阅读说明：本技术 一种提高甲硫氨酸产量的方法 (Method for improving methionine yield ) 是由 赵嫚 魏磊 应向贤 汪钊 于 2019-11-13 设计创作，主要内容包括：本发明公开了一种提高甲硫氨酸产量的方法,所述方法为：将胱硫醚-γ-合酶基因、同型半胱氨酸甲基转移酶基因或甲硫氨酸甲基转移酶基因中的一种或多种导入宿主菌构建重组基因工程菌,将重组基因工程菌发酵培养,获得含甲硫氨酸发酵液,发酵液分离纯化获得甲硫氨酸。本发明方法通过增加甲硫氨酸合成过程中甲基的供应和高半胱氨酸的含量,其中工程菌E.coli BL21(DE3)pBlunt-E1-CGS-MMT-HMT发酵产生甲硫氨酸的产量是0.3g/L,相对于野生型E.coli BL21(DE3)菌株,甲硫氨酸的产量增加了9倍,显著提高了反应效率。(The invention discloses a method for improving methionine yield, which comprises the following steps: introducing one or more of cystathionine-gamma-synthase gene, homocysteine methyltransferase gene or methionine methyltransferase gene into host bacteria to construct recombinant genetic engineering bacteria, fermenting and culturing the recombinant genetic engineering bacteria to obtain methionine-containing fermentation liquor, and separating and purifying the fermentation liquor to obtain methionine. According to the method, the methyl supply and the content of homocysteine are increased in the methionine synthesis process, wherein the yield of methionine generated by fermentation of the engineering bacteria E.coli BL21(DE3) pBlunt-E1-CGS-MMT-HMT is 0.3g/L, and compared with the wild E.coli BL21(DE3) strain, the yield of methionine is increased by 9 times, and the reaction efficiency is obviously improved.)

1. A method for increasing the production of methionine, comprising: introducing one or more of cystathionine-gamma-synthase gene, homocysteine methyltransferase gene or methionine methyltransferase gene into host bacteria to construct recombinant genetic engineering bacteria, fermenting and culturing the recombinant genetic engineering bacteria to obtain methionine-containing fermentation liquor, and separating and purifying the fermentation liquor to obtain methionine.

2. The method for increasing the production of methionine as claimed in claim 1, wherein the host bacterium is E.coli BL21(DE 3).

3. The method for increasing the production of methionine according to claim 1, wherein the cystathionine- γ -synthase gene has a nucleotide sequence represented by SEQ ID No. 1; the nucleotide sequence of the homocysteine methyltransferase gene is shown in SEQ ID No. 3; the nucleotide sequence of the methionine methyltransferase gene is shown as SEQ ID No. 5.

4. The method for increasing the production of methionine according to claim 1, wherein the recombinant genetically engineered bacterium is constructed according to one of the following methods: (1) single enzyme system: inserting cystathionine-gamma-synthase encoding gene, homocysteine methyltransferase gene and methionine methyltransferase gene into plasmid pBlunt-E1 respectively to obtain recombinant plasmid pBlunt-E1-CGS, pBlunt-E1-HMT and pBlunt-E1-MMT; respectively introducing the recombinant plasmids into Escherichia coli E.coli BL21(DE3) to obtain corresponding engineering bacteria E.coli BL21(DE3) pBlunt-E1-CGS, E.coli BL21(DE3) pBlunt-E1-HMT and E.coli BL21(DE3) pBlunt-E1-MMT; (2) two-enzyme system: simultaneously, the homocysteine methyltransferase gene and the methionine methyltransferase gene are inserted into a plasmid pBlunt-E1 in series to recombine the plasmid pBlunt-E1-HMT-MMT; introducing the recombinant plasmid into Escherichia coli E.coli BL21(DE3) to obtain recombinant gene engineering bacteria E.coli BL21(DE3) pBlunt-E1-HMT-MMT; (3) three-enzyme system: cystathionine-gamma-synthase gene, homocysteine methyltransferase gene and methionine methyltransferase gene are inserted into plasmid pBlunt-E1 in series to obtain recombinant plasmid pBlunt-E1-CGS-HMT-MMT, and the recombinant plasmids are respectively introduced into escherichia coli to obtain recombinant gene engineering bacteria E.coli BL21(DE3) pBlunt-E1-CGS-HMT-MMT.

5. The method for increasing the production of methionine according to claim 1, wherein the genetically engineered recombinant bacterium is a genetically engineered recombinant bacterium co-expressing cystathionine- γ -synthase and homocysteine methyltransferase and methionine methyltransferase.

6. The method for increasing the production of methionine as claimed in claim 1, wherein the fermentation culture method of the recombinant genetically engineered bacteria comprises: inoculating the engineering bacteria into a fermentation culture medium, performing fermentation culture at 28 ℃ and 180rpm to obtain a fermentation broth containing methionine, centrifuging the fermentation broth, and separating and purifying to obtain methionine; the final concentration of the fermentation medium is as follows: glucose 20g/L, NaS₂O₃16g/L, 2g/L yeast powder, KH₂PO₄1g/L，MgSO₄1g/L，CaCO₃10g/L，FeSO₄0.01g/L，MnSO₄0.01g/L，ZnSO₄0.01g/L, water as solvent, and natural pH.

7. The method for increasing the production of methionine as claimed in claim 1, wherein the recombinant genetically engineered bacteria are induced and cultured before fermentation, specifically as follows: inoculating the recombinant genetically engineered bacteria into LB liquid culture medium containing 100 mug/mL ampicillin at final concentration, culturing overnight at 37 ℃ and 200rpm, inoculating the culture into LB liquid culture medium containing 100 mug/mL ampicillin at an inoculum size of 1-2% by volume, and culturing at 37 ℃ and 200rpm to OD₆₀₀0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM into the culture, carrying out induction culture at 25 ℃ and 140rpm for 14h, then centrifugally collecting wet thalli at 4 ℃ and 5000rpm, washing the wet thalli by using a fermentation medium for heavy suspension for 2 times, and inoculating the wet thalli to the fermentation medium in an amount of 1g/50ml for fermentation.

(I) technical field

The invention relates to an application method of a multienzyme combination from a plant methionine synthetic pathway in fermentation synthesis of methionine in microorganisms, belonging to the fields of genetic engineering and biological fermentation.

(II) background of the invention

Methionine (Met) is an essential amino acid in humans and must be obtained from food. Methionine has important physiological and biochemical functions in the aspects of protein translation initiation, metabolism regulation, important compound precursors and the like in organisms. Plants and microorganisms can synthesize methionine via the de novo pathway, the aspartate synthetic pathway, and plants can also provide methionine in large amounts via the methyl cycle during plant seed development via a complementary pathway, the S-methyl methionine cycle (SMM cycle). Cystathionine-gamma-synthase (CGS) in plants catalyzes O-acetyl-L-homoserine and cysteine to synthesize cystathionine, and further synthesize homocysteine, which is a rate-limiting enzyme for methionine synthesis and is subjected to feedback inhibition by products Met and S-adenosylmethionine (SAM). Homocysteine Methyltransferase (HMT) and Methionine Methyltransferase (MMT) are two key enzymes in the SMM cycle. HMT converts homocysteine to 2 molecules of methionine when SMM or SAM acts as a methyl donor; MMT synthesizes methionine and homocysteine together into SMM, thereby achieving SMM circulation. The SMM cycle can achieve a large supply of Met required for plant seed development in a short period of time.

The methionine synthesis pathway has been analyzed, but methionine synthesis in vivo is generally low, and thus methionine is synthesized industrially essentially by a chemical method. The chemical synthesis of Met mainly uses acrolein, methyl mercaptan and hydrocyanic acid as raw materials to synthesize racemate (D-and L-Met) of Met, and L-Met is obtained by racemate separation. Although the chemical method is mature, the problems of toxic synthesis raw materials, high energy consumption, complex process and the like still exist. Currently, studies on the biosynthesis of Met are actively being explored. The biological method for synthesizing Met comprises two methods, namely enzymatic synthesis and biological fermentation. At present, the enzymatic synthesis yield is relatively high, about 40g/L, but the process is relatively complex, i.e., homoserine is synthesized as an intermediate product through fermentation, and then the enzyme, homoserine produced by fermentation cells and a substrate, methyl mercaptan, are catalyzed to synthesize Met. The biological fermentation method mainly focuses on the modification of the self Met synthesis routes of escherichia coli, corynebacterium glutamicum and the like, and the Met is directly generated by glucose fermentation, wherein the specific modification routes comprise the synthesis of aspartic acid, homocysteine and the like which are synthetic precursors of the Met, and the feedback inhibition of key enzymes in the synthesis routes is relieved, for example: aspartokinase, cystathionine-gamma-synthase, etc., decrease the synthesis of competitive pathways such as lysine and threonine, decrease the synthesis of downstream product S-methyl methionine, etc. The current Huang et al 2018 recent research shows that the fermentation level of methionine has reached 16.86g/L through modification of the whole metabolic pathway including the reducing power NADPH pathway, the aspartic acid precursor synthesis pathway, the competitive lysine and threonine synthesis pathway and the methionine synthesis internal pathway in a plurality of steps and continuous fermentation in a fermentation tank (the continuous fermentation can improve the yield by more than 10-20 times compared with the shake flask), but the research is only limited to modification of the escherichia coli internal synthesis pathway and over-expression of enzyme. Although the biological fermentation method has the advantages of simple method, low energy consumption and green and pollution-free process, the method has a certain distance from the industrial production.

At present, no report that the biological method or the enzymatic method applies the methionine synthetic pathway CGS and SMM circulating key enzyme in plants to the microorganisms to ferment and synthesize methionine is found.

Disclosure of the invention

The invention provides a new thought method for improving the yield of methionine on the basis of the existing biological method for synthesizing methionine, and particularly relates to a method for improving the yield of methionine by applying a multi-enzyme combination which is unique to a plant methionine synthesis path to a microorganism, improving the content of methionine in the microorganism through overexpression of specific enzymes in a plant, and simultaneously improving the content of methionine by converting a synthesis precursor of threonine in the microorganism into methionine.

The technical scheme adopted by the invention is as follows:

the invention provides a method for improving methionine yield, which comprises the following steps: introducing one or more of cystathionine-gamma-synthase gene, homocysteine methyltransferase gene or methionine methyltransferase gene into host bacteria to construct recombinant genetic engineering bacteria, fermenting and culturing the recombinant genetic engineering bacteria to obtain methionine-containing fermentation liquor, and separating and purifying the fermentation liquor to obtain methionine. The host bacterium is preferably E.coli BL21(DE 3).

The cystathionine-gamma-synthase (CGS) gene is derived from Arabidopsis thaliana (Arabidopsis thaliana), the nucleotide sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2. The Homocysteine Methyltransferase (HMT) gene is derived from soybean (Glycine max), the nucleotide sequence is shown as SEQ ID No.3, and the amino acid sequence is shown as SEQ ID No. 4. The Methionine Methyltransferase (MMT) gene is derived from soybean (Glycine max), the nucleotide sequence is shown as SEQ ID No.5, and the amino acid sequence is shown as SEQ ID No. 6. Wherein the homocysteine methyltransferase and the methionine methyltransferase are enzymes in the SMM cycle of soybean (Glycine max).

The recombinant gene engineering bacteria are constructed according to one of the following methods: (1) single enzyme system: inserting cystathionine-gamma-synthase gene, homocysteine methyltransferase gene and methionine methyltransferase gene into plasmid pBlunt-E1 to obtain recombinant plasmid pBlunt-E1-CGS, pBlunt-E1-HMT and pBlunt-E1-MMT; respectively introducing the recombinant plasmids into Escherichia coli E.coli BL21(DE3) to obtain corresponding engineering bacteria E.coli BL21(DE3) pBlunt-E1-CGS, E.coli BL21(DE3) pBlunt-E1-HMT and E.coli BL21(DE3) pBlunt-E1-MMT; (2) two-enzyme system: simultaneously, the homocysteine methyltransferase gene and the methionine methyltransferase gene are inserted into a plasmid pBlunt-E1 in series to recombine the plasmid pBlunt-E1-HMT-MMT; introducing the recombinant plasmid into Escherichia coli E.coli BL21(DE3) to obtain recombinant gene engineering bacteria E.coli BL21(DE3) pBlunt-E1-HMT-MMT; (3) three-enzyme system: cystathionine-gamma-synthase gene, homocysteine methyltransferase gene and methionine methyltransferase gene are inserted into plasmid pBlunt-E1 in series to obtain recombinant plasmid pBlunt-E1-CGS-HMT-MMT, and the recombinant plasmids are respectively introduced into escherichia coli to obtain recombinant gene engineering bacteria E.coli BL21(DE3) pBlunt-E1-CGS-HMT-MMT.

The invention preferably uses recombinant genetic engineering bacteria which co-express cystathionine-gamma-synthase, homocysteine methyltransferase and methionine methyltransferase to carry out biological fermentation to synthesize methionine. CGS provides a large amount of homocysteine precursor for methionine synthesis in the same reaction system, and HMT and SMM realize methyl cycle in methionine synthesis process.

The fermentation culture method of the recombinant gene engineering bacteria comprises the following steps: inoculating the engineering bacteria into a fermentation culture medium, performing fermentation culture (preferably shake flask fermentation for 48h) at 28 ℃ and 180rpm to obtain a fermentation broth containing methionine, centrifuging the fermentation broth, and separating and purifying to obtain methionine; the final concentration of the fermentation medium is as follows: glucose 20g/L, NaS₂O₃16g/L, 2g/L yeast powder, KH₂PO₄1g/L，MgSO₄1g/L，CaCO₃10g/L, FeSO40.01g/L, MnSO40.01g/L and ZnSO40.01g/L, wherein the solvent is water, and the pH is natural.

The recombinant gene engineering bacteria of the invention are firstly induced and cultured before fermentation, and specifically comprise the following steps: inoculating the recombinant genetically engineered bacteria into LB liquid culture medium containing 100 mug/mL ampicillin at final concentration, culturing overnight at 37 deg.C and 200rpm, inoculating the culture with 1-2% volume concentration into 50mL LB liquid culture medium containing 100 mug/mL ampicillin, and culturing at 37 deg.C and 200rpm until OD₆₀₀0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM into the culture, carrying out induction culture at 25 ℃ and 140rpm for 14h, then centrifugally collecting wet thalli at 4 ℃ and 5000rpm, washing the wet thalli by using a fermentation medium for heavy suspension for 2 times, and inoculating the wet thalli to the fermentation medium in an amount of 1g/50ml for fermentation.

The invention modifies two steps in the methionine synthetic pathway, firstly, the integration of the methionine synthetic pathway of plants and microorganisms is realized, specific HMT and MMT genes in the plants are over-expressed in the microorganisms, the application of methyl cycle in the plants in the microorganisms is realized, and the methyl cycle is improved; secondly, CGS in plants simultaneously utilizes threonine synthesis precursors in microorganisms, thus partially switching threonine synthesis flux to methionine synthesis and increasing methionine production.

Compared with the prior art, the invention has the following beneficial effects: conventional methods essentially involve metabolic engineering of the microorganism itself, including increasing methionine precursor levels, decreasing the carbon flux of methionine competing pathways, decreasing methionine metabolism, and increasing transport of methionine out of the transport protein. The invention provides a new idea for synthesizing methionine by using a multi-enzyme combination in plants in microbial metabolism methionine synthesis. According to the method, the methyl supply and the content of homocysteine are increased in the methionine synthesis process, wherein the yield of methionine generated by fermentation of the engineering bacterium E.coli BL21(DE3) pBlunt-E1-CGS-MMT-HMT is 0.3g/L, and compared with a wild E.coli BL21(DE3) strain, the yield of methionine is increased by 9 times, and the reaction efficiency is obviously improved.

(IV) description of the drawings

FIG. 1 is a schematic diagram of the modification of the methionine synthetic pathway; CGS, cystathionine-gamma-synthetase derived from engineered Arabidopsis thaliana; HMT, homocysteine methyltransferase from Glycine max; MMT, methionine methyltransferase derived from Glycine max.

FIG. 2 is a schematic diagram of the construction of expression vectors for single, double and triple enzymes; the single enzymes pBlunt-E1-CGS, pBlunt-E1-HMT and pBlunt-E1-MMT; the double enzyme pBlunt-E1-MMT-HMT; three enzymes: pBlunt-E1-CGS-MMT-HMT.

FIG. 3 is a SDS-PAGE gel of genetically engineered bacteria expressing cystathionine-gamma-synthetase encoding gene CGS derived from Arabidopsis thaliana, homocysteine methyltransferase encoding gene HMT derived from Glycine max, and methionine methyltransferase encoding gene MMT of single enzyme, double enzyme, and triple enzyme systems. Wherein the Lane 1 is the Control empty strain E.coliBL21(DE3), the Lane 7 is the thallus before induction by the Control empty vector E.coliBL21(DE3) pBlunt-E1, the Lane 2 is the thallus after induction by the Control empty vector E.coliBL21(DE3) pBlunt-E1, the Lane 3 is the thallus after induction by the E.coliBL21(DE3) pBlunt-E1-CGS, the Lane 4 is the thallus after induction by the E.coliBL21(DE3) pBlunt-E1-HMT, the Lane 5 is the thallus after induction by the E.coliBL21(DE3) pBlunt-E1-MMT, the Lane 6 is the standard molecular weight, the Lane 8 is the E.coliBL21(DE3) pBlunt-E1-HMT-E-MMT, and the Lane 9 is the Lane 3-MMT after induction by the DE 3524-MMT (DE 3).

FIG. 4 liquid chromatography analysis of methionine standard sample of example 1.

FIG. 5 comparison of the titers of methionine produced by fermentation in E.coli for the single, double and triple enzyme systems, each set corresponding to 12, 24, 36 and 48h of fermentation time, respectively.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

composition of LB medium: 5g/L of yeast extract, 10g/L of peptone, 10g/L of NaCl and water as a solvent, wherein the pH value is natural.

The fermentation medium comprises the following components: glucose 20g/L, NaS₂O₃16g/L, 2g/L yeast powder, KH₂PO₄1g/L，MgSO₄1g/L，CaCO₃10g/L，FeSO₄0.01g/L，MnSO₄0.01g/L，ZnSO₄0.01g/L, water as solvent, and natural pH.