CN109679979B

CN109679979B - Recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and production method of alpha-ketoglutaric acid

Info

Publication number: CN109679979B
Application number: CN201910030458.4A
Authority: CN
Inventors: 王苓; 李晚军; 伍万兵; 曾帅; 刘鑫
Original assignee: Sichuan Jisheng Biopharmaceutical Co ltd
Current assignee: Sichuan Jisheng Biopharmaceutical Co ltd
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2023-03-17
Anticipated expiration: 2039-01-11
Also published as: CN109679979A

Abstract

The invention provides a recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and a production method of alpha-ketoglutaric acid, belonging to the fields of molecular biotechnology and enzyme engineering. The recombinant vector for expressing the L-glutamate oxidase and the catalase improves the capability of converting substrates into the alpha-ketoglutarate by the L-glutamate oxidase and the catalase by inserting the regulation sequence SD for regulating the expression of the L-glutamate oxidase gene LGOX and the catalase gene CAT into the vector, and has better practical use value.

Description

Recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and production method of alpha-ketoglutaric acid

Technical Field

The invention relates to the fields of molecular biotechnology and enzyme engineering, in particular to a recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and a production method of alpha-ketoglutaric acid.

Background

Alpha-ketoglutarate is a key node of tricarboxylic acid cycle and amino acid metabolism of organisms, can generate glutamic acid by adding ammonia or transaminase through glutamate dehydrogenase, is widely applied to the fields of diet, medical treatment, cosmetics, fine chemical industry and the like, even is used as an auxiliary supplement for relieving ammonia accumulation injury in the field of sports, has higher market demand than supply and higher price. At present, three methods are mainly used for synthesizing alpha-ketoglutaric acid: organic chemical synthesis, fermentation accumulation and enzymatic conversion.

The traditional organic synthesis methods comprise acyl nitrile hydrolysis and oxalic acid ethyl ester hydrolysis, which can be achieved in several steps, are complex and cause serious pollution; the fermentation accumulation method has made a great breakthrough, alpha-ketoglutaric acid is accumulated in the glucose culture of Torulopsis glabrata, and the content of alpha-ketoglutaric acid reaches 43.7g/L after 64 hours; when the yarrowia lipolytica is cultured by using the glycerol, the yield reaches 186g/L after 117h, although the yield is high, other heteroacid is more, the separation and purification difficulty is high, the fermentation period is too long, and the industrial production is not realized.

Disclosure of Invention

The embodiment of the application provides a recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and a production method of alpha-ketoglutaric acid, and the recombinant vector with high expression efficiency is constructed, so that the production is convenient; engineering bacteria are obtained through the recombinant vector, whole-cell catalysis is facilitated, and the yield of alpha-ketoglutaric acid can be improved through a method for catalytic conversion of the engineering bacteria.

In a first aspect of the present application, there is provided a recombinant vector expressing L-glutamate oxidase and catalase, the recombinant vector comprising an L-glutamate oxidase gene LGOX expressing L-glutamate oxidase, a catalase gene CAT expressing catalase, and a regulatory sequence SD regulating the expression of the L-glutamate oxidase gene LGOX and the catalase gene CAT; the base sequence of the L-glutamic acid oxidase gene LGOX is shown as SEQ ID No.1, the base sequence of the catalase gene CAT is shown as SEQ ID No.2, and the base sequence of the regulatory sequence SD is shown as SEQ ID No. 3.

The principle of converting glutamate to alpha-ketoglutarate by co-expressing L-glutamate oxidase and catalase is as follows:

the L-glutamate oxidase is used for converting glutamate into alpha-ketoglutaric acid, hydrogen peroxide is simultaneously generated, the concentration of the hydrogen peroxide is increased along with the reaction, the reaction is inhibited, and therefore, the hydrogen peroxide is degraded through the action of catalase, water and oxygen are generated, and the reaction is continuously and effectively carried out.

In the embodiment, the regulation sequence SD for regulating the expression of the L-glutamic acid oxidase gene LGOX and the catalase gene CAT in the expression vector has a ribosome binding site and regulates the expression of the L-glutamic acid oxidase gene LGOX and the catalase gene CAT, so that the matching balance of the expression quantities of the L-glutamic acid oxidase and the catalase is achieved, the better reaction rate is realized, and the yield of the alpha-ketoglutaric acid is improved.

In a second aspect of the present application, there is provided the use of the above recombinant vector expressing L-glutamate oxidase and catalase for the preparation of α -ketoglutarate.

In a third aspect of the application, an engineering bacterium for expressing L-glutamate oxidase and catalase is provided, the engineering bacterium comprises an L-glutamate oxidase gene LGOX for expressing the L-glutamate oxidase, a catalase gene CAT for expressing the catalase, and a regulation sequence SD for regulating the expression of the L-glutamate oxidase gene LGOX and the catalase gene CAT; the base sequence of the L-glutamic acid oxidase gene LGOX is shown as SEQ ID No.1, the base sequence of the catalase gene CAT is shown as SEQ ID No.2, and the base sequence of the regulatory sequence SD is shown as SEQ ID No. 3.

In the fourth aspect of the application, the application of the engineering bacteria for expressing L-glutamate oxidase and catalase in the preparation of alpha-ketoglutaric acid is provided.

In a fifth aspect of the present application, there is provided a method for producing α -ketoglutaric acid, the method comprising the steps of:

constructing a recombinant vector for expressing L-glutamate oxidase and catalase, and transforming the recombinant vector into competent cells to obtain engineering bacteria;

the engineering bacteria are subjected to primary culture to obtain fermentation thalli;

inoculating the fermentation thalli into reaction liquid of a bioreactor for reaction to produce the alpha-ketoglutaric acid.

In the embodiment, the alpha-ketoglutaric acid can be produced and obtained by constructing a recombinant vector for co-expressing L-glutamate oxidase and catalase (the skeleton vector of the recombinant vector can be selected in various ways, and the invention is not particularly limited), transforming the operation vector into a competent cell (the competent cell can be selected in various ways, and the invention is not particularly limited) to obtain an engineering bacterium capable of co-expressing L-glutamate oxidase and catalase, then obtaining thalli through amplification culture, and using the thalli for whole-cell catalytic reaction.

In some embodiments of the foregoing fifth aspect, the primary culture comprises a primary culture and a secondary culture, the primary culture is performed at a temperature of 37 ℃ and at a speed of 220-250rpm for 8-12 hours; the primary culture is carried out until OD600 is 3.8-4.1, and the secondary culture is carried out under the temperature condition of 37 ℃ and the rpm of 200-235 for 4-5h.

In this example, sufficient cells can be obtained quickly by the stepwise amplification culture to perform the catalytic reaction.

In some embodiments of the foregoing fifth aspect, the secondary culture further comprises a supplementary culture, wherein the supplementary culture is to transfer the secondary culture bacterial liquid to a fermentation medium for fermentation, the pH and/or dissolved oxygen of the fermentation medium is increased, the supplementary culture is added, the temperature is reduced to 29-30 ℃ when the OD600 reaches 22-28, and IPTG (isopropyl thiogalactoside) with the concentration of 0.1-0.2mM is added for induction culture for 11-14h to obtain the zymophyte.

After secondary culture, nutrient solution is continuously supplemented and cultured to obtain high-concentration thalli, a certain concentration of the thalli can be maintained in a bioreactor, and the reaction efficiency is improved; and a large amount of protein is expressed and generated by adding IPTG for induction, thalli can accumulate to generate a large amount of L-glutamate oxidase and catalase, and alpha-ketoglutaric acid is generated through enzyme catalytic reaction.

In some embodiments of the foregoing fifth aspect, the primary medium is LB medium, the secondary medium is TB medium, and the fermentation medium comprises 28g/L yeast extract, 15g/L peptone, 10g/L glycerol, 16.4g/L dipotassium hydrogenphosphate trihydrate, 2.3g/L potassium dihydrogenphosphate, 0.3g/L magnesium sulfate heptahydrate, and 0.1g/L ferric ammonium citrate; the feed medium contains 100g/L of yeast extract, 30g/L of peptone and 400g/L of glycerol.

In some embodiments of the fifth aspect, the feed/liquid ratio of the fermentation broth to the reaction solution is 20 to 35g/L.

The thalli generated by fermentation contains a large amount of L-glutamate oxidase and catalase, and the reaction speed can be increased through the proper proportion of the fermentation thalli and the reaction liquid and the catalytic reaction of the L-glutamate oxidase and the catalase, so that the phenomenon that the production efficiency is influenced by long-time fermentation caused by too low fermentation thalli is avoided.

In some embodiments of the foregoing fifth aspect, the reaction solution includes glutamate, the reaction solution of the bioreactor is reacted at a temperature of 30 ℃,260 to 320rpm, and the pH is controlled to 6.5 to 7.0.

Glutamate (including sodium glutamate, potassium glutamate and the like) is added into the fermentation liquor as a reaction substrate, and the alpha-ketoglutaric acid can be generated through reaction.

Compared with the prior art, the invention has the beneficial effects that: the recombinant vector for expressing the L-glutamate oxidase and the catalase improves the capability of converting substrates into alpha-ketoglutarate by the L-glutamate oxidase and the catalase by inserting the regulation sequence SD for regulating the expression of the L-glutamate oxidase gene LGOX and the catalase gene CAT into the vector, and has better practical use value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below. It is appreciated that the following drawings depict only certain embodiments of the invention and are therefore not to be considered limiting of its scope. It is obvious to a person skilled in the art that other relevant figures can also be derived from these figures without inventive effort.

FIG. 1 is a view showing the structure of a recombinant vector of 1 according to an experimental example of the present invention;

FIG. 2 is a photograph showing the results of SDS-PAGE protein electrophoresis analysis of the expression amounts of L-glutamate oxidase and catalase provided in Experimental example 1 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a recombinant vector and an engineering bacterium for expressing L-glutamate oxidase and catalase, wherein the recombinant vector comprises an L-glutamate oxidase gene LGOX for expressing the L-glutamate oxidase, a catalase gene CAT for expressing the catalase and a regulation sequence SD for regulating the expression of the L-glutamate oxidase gene LGOX and the catalase gene CAT; the base sequence of the L-glutamic acid oxidase gene LGOX is shown as SEQ ID No.1, the base sequence of the catalase gene CAT is shown as SEQ ID No.2, and the base sequence of the regulatory sequence SD is shown as SEQ ID No. 3.

The recombinant vector and engineering bacteria for expressing L-glutamate oxidase and catalase are obtained by the following steps:

1.1, optimizing a full-length sequence of an L-glutamic acid oxidase gene according to Streptomyces afghaniensis 772, removing a signal peptide of the sequence to obtain the L-glutamic acid oxidase gene, designing an amplification primer to amplify the L-glutamic acid oxidase gene, connecting the L-glutamic acid oxidase gene to a T vector, and constructing to obtain a T-LGOX vector;

1.2 optimizing according to a bacillus pumilus catalase gene to obtain a catalase gene CAT, designing a primer sequence, designing a regulatory sequence SD onto the primer, and amplifying to obtain an SD sequence-catalase gene CAT and connecting the SD sequence-catalase gene CAT to a pUC57 vector to obtain a pUC57-SD-CAT vector;

1.3 selecting proper endonuclease double-restriction digestion T-LGOX vector, pUC57-SD-CAT vector and pET32a vector skeleton system; recovering enzyme-digested fragments, and connecting the enzyme-digested fragments with double enzyme-digested fragments by using T4 ligase to obtain pET32a-LGOX-SD-CAT recombinant vector for expressing L-glutamate oxidase and catalase, wherein the structural diagram of the vector is shown in figure 1;

1.4 transforming DH5 alpha competent cells with the obtained pET32a-LGOX-SD-CAT recombinant vector, extracting plasmids and transferring the plasmids into Escherichia coli BL21 (DE 3) to obtain an engineering bacterium pET32a-LGOX-SD-CAT/BL21 (DE 3) for expressing L-glutamate oxidase and catalase.

Transforming the recombinant vector into escherichia coli BL21 (DE 3) competent cells, plating for 15h, selecting a single colony to culture in an LB test tube for 9h, transferring the single colony to a TB culture medium for induction culture for 16h, detecting the expression quantity through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), selecting bacteria with both enzymes normally expressed, namely successfully obtaining the engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3), and storing a plurality of permanent bacteria glycerol tubes in an ultra-low temperature refrigerator for later use, wherein the results are shown in figure 2.

Thus, pET32a-LGOX-SD-CAT recombinant vectors or engineered bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) can be used to co-express L-glutamate oxidase and catalase.

Example 2

This example provides a method for producing α -ketoglutaric acid, which comprises the following steps:

1.1 construction of pET32a-LGOX-SD-CAT recombinant vector and engineered bacterium pET32a-LGOX-SD-CAT/BL21 (DE 3) by the method of reference example 1;

1.2 inoculating the engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) into a primary culture medium LB to carry out a primary culture stage of primary culture, wherein the primary culture condition is that the primary culture is carried out for 12 hours at 220rpm under the temperature condition of 37 ℃; to an OD600 of 3.8;

1.3 transferring the bacterial liquid of the primary culture to a secondary culture medium TB for continuous secondary culture stage culture of the primary culture, wherein the secondary culture condition is that the bacterial liquid of the primary culture is cultured for 4 hours at 200rpm under the temperature condition of 37 ℃;

1.4 inoculating the secondary cultured thalli into a fermentation tank added with a fermentation medium (the fermentation medium contains 28g/L of yeast extract, 15g/L of peptone, 10g/L of glycerol, 16.4g/L of dipotassium phosphate trihydrate, 2.3g/L of potassium dihydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate and 0.1g/L of ferric ammonium citrate) for culturing for 4-5h at 37 ℃;

1.5 when the pH and dissolved oxygen in the fermentation tank begin to rise, adding a supplemented medium (the supplemented medium contains 100g/L yeast extract, 30g/L peptone and 400g/L glycerol), when the thallus concentration OD600 reaches 28, cooling, performing induced culture with 0.1mM IPTG for 14h, and collecting the thallus for later use;

1.6 taking 150g of the thalli collected in the step 1.5, adding the thalli into 7.5L of reaction liquid according to the feed-liquid ratio of 20g/L, wherein the reaction liquid contains 1370.4g of monosodium glutamate (L-sodium glutamate), and introducing sterile air for transformation and culture at the temperature of 30 ℃ and the rpm of 260;

1.7 controlling the pH value to be 6.5-7.0 by using 4mol/L sulfuric acid, detecting that the reaction solution does not contain L-sodium glutamate, and the dissolved oxygen is more than 50%, and finishing the reaction.

Example 3

1.2 inoculating the engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) into a primary culture medium LB to carry out a primary culture stage of primary culture, wherein the primary culture condition is that the primary culture is carried out for 8 hours at 250rpm under the temperature condition of 37 ℃; to an OD600 of 4.1;

1.3 transferring the bacterial liquid of the primary culture to a secondary culture medium TB for continuous secondary culture stage culture of the primary culture, wherein the secondary culture condition is culture at 235rpm for 5h at the temperature of 37 ℃;

1.4 inoculating the secondary cultured thalli into a fermentation tank added with a fermentation medium (the fermentation medium contains 28g/L of yeast extract, 15g/L of peptone, 10g/L of glycerol, 16.4g/L of dipotassium phosphate trihydrate, 2.3g/L of potassium dihydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate and 0.1g/L of ferric ammonium citrate) to culture for 4-5h at 37 ℃;

1.5 when the pH value and dissolved oxygen in the fermentation tank begin to rise, adding a supplemented medium (the supplemented medium contains 100g/L yeast extract, 30g/L peptone and 400g/L glycerol), when the thallus concentration OD600 reaches 22, cooling, performing induced culture with 0.2mM IPTG for 11h, and collecting the thallus for later use;

1.6 taking 262.5g of the thalli collected in the step 1.5, adding the thalli into 7.5L of reaction liquid according to the feed-liquid ratio of 35g/L, wherein the reaction liquid contains 1370.4g of monosodium glutamate (L-sodium glutamate), and introducing sterile air for transformation and culture at the temperature of 30 ℃ and the rpm of 320;

Example 4

1.2 inoculating the engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) into a primary culture medium LB to carry out a primary culture stage of primary culture, wherein the primary culture condition is that the primary culture is carried out for 9 hours at 240rpm under the temperature condition of 37 ℃; to an OD600 of 4;

1.3 transferring the bacterial liquid of the primary culture to a secondary culture medium TB for continuous secondary culture stage culture of the primary culture, wherein the secondary culture condition is that the bacterial liquid is cultured for 5 hours at 220rpm under the temperature condition of 37 ℃;

1.6 taking 200g of the thalli collected in the step 1.5, adding the thalli into 7.5L of reaction liquid according to the feed-liquid ratio of 26.67g/L, wherein the reaction liquid contains 1370.4g of monosodium glutamate (L-sodium glutamate), and introducing sterile air for transformation and culture at the temperature of 30 ℃ and the rpm of 300;

1.7 controlling the pH value to be 6.5-7.0 by using 4mol/L sulfuric acid, detecting that the reaction solution does not contain L-sodium glutamate, and the dissolved oxygen is more than 50 percent, and finishing the reaction.

Experimental example 1

pET32a-LGOX-SD-CAT recombinant vectors and engineered bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) were constructed by the method of reference example 1.

Culturing engineering bacteria: inoculating engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) in a first-level culture medium under a sterile environment, culturing for 9h under the conditions of 37 ℃ and 240rpm of rotation speed, inoculating the engineering bacteria into a second-level culture medium (10 percent of inoculum concentration) after OD600 reaches 3-4, and culturing for 4-5h under the conditions of 37 ℃ and 220rpm of rotation speed; then inoculating the strain into a fermentation tank for culture at 37 ℃ (inoculum size is 10%), starting feeding when pH and dissolved oxygen rise, starting cooling to 30 ℃ when the bacterial concentration OD600 reaches 22-28 ℃, adding 0.1mM inducer IPTG for induction culture for 12 hours, putting the strain into the tank, and collecting the strain for later use.

Transformation experiments: 200g of wet thallus is weighed and suspended in 7.5L of water, poured into a 10L bioreactor, 1370.4g of monosodium glutamate is weighed and gradually put into the bioreactor, the temperature is 30 ℃, the rotation speed is 300rpm, sterile air is introduced for conversion, and the pH value is controlled to be 6.5-7.0 by 4mol/L of sulfuric acid. The conversion is carried out for 24-28h, the L-sodium glutamate is detected by TLC and then put into a tank, the dissolved oxygen is more than 50 percent, and the conversion is finished. HPLC detection shows that the content of alpha-ketoglutaric acid is 123.37g/L, and the conversion rate is 93.25%.

Experimental example 2

Culturing engineering bacteria: inoculating engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) in a primary culture medium in a sterile environment, culturing for 9h under the conditions of 37 ℃ and 240rpm, inoculating the OD600 into a secondary culture medium (10 percent of inoculum size) after reaching 3-4, and culturing for 4-5h under the conditions of 37 ℃ and 220 rpm; then inoculating the strain into a fermentation tank for culture at 37 ℃ (the inoculation amount is 10%), starting feeding when the pH and dissolved oxygen rise, starting cooling to 30 ℃ when the bacterium concentration OD600 reaches 22-28 ℃, adding 0.15mM inducer IPTG for induction culture for 12 hours, putting the strain into the tank, and collecting the strain for later use.

Transformation experiments: measuring 1.5L of zymocyte liquid, adding deionized water to constant volume of 7.5L, pouring into a 10L bioreactor, weighing 1370.4g of monosodium glutamate, gradually adding into the bioreactor, converting at 30 ℃,300rpm by introducing sterile air, and controlling the pH value to 6.5-7.0 by using 4mol/L sulfuric acid. The conversion is carried out for 24-28h, the L-sodium glutamate is detected by TLC and then put into a tank, the dissolved oxygen is more than 50 percent, and the conversion is finished. HPLC detection shows that the content of alpha-ketoglutaric acid is 120.4g/L, and the conversion rate is 91.03%.

Experimental example 3

Culturing engineering bacteria: inoculating engineering bacteria pET32a-LGOX-SD-CAT/BL21 (DE 3) in a primary culture medium in a sterile environment, culturing for 9h under the conditions of 37 ℃ and 240rpm, inoculating the OD600 into a secondary culture medium (10 percent of inoculum size) after reaching 3-4, and culturing for 4-5h under the conditions of 37 ℃ and 220 rpm; then inoculating the strain into a fermentation tank for culture at 37 ℃ (the inoculation amount is 10%), starting feeding when the pH and dissolved oxygen rise, starting cooling to 30 ℃ when the bacterium concentration OD600 reaches 22-28 ℃, adding 0.20mM inducer IPTG for induction culture for 12 hours, putting the strain into the tank, and collecting the strain for later use.

Transformation experiments: measuring 1.7L of zymocyte liquid, adding deionized water to constant volume of 7.5L, pouring into a 10L bioreactor, weighing 1370.4g of monosodium glutamate, gradually adding into the bioreactor, converting at 30 ℃,300rpm by introducing sterile air, and controlling the pH value to 6.5-7.0 by using 4mol/L sulfuric acid. The conversion is carried out for 24-28h, the L-sodium glutamate is detected by TLC and then put into a tank, the dissolved oxygen is more than 50 percent, and the conversion is finished. HPLC detection shows that the content of alpha-ketoglutaric acid is 126.3g/L, and the conversion rate is 95.46%.

After-treatment, 98.5% alpha-ketoglutaric acid crystal is obtained, and the total yield is 80-85%.

In summary, the recombinant vector and the engineering bacteria for expressing L-glutamate oxidase and catalase, and the application and the method for producing alpha-ketoglutaric acid provided by the embodiments of the present invention obtain a recombinant vector capable of efficiently expressing glutamate, and through transforming cells, obtain an efficient expression system, and through fermentation, efficiently transform glutamate and convert glutamate into alpha-ketoglutaric acid, and improve the efficiency of transforming substrate glutamate into alpha-ketoglutaric acid, and improve the yield and recovery rate of the product.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> Sichuan Ji Cheng biological medicine Limited

<120> recombinant vector for expressing L-glutamate oxidase and catalase, engineering bacteria and application thereof, and alpha-ketopentosan

Process for the production of diacids

<170> PatentIn version 3.5

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tacgctccgg ttcacaacaa ccagcaggac ggtttcatga ccaccacccg tccgtctggt 1140

cacatcaact acgaaccgaa ccgttacgac gaccagccga aagaaaaccc gcactacaaa 1200

gaatctgaac aggttctgca cggtgaccgt atggttcgtc agaaaatcga aaaaccgaac 1260

gacttcaaac aggctggtga aaaataccgt tcttactctg aagaagaaaa acaggctctg 1320

atcaaaaacc tgaccgctga cctgaaagac gttaacgaca aaaccaaact gctggctatc 1380

tgcaacttct accgtgctga cgaagactac ggtcagcgtc tggctgactc tctgggtgtt 1440

gacatccgtt cttacctgca gggttctatg aaataa 1476

<210> 3

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 3

agatctggaa ggaatacaaa 20

Claims

1. A method for producing α -ketoglutaric acid, wherein the method comprises the steps of:

constructing a recombinant vector for expressing L-glutamate oxidase and catalase, and transforming competent cells into the recombinant vector to obtain engineering bacteria;

the engineering bacteria are subjected to primary culture to obtain fermentation bacteria;

inoculating the fermentation thalli into a reaction solution of a bioreactor for reaction to produce alpha-ketoglutaric acid;

wherein the recombinant vector comprises a L-glutamic acid oxidase gene LGOX for expressing L-glutamic acid oxidase, a catalase gene CAT for expressing catalase, and a regulatory sequence SD for regulating the expression of the L-glutamic acid oxidase gene LGOX and the catalase gene CAT; the base sequence of the L-glutamic acid oxidase gene LGOX is shown in SEQ ID No.1, the base sequence of the catalase gene CAT is shown in SEQ ID No.2, and the base sequence of the regulatory sequence SD is shown in SEQ ID No. 3;

the primary culture comprises primary culture and secondary culture, wherein the primary culture is carried out for 8-12h at 220-250rpm under the temperature condition of 37 ℃; the primary culture is carried out until the OD600 is 3.8-4.1, and the secondary culture is carried out under the temperature condition of 37 ℃ and the culture speed of 200-235rpm for 4-5h;

after secondary culture, performing supplementary culture, namely transferring the bacterial liquid of the secondary culture to a fermentation culture medium for fermentation, increasing the pH and/or dissolved oxygen of the fermentation culture medium, adding a supplementary culture medium, performing culture until OD600 reaches 22-28, cooling to 29-30 ℃, adding 0.1-0.2mM IPTG (isopropyl thiogalactoside) for induction culture for 11-14h to obtain zymophyte;

the primary culture medium is an LB culture medium, the secondary culture medium is a TB culture medium, and the fermentation culture medium contains 28g/L of yeast extract, 15g/L of peptone, 10g/L of glycerol, 16.4g/L of dipotassium phosphate trihydrate, 2.3g/L of potassium dihydrogen phosphate, 0.3g/L of magnesium sulfate heptahydrate and 0.1g/L of ferric ammonium citrate; the supplementary culture medium contains 100g/L of yeast extract, 30g/L of peptone and 400g/L of glycerol;

the feed-liquid ratio of the fermentation thalli to the reaction liquid is 20-35g/L;

the reaction solution includes glutamate, the temperature of the reaction solution of the bioreactor is 30 ℃,260-320rpm, and the pH is controlled to 6.5-7.0.