in this step are shown in Table 7.

[0115] In this embodiment, following the above method, six recombinant vectors were constructed by changing the combination of L-glutamate oxidase and catalase from different sources. Each recombinant vector was transformed into Escherichia coli BL21, resulting in six strains. After the strains produced positive colonies, they were inoculated into shake tubes containing LB medium and cultured at 37°C for 12 h for primary activation culture. Then, the bacterial solution obtained from the primary activation culture was transferred to a 250 mL Erlenmeyer flask containing 50 mL of LB medium at a 2% volume inoculation rate for further activation culture. The strains were cultured at 37°C until the logarithmic growth phase (OD600 reached 0.6–0.8). Isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM (the concentration of IPTG in the bacterial solution immediately after IPTG addition) was added, and the strains were then induced to grow at 25°C and 220 rpm for 8 h to ensure that the L-glutamate oxidase gene and catalase gene were fully expressed in the strains. After induction culture, sodium glutamate with a final concentration of 80 g / L was added to the bacterial culture as a substrate for whole-cell catalytic fermentation. Whole-cell catalytic fermentation was carried out at 32℃. After 12 h, the yield of 2-oxoglutarate was detected by high performance liquid chromatography.

[0116] like Figure 2As shown in the figure, the OD600 value is the OD600 value of the bacterial culture after the induction culture is completed. Among the six strains, the strain carrying pET22b-MiCat-LgoX had the highest 2-oxoglutaric acid yield, at 8.99 g / L, with a product molar conversion rate of 14.9% (the product molar conversion rate is calculated as: the amount of product / the amount of substrate × 100%). This indicates that the combination of L-glutamate oxidase LgoX from *Streptomyces molybdenum* and catalase MiCat from *Micrococcus* has the best effect.

[0117] The experimental parameters and results in this embodiment are shown in Table 1.

[0118] Table 1. Screening results of different combinations of L-glutamate oxidase and catalase in Example 2.

[0119]

[0120] The results in the table show that the yield of 2-oxoglutarate varies significantly depending on the combination of L-glutamate oxidase and catalase, and according to... Figure 2 It was found that the OD600 values ​​of the bacterial cultures after whole-cell catalytic fermentation showed some differences. A higher OD600 value indicates a lower inhibition of the strain by hydrogen peroxide. Overall, all bacterial cultures had relatively high OD600 values, indicating that various catalases could effectively mitigate the toxicity of hydrogen peroxide. However, it is noteworthy that the pET22b-BosCat-StLgoX group, which had the highest OD600 value, had a very low 2-oxoglutaric acid yield, less than half that of the pET22b-MiCat-StLgoX group, which also contained the StLgoX enzyme. Furthermore, the pET22b-BosCat-LgoX and pET22b-MiCat-LgoX groups differed only in the type of catalase; their OD600 values ​​were similar, but their 2-oxoglutaric acid yields differed significantly.

[0121] The above results indicate that the differences in 2-oxoglutarate production among different groups are not simply the result of the superposition of "L-glutamate oxidase catalytic capacity + catalase catalytic capacity". Combining specific L-glutamate oxidase and catalase can achieve a "1+1>2" effect.

[0122] Example 3: Change of induction type and screening of host bacteria

[0123] Currently, commercially available pET22b plasmids use isopropyl-β-D-thiogalactoside (IPTG) as an inducer to induce the expression of the gene encoding the target enzyme carried on the pET22b plasmid. High concentrations of IPTG are toxic to the host bacteria, potentially causing cellular stress, affecting protein folding and activity, and even increasing the probability of inclusion body formation. Furthermore, trace amounts of IPTG may remain during the subsequent isolation and purification of 2-oxoglutaric acid. Moreover, inducing the expression of related genes in genetically engineered bacteria using IPTG is costly and not conducive to industrial production. Therefore, the pET22b plasmid was modified by replacing the IPTG-inducible form (recombinant vector) with an arabinose-inducible form. Controlling exogenous protein expression through the arabinose-inducible system reduces toxicity to host cells, increases protein expression levels, and allows arabinose to be utilized by the bacterial strain, minimizing residue and reducing cost.

[0124] The lacI operon on the original pET22b is replaced with the arabinose operon to achieve a change in induction type. Specifically, using pET22b-araC-R (SEQ ID No. 20) and pET22b-LgoX-F (SEQ ID No. 15) as primers and pET22b-MiCat-LgoX as a template, a linearized pET22b-MiCat-LgoX plasmid (which no longer contains the lacI operon fragment) was amplified; using araC-MiCat-R (SEQ ID No. 22) and araC-F (SEQ ID No. 21) as primers and E. coli genome as a template, an arasugar operon fragment was amplified; then, referring to the homologous recombination method used in Example 2 for inserting LgoX into the pET22b-MiCat plasmid, homologous recombination was performed between the amplified arasugar operon fragment and the linearized pET22b-MiCat-LgoX plasmid to obtain the arasugar-inducible recombination vector pET22b-MiCat-LgoX-ara. For confirmatory sequencing of pET22b-MiCat-LgoX-ara, colony PCR was performed using primers pET22b-araC-yz-F (SEQ ID No. 23) and araC-yz-R (SEQ ID No. 24), followed by sequencing, using the same method as in Example 2.

[0125] The correctly sequenced pET22b-MiCat-LgoX-ara was transformed into *E. coli* Nissle1917 (pMUT1 and pMUT2 knocked out), W3110, MG1655, and BW25113, respectively, to obtain genetically engineered bacteria. These four genetically engineered bacteria were activated and cultured according to the method described in Example 2. After activation, 2 g / L of arabinose was added to the bacterial culture, and then the four genetically engineered bacteria were induced and cultured at 25°C and 220 rpm for 8 h. After induction, the bacterial cells were collected by centrifugation at 8000 rpm for 10 min to obtain the induced genetically engineered bacteria.

[0126] Genetically engineered bacteria were inoculated into a whole-cell catalytic fermentation system (an aqueous solution containing 150 g / L monosodium glutamate). The inoculation amount of the genetically engineered bacteria was 0.03 g / mL, calculated as wet cells. Whole-cell catalytic fermentation was carried out at 32℃ for 12 h, and the yield of 2-oxoglutaric acid was detected by high-performance liquid chromatography (HPLC). The results showed that Nissle1917 exhibited the best performance, with a 2-oxoglutaric acid yield of 73.7 g / L and a molar conversion rate of 57.1%. Figure 3 As shown. Therefore, Nissle1917 (with pMUT1 and pMUT2 plasmids knocked out) was subsequently selected as the host bacterium.

[0127] The experimental parameters and results in this embodiment are shown in Table 2.

[0128] Table 2. Screening results of different host bacteria in Example 3

[0129]

[0130] The results in the table show that, without knocking out the endogenous plasmids pMUT1 and pMUT2, different strains produce 2-oxoglutaric acid differently. Therefore, the selection of the host strain is a crucial step in constructing genetically engineered bacteria capable of efficiently producing 2-oxoglutaric acid.

[0131] Example 4 Optimization of Induction Conditions

[0132] In the E. coli recombinant protein expression system, induction conditions affect the translation, transport, and folding processes of proteins, thereby influencing the expression level of the target enzyme within the host bacteria and the whole-cell catalytic fermentation effect. Therefore, the induction concentration, induction time, and induction temperature of arabinose were optimized.

[0133] Unless otherwise specified, the following basic culture method was used in the following optimization process: Positive single colonies were picked and inoculated into LB liquid medium, and initially activated at 37°C for 12 h; the bacterial culture obtained after initial activation was transferred to a 250 mL Erlenmeyer flask containing 50 mL of LB medium at a 2% volume inoculation rate, and further activated at 37°C until the OD600 reached 0.6 to 0.8, then arabinose was added to begin induction culture; after induction culture, the induced genetically engineered bacteria were collected according to the method in Example 3, and whole-cell catalytic fermentation was performed according to the whole-cell catalytic fermentation method in Example 3. The genetically engineered bacteria used was Nissle1917 transformed with pET22b-MiCat-LgoX-ara (pMUT1 and pMUT2 were knocked out).

[0134] (1) Optimization of inducer concentration

[0135] Under the same conditions as in Example 3, at the end of the expansion and activation culture, arabinose was added to the bacterial culture at final concentrations (arabinose addition / bacterial culture volume) of 1 g / L, 2 g / L, and 3 g / L, respectively, followed by induction culture. The results showed that when 3 g / L of arabinose was added, the host bacterium transformed into pET22b-MiCat-LgoX-ara (i.e., the genetically engineered bacterium) produced the highest yield of 2-oxoglutaric acid, at 65.37 g / L, with a molar conversion rate of 51%.

[0136] (2) Optimization of induction culture time

[0137] Under the same conditions as in Example 3, the induced cultured genetically engineered bacteria were collected after 6 h, 8 h, and 10 h of induction culture, respectively, and then subjected to whole-cell catalytic fermentation. The results showed that the induced cultured genetically engineered bacteria obtained after 8 h of induction culture produced a higher yield of 2-oxoglutaric acid during whole-cell catalytic fermentation than those induced for 6 h and 10 h. The 2-oxoglutaric acid yield of the genetically engineered bacteria after 8 h of induction culture was 50.2 g / L, with a molar conversion ratio of 39.2%.

[0138] (3) Optimization of induction temperature

[0139] Under the same conditions as in Example 3, induction culture was carried out at 20℃, 25℃, and 28℃, respectively. The results showed that different induction culture temperatures had no significant effect on the yield of 2-oxoglutaric acid, with the highest yield being 51.56 g / L and a molar conversion rate of 40%. Considering energy costs, 28℃ was selected as the preferred induction culture temperature for subsequent induction culture.

[0140] (4) Secondary induction

[0141] Since arabinose is utilized by genetically engineered bacteria, its induction may weaken in the later stages of induction culture. Therefore, after the initial addition of 3 g / L arabinose and 4 h of induction culture, a second induction was performed by adding 2 g / L arabinose. The control group (those without additional arabinose, who only added 3 g / L arabinose after the expansion and activation culture and at the start of the induction culture) was used. The results showed that the yield of 2-oxoglutaric acid after the second induction was slightly higher than that of the control group without the second induction. Figure 4 The bottom right corner shows the results of the secondary induction experiment. CK represents the control group (corresponding to 4-11 in Table 3), and ara represents the treatment group supplemented with arabinose.

[0142] like Figure 4 The relevant experimental results are presented. Combined with... Figure 4 The results given indicate that the optimal induction culture conditions are as follows: 3 g / L arabinose is added at the beginning of the induction culture, and the culture is induced at 28℃ for a total induction culture time of 8 h. 2 g / L arabinose is added after 4 h of induction (that is, after half of the induction culture time).

[0143] The experimental parameters and results in this embodiment are shown in Table 3.

[0144] Table 3 Results of optimization of induction culture conditions in Example 4

[0145]

[0146] Table 3 shows that arabinose induction conditions significantly affect the ability of genetically engineered bacteria to produce 2-oxoglutaric acid. In the optimization of inducer concentration, when the final arabinose concentration was 3 g / L, the yield and molar conversion rate of 2-oxoglutaric acid were higher than those at 1 g / L and 2 g / L, indicating that higher concentrations of arabinose are more conducive to the expression of the target enzyme. In the optimization of induction time, a total induction culture time of 8 h was better than 6 h and 10 h, indicating that too short an induction time is not conducive to sufficient enzyme expression, while too long an induction time may affect the whole-cell catalytic fermentation effect. In the optimization of induction temperature, the results at 20℃, 25℃, and 28℃ showed little difference. Furthermore, under the condition of initially adding 3 g / L arabinose and then supplementing with 2 g / L arabinose after 4 h of induction, the yield and molar conversion rate of 2-oxoglutaric acid were further improved, superior to the control group that only added arabinose once.

[0147] Example 5: Soluble expression of MBP-promoting enzyme with added solubility tag

[0148] When using *E. coli* as the host cell for enzyme expression, its rapid transcription and translation rates coupled with relatively low folding efficiency lead to the formation of inclusion bodies, thus affecting the expression and activity of the target enzyme. Adding a solubilizing tag to the N-terminus of the enzyme can improve protein soluble expression, reduce inclusion body formation, and promote proper protein folding and increased expression levels. Therefore, this embodiment selects MBP (maltose-binding protein) as the solubilizing tag. The nucleotide sequence of the MBP is shown in SEQ ID No. 7.

[0149] Based on the optimal host cells and induction culture conditions determined in Examples 3 and 4, the MBP tag was ligated to the N-terminus of L-glutamate oxidase (to fuse the MBP tag with the L-glutamate oxidase for expression), constructing a recombinant expression system with a lysis-promoting tag, and compared in parallel with a control strain without the lysis-promoting tag. Specifically, using pET22b-MiCat-LgoX-ara as a template, PCR was performed using primers MiCat-LgoX-MBP-R (SEQ ID No. 25) and MBP-F (SEQ ID No. 26) to obtain the linearized pET22b-MiCat-LgoX-ara plasmid. Using primers MBP-R (SEQ ID No. 27) and MiCat-LgoX-MBP-F (SEQ ID No. 28) as primers, PCR amplification was performed using the artificially synthesized MBP tag sequence (SEQ ID No. 7) as a template. The amplified product was then homologously recombinated with the linearized pET22b-MiCat-LgoX-ara plasmid. The recombination product was transformed into Escherichia coli JM109. Single colonies of the transformed bacteria were PCR-mediated using primers MBP-yz-F (SEQ ID No. 29) and MiCat-yz-R (SEQ ID No. 30). Positive colonies with the correct bands were selected, plasmids were extracted, and sequenced. The plasmid with the correct sequence (i.e., pET22b-MiCat-MBP-LgoX-ara) was retained and transformed into the host bacterium Nissle1917 (pMUT1 and pMUT2 were knocked out).

[0150] Whole-cell catalytic fermentation was performed on two strains (pET22b-MiCat-LgoX-ara and pET22b-MiCat-MBP-LgoX-ara, respectively). The results showed that the production of 2-oxoglutaric acid increased by 13.7 g / L after adding the MBP tag compared with the control strain without the solubilizing tag.

[0151] Further analysis using SDS-PAGE gel electrophoresis revealed protein expression in the supernatant and precipitate after induction by strains with and without the MBP tag. Results showed that the concentration of the target protein in the supernatant was higher than that in the precipitate after MBP tagging. Furthermore, compared to the control strain without the solubilization tag, the L-glutamate oxidase band was significantly enhanced in the strain with the solubilization tag, indicating that MBP tagging significantly promoted the soluble expression of L-glutamate oxidase, and this enhanced soluble expression of L-glutamate oxidase is beneficial for increasing the yield of 2-oxoglutamate. Figure 5 In the diagram, 1 represents the supernatant of the strain without the addition of the soluble tag, 2 represents the precipitate of the strain without the addition of the soluble tag, 3 represents the supernatant of the strain with the addition of the soluble tag, and 4 represents the precipitate of the strain with the addition of the soluble tag. The unit of the marker fragment size values ​​marked on the left is kDa.

[0152] The experimental parameters and results in this embodiment are shown in Table 4.

[0153] Table 4. Effects of MBP tag on enzyme soluble expression and catalytic performance in Example 5.

[0154]

[0155] The above results indicate that adding an MBP solubilizing tag to the N-terminus of L-glutamate oxidase can promote the soluble expression level of L-glutamate oxidase, thereby improving the yield of 2-oxoglutarate and the molar conversion rate of the product.

[0156] Example 6 Optimization of Whole-Cell Catalytic Fermentation Conditions

[0157] The reaction conditions of whole-cell catalytic fermentation, such as pH, buffer system, and mass transfer efficiency, affect substrate and product inhibition, cytotoxicity, and the level of side reactions, thereby influencing the yield and conversion rate of 2-oxoglutaric acid. Therefore, the pH, buffer system, and mass transfer efficiency in the whole-cell catalytic fermentation process were optimized.

[0158] (1) pH optimization

[0159] Whole-cell catalytic fermentation was carried out under conditions of pH 5, 6, 7, 8, and 9, respectively. Results ( Figure 6 As shown in the top left, the effect is better under neutral and weakly alkaline conditions (pH 7~9), so the pH of the whole-cell catalytic fermentation system was subsequently set to 8.

[0160] During pH optimization, the pH of the whole-cell catalytic fermentation system was adjusted by hydrochloric acid or sodium hydroxide. Whole-cell catalytic fermentation was carried out in a 250 mL Erlenmeyer flask with a substrate of 150 g / L sodium glutamate. The inoculum size of the engineered bacteria was 0.03 g / mL. The whole-cell catalytic fermentation temperature was 32℃ and the whole-cell catalytic fermentation time was 12 h.

[0161] (2) Optimization of buffer system

[0162] In this part of the experiment, whole-cell catalytic fermentation systems were prepared using water (as in other examples), PBS, and MES, respectively, and whole-cell catalytic fermentation was carried out. Results ( Figure 6 The top right image shows that PBS was the best buffer solution, so PBS was subsequently chosen as the buffer system for whole-cell catalytic fermentation.

[0163] When optimizing the buffer system, the pH of the prepared whole-cell catalytic fermentation system was 8. Whole-cell catalytic fermentation was carried out in a 250 mL Erlenmeyer flask with a substrate of 150 g / L sodium glutamate; the inoculum size of the engineered bacteria was 0.03 g / mL; the whole-cell catalytic fermentation temperature was 32℃ and the whole-cell catalytic fermentation time was 12 h.

[0164] (3) Optimization of mass transfer conditions

[0165] The fermentation process was changed from a 250 mL ordinary conical flask to a 250 mL baffle flask. Results ( Figure 6 (Lower left) This shows that the system mixes better and has higher mass transfer efficiency when using a 250 mL baffle bottle. The yield of 2-oxoglutaric acid reaches 84.1 g / L and the product conversion rate is 65%, as shown in 6-1 of Table 5.

[0166] When optimizing mass transfer conditions, a whole-cell catalytic fermentation system was prepared with water. The pH of the whole-cell catalytic fermentation system was 8, the substrate was 150 g / L sodium glutamate, the inoculum size of the engineered bacteria was 0.03 g / mL, the whole-cell catalytic fermentation temperature was 32℃, and the whole-cell catalytic fermentation time was 12 h.

[0167] (4) Effect of waaA gene knockout on catalytic performance

[0168] To further improve biosafety and reduce endotoxin accumulation, the waaA gene (SEQ ID No. 1) in the genome of the host bacterium (Nissle1917, where both pMUT1 and pMUT2 were knocked out) was knocked out. The method for knocking out the waaA gene was the same as that for knocking out pMUT1 and pMUT2 in Example 1. Specifically: using waaA-1-F / waaA-1-R (SEQ ID No. 35 / SEQ ID No. 36) or waaA-2-F / waaA-2-R as primers (SEQ ID No. 37 / SEQ ID No. 38), and using the pTarget plasmid from Example 1 as a template, the first 20 bp sequence of the waaA gene PAM site (such as waaA-1 shown in SEQ ID No. 46 or waaA-2 shown in SEQ ID No. 47) is introduced into the pTarget plasmid as a guide signal for sgRNA. The constructed pTarget-waaA-1 and / or pTarget-waaA-2 are sent for sequencing verification, and plasmids with correct sequencing are reserved for subsequent operations. Using *E. coli* Nissle1917 as a template, upstream and downstream homologous arms were amplified by PCR using two pairs of primers (waaA-up-F / waaA-up-R, sequences shown in SEQ ID No. 31 / SEQ ID No. 32; waaA-down-F / waaA-down-R, sequences shown in SEQ ID No. 33 / SEQ ID No. 34). Using the upstream and downstream homologous arms as templates, the two homologous arms were ligated by overlap extension PCR using waaA-up-F / waaA-down-R primers. Agarose gel electrophoresis was then performed to verify the band size. PCR products with correct band sizes were purified and recovered to obtain the homologous recombination donor fragment for waaA gene deletion knockout. The pTarget-waaA plasmid (pTarget-waaA-1 or pTarget-waaA-2) and this homologous recombination donor fragment were introduced into a strain carrying pCas. The specific sequences of the primers used in this step are shown in Table 7.

[0169] Whole-cell catalytic fermentation was performed according to the conditions described in Tables 5, 6-2 and 6-3, with 6-2 serving as the control group. Results of whole-cell catalytic fermentation validation ( Figure 6 (Bottom right) This shows that the yield of 2-oxoglutaric acid after waaA knockout was 72.3 g / L, which was 7.8 g / L higher than the control. In Table 5, 6-2 represents the control group data, and 6-3 represents the data from the waaA knockout genome.

[0170] (5) Optimal combination of conditions verification

[0171] The optimal combination of conditions was obtained as follows: 3 g / L arabinose was added at the beginning of the induction culture, and the culture was induced at 28℃ for a total induction culture time of 8 h. 2 g / L arabinose was added after 4 h of induction (that is, after half of the induction culture time).

[0172] For whole-cell catalytic fermentation, the whole-cell catalytic fermentation system was prepared using PBS buffer; the pH of the whole-cell catalytic fermentation system was 8; the inoculum amount of the engineered bacteria in the whole-cell catalytic fermentation system was calculated based on the wet cell mass, and the inoculum amount of the engineered bacteria was 0.03 g / mL; the whole-cell catalytic fermentation time was 12 h; the whole-cell catalytic fermentation temperature was 32℃; and the whole-cell catalytic fermentation was carried out in a baffle flask.

[0173] Whole-cell catalytic fermentation was performed using the strain Nissle1917 ΔwaaA / pET22b-MiCat-MBP-LgoX-ara. With 200 g / L monosodium glutamate as the substrate, 179.2 g / L 2-oxoglutarate was produced, achieving a 100% conversion rate; with 80 g / L glutamate as the substrate, 79.4 g / L 2-oxoglutarate was produced, also achieving a 100% conversion rate.

[0174] The experimental parameters in this embodiment are shown in Table 5.

[0175] Table 5. Optimization results of some whole-cell catalytic fermentation conditions in Example 6

[0176]

[0177] Example 7

[0178] Using pXMJ19 as the starting plasmid, MiCat and LgoX were ligated into the pXMJ19 plasmid, and the lacI operator of pXMJ19 was replaced with an arabinose operator. An MBP tag was added to the N-terminus of LgoX to construct the plasmid pXMJ19-MiCat-MBP-LgoX-ara. Specifically, using the empty vector pXMJ19 as a template and pXMJ19-R / pXMJ19-F (SEQ ID No. 40 / SEQ ID No. 42) as primers, the linearized pXMJ19 plasmid was amplified. Using pXMJ19-ara-CR / pXMJ19-ara-CF (SEQ ID No. 41 / SEQ ID No. 39) as primers, and the pET22b-MiCat-MBP-LgoX-ara gene constructed in the above examples as a template, a MiCat-MBP-LgoX-ara gene fragment with homologous sequences was amplified. The fragment was purified using a DNA purification kit, and then ligated using the ClonExpress® kit to obtain the recombinant vector pXMJ19-MiCat-MBP-LgoX-ara. This recombinant vector was transformed into Escherichia coli JM109, and colony PCR was performed using LogX-yz-F / pXMJ19-yz-R (SEQ ID No. 18 / SEQ ID No. 43) as verification primers. Colonies with PCR bands of approximately 500 bp were selected, plasmids were extracted, and sequencing was performed to verify the correct recombinant vector pET22b-MiCat-MBP-LgoX-ara.

[0179] The pXMJ19-MiCat-MBP-LgoX-ara plasmid was transformed into Corynebacterium glutamicum ATCC13023, and induced culture and whole-cell catalytic fermentation were carried out according to the optimal combination of conditions obtained in Example 6. When using 200 g / L monosodium glutamate as substrate, 143.9 g / L 2-oxoglutarate was produced, with a conversion rate of 83.3%; when using 80 g / L glutamate as substrate, 71.6 g / L 2-oxoglutarate was produced, with a conversion rate of 90.1%.

[0180] The experimental parameters in this embodiment are shown in Table 6.

[0181] Table 6 Catalytic results of the Corynebacterium glutamicum system in Example 7

[0182]

[0183] The sequences of the primers used in Examples 1 to 7 above are shown in Table 7.

[0184] Table 7

[0185]

[0186] The N20 sequences involved are shown in Table 8:

[0187] Table 8

[0188]

[0189] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A genetically engineered bacterium producing 2-oxoglutaric acid, characterized in that, The genetically engineered bacteria contain the L-glutamate oxidase gene and the catalase gene; the genetically engineered bacteria are prokaryotes.

2. The genetically engineered bacterium according to claim 1, characterized in that, The lipopolysaccharide synthesis-related genes of the prokaryotic bacteria were knocked out.

3. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria also contain inducible expression regulatory elements; these elements are used to regulate the expression of the L-glutamate oxidase gene and catalase gene in the genetically engineered bacteria in the presence of an inducer.

4. The genetically engineered bacteria according to claim 3, characterized in that, The inducible expression regulatory element is an arabinose inducible expression regulatory element, and the inducer is arabinose.

5. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria also contain a nucleotide sequence encoding an MBP tag, which is fused with L-glutamate oxidase and expressed such that the MBP tag in the resulting fusion protein is located at the N-terminus of the L-glutamate oxidase.

6. The genetically engineered bacterium according to claim 1, characterized in that, The L-glutamate oxidase gene is derived from *Streptomyces mobaraensis*, and the catalase gene is derived from *Micrococcus*.

7. The genetically engineered bacterium according to claim 6, characterized in that, The amino acid sequence encoded by the L-glutamate oxidase gene is shown in SEQ ID No. 48; the amino acid sequence encoded by the catalase gene is shown in SEQ ID No. 49; preferably, the nucleotide sequence of the L-glutamate oxidase gene is shown in SEQ ID No. 2, and the nucleotide sequence of the catalase gene is shown in SEQ ID No.

5.

8. The genetically engineered bacteria according to any one of claims 1-7, characterized in that, The prokaryotic bacteria are Escherichia coli or Corynebacterium glutamicum, preferably Escherichia coli Nissle1917.

9. The genetically engineered bacterium according to claim 8, characterized in that, Both the endogenous plasmid pMUT1 and endogenous plasmid pMUT2 of the *E. coli* were knocked out.

10. The genetically engineered bacteria according to any one of claims 2-9, characterized in that, The gene involved in the knocked-out lipopolysaccharide synthesis is the waaA gene.

11. The genetically engineered bacterium according to claim 10, characterized in that, The prokaryotic bacterium whose waaA gene was knocked out was Escherichia coli Nissle1917, and both the endogenous plasmids pMUT1 and pMUT2 of Escherichia coli Nissle1917 were knocked out.

12. The genetically engineered bacteria according to any one of claims 1 to 11, characterized in that, The L-glutamate oxidase gene and catalase gene are carried by a recombinant vector; preferably, the starting plasmid of the recombinant vector is pET22b or pXMJ19.

13. The use of the genetically engineered bacteria according to any one of claims 1-12 in the preparation of 2-oxoglutaric acid.

14. A method for producing 2-oxoglutaric acid, characterized in that, Inoculate a whole-cell catalytic fermentation system containing glutamic acid or glutamate with any of the genetically engineered bacteria described in claims 1-11, and carry out whole-cell catalytic fermentation to obtain 2-oxoglutaric acid.

15. The production method according to claim 14, characterized in that, When the genetically engineered bacteria contain arabinose-inducible expression regulatory elements, arabinose is used to induce the expression of L-glutamate oxidase and catalase in the genetically engineered bacteria before inoculating them into the whole-cell catalytic fermentation system.

16. The production method according to claim 15, characterized in that, When inducing the expression of L-glutamate oxidase and catalase in the genetically engineered bacteria containing arabinose-inducible expression regulatory elements, arabinose is added to the bacterial culture containing the genetically engineered bacteria for the first time to start the induction culture, and arabinose is added a second time during the induction culture; after the induction culture is completed, the bacterial culture is separated into solid and liquid components, and the genetically engineered bacteria are collected.

17. The production method according to claim 16, characterized in that, The induced culture has one or more of the following characteristics: 1) The induction culture temperature is 20–28℃; 2) The induction culture time is 6–10 h; 3) When arabinose is added to the bacterial solution for the first time, the OD600 value of the genetically engineered bacteria in the bacterial solution is 0.6 to 0.8; 4) After half of the induction time has been reached, add arabinose to the bacterial culture for the second time; 5) Each time arabinose is added to the bacterial solution, it shall be added at a ratio of arabinose mass to bacterial solution volume of 1 to 5 g / L.

18. The production method according to claim 14, characterized in that, Whole-cell catalytic fermentation methods have one or more of the following characteristics: 1) The whole-cell catalytic fermentation system is prepared by dispersing glutamic acid or glutamate in water or a buffer solution; the buffer solution is PBS buffer or MES buffer; preferably, the buffer solution is PBS buffer. 2) The whole-cell catalytic fermentation system contains 80–200 g / L of glutamic acid or glutamate; 3) The pH of the whole-cell catalytic fermentation system is 5-9; preferably, the pH of the whole-cell catalytic fermentation system is 7-9; 4) In the whole-cell catalytic fermentation system, the inoculum size of the induced cultured genetically engineered bacteria is calculated based on the wet cell mass; the inoculum size of the induced cultured genetically engineered bacteria is 0.01–0.50 g / mL; 5) Whole-cell catalytic fermentation time is 4–20 h; 6) The temperature for whole-cell catalytic fermentation is 28–37 °C; 7) Whole-cell catalytic fermentation is carried out in a baffle flask.

19. The use of the production method according to any one of claims 14-18 in the production of 2-oxoglutaric acid.

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