阅读说明：本技术 一种基于酶促拆分法的混菌发酵产d-赖氨酸 (Enzymatic resolution method-based D-lysine produced by mixed fermentation ) 是由 陈可泉 许晟 王昕� 冯娇 于 2020-03-13 设计创作，主要内容包括：本发明公开了一种基于酶促拆分法的混菌发酵产D-赖氨酸。该方法包括以下步骤：构建重组菌株E.coli BL-pTrc99a-DapA；构建E.coli BL-pET28a-LYR葡萄糖代谢缺陷型菌株；选取重组菌株E.coli BL-pTrc99a-DapA和E.coli BL-pET28a-LYR加入发酵培养基中,葡萄糖和甘油为碳源,生产DL-赖氨酸,最后采用含有赖氨酸脱羧酶的粗酶液将DL-赖氨酸中L-赖氨酸转化为1,5-戊二胺。与现有技术相比,降低了生产成本,终产物D-赖氨酸的产量提高。相比于利用E.coli BL-pET28a-LYR单一发酵生产,D-赖氨酸的摩尔收率提高了35%。(The invention discloses a method for producing D-lysine by mixed fermentation based on an enzymatic resolution method, which comprises the following steps of constructing a recombinant strain E.coli B L-pTrc 99a-DapA, constructing an E.coli B L-pET 28 a-L0 YR glucose metabolism defective strain, selecting the recombinant strain E.coli B L-pTrc 99-DapA and E.coli B L-pET 28 a-L YR, adding glucose and glycerol as carbon sources into a fermentation culture medium, producing D L-lysine, and finally adopting crude enzyme liquid containing lysine decarboxylase to convert L-lysine in D L-lysine into 1, 5-pentanediamine.)

1. The method for producing D-lysine by mixed fermentation based on an enzymatic resolution method is characterized by comprising the following steps:

step 1, constructing a recombinant strain E.coli B L-pTrc 99 a-DapA;

step 2, constructing a glucose metabolism defective strain of E.coli B L-pET 28 a-L YR;

step 3, selecting recombinant strains E.coli B L-pTrc 99a-DapA and E.coli B L-pET 28 a-L YR, adding the recombinant strains into a fermentation culture medium, using glucose and glycerol as carbon sources, culturing until OD is about 0.6, inducing by IPTG, and continuously culturing to produce D L-lysine;

and 4, converting L-lysine in the D L-lysine into 1, 5-pentanediamine by using crude enzyme liquid containing lysine decarboxylase, thereby obtaining the D-lysine.

2. The method for producing D-lysine by mixed fermentation based on the enzymatic resolution method in the step 1 is characterized in that E.coli B L-pTrc 99a-DapA in the step 1 is constructed by selecting primers, copying dihydropicolinate synthase on Escherichia coli MG1655 through PCR, connecting the primers with a vector pTrc99a, transferring the vectors into a cloning vector Trans1-T1, primarily screening through a L B plate, selecting points, performing colony PCR verification, and sequencing.

3. The process for producing D-lysine by mixed fermentation based on enzymatic resolution as claimed in claim 1, wherein the E.coli B L-pET 28 a-L YR glucose metabolism deficient strain in step 1 is obtained by introducing a strain derived from a microorganism belonging to the genus EscherichiaP. mirabilisThe gene L YR of BCRC10725 was codon-optimized and then transferred to pET28a synthesized by Jinwei Zhi CoNcoI /XhoTransferring a cloning vector Trans1-T1 between the I enzyme cutting sites, primarily screening by a L B plate, selecting the sites, carrying out colony PCR verification and sequencing.

4. The method for producing D-lysine by mixed fermentation based on enzymatic resolution as claimed in claim 1, wherein the E.coli B L-pET 28 a-L YR glucose metabolism deficient strain in step 2 is constructed by knocking out phosphotransferase system (PTS) of Escherichia coli B L21 (DE3) by Crispr-Cas9 editing technologyptsG、ptsH、ptsI、crrAndglkthe gene coded protein is obtained by transforming the constructed plasmid pET28 a-L YR into the competence of B L21 (DE3) of glucose metabolism defect, thus obtaining the E.coli B L-pET 28 a-L YR glucose metabolism defect strain.

5. The D-lysine produced by mixed fermentation based on enzymatic resolution method as claimed in claim 1, wherein OD ratio of seed liquid cells added with recombinant strains E.coli B L-pTrc 99a-DapA and E.coli B L-pET 28 a-L YR in step 3 is 3: 1.

6. The D-lysine produced by mixed fermentation based on the enzymatic resolution method of claim 1, wherein the molar ratio of the added glucose to the added glycerol in the step 3 is 6: 1.

7. the D-lysine produced by mixed fermentation based on the enzymatic resolution method of claim 1, wherein the induction temperature in step 3 is 35 ℃.

8. The process for producing D-lysine by mixed fermentation based on the enzymatic resolution method of claim 1, wherein the final concentration of the crude enzyme solution containing lysine decarboxylase in step 4 is 1mg/m L.

Technical Field

The invention relates to the technical field of D-lysine preparation, and particularly relates to a method for producing D-lysine by mixed fermentation based on an enzymatic resolution method.

Background

The D-lysine is stable at normal temperature and pressure, is needle-shaped or sheet-shaped crystal, is soluble in water, acid and slightly soluble in chloroform, diethyl ether and ethanol, has better antibacterial property and more important application in medicine, can be used as a precursor to participate in the synthesis of luteinizing hormone releasing hormone analogues, can inhibit the glycosylation of some proteins which are not enzymes, and can prevent some complications of diabetes mellitus.

The method for preparing D-lysine by adopting the biological enzyme racemization has the advantages of mild reaction conditions, strong stereoselectivity, high catalytic efficiency and the like, and the method becomes an important way for resolving or synthesizing chiral compounds.

Enzymatic resolution refers to the resolution of chiral compounds using enzymes isolated from microbial cells or directly using live cells of microorganisms containing the enzymes, the enzymatic resolution is unique in that the active center of the enzyme protein is an asymmetric environment composed of L-amino acidsOne enantiomer in the D-lysine is combined and subjected to a certain reaction to be changed into another compound with a property which is greatly different from that of the enantiomer, so that the aim of separating the two enantiomers is fulfilled, scientific research workers in China also make a contribution in related fields, for example, Qian Shao pine and the like firstly acetylate (D L) -theanine to generate (D L) -N-acetyl theanine, then aspergillus oryzae aminoacylase is used for separating (D L) -N-acetyl theanine to obtain L-theanine with the yield of 85 percent and the optical purity of 98 percent, Huangguanhua and the like complete the process of preparing D-phenylalanine by enzymatic separation of (D L) -phenylalanine by selective hydrolysis of N-phenylacetyl- (D L) -phenylalanine in the presence of immobilized penicillin acylase (IPA-750), the yield of 67 percent and the optical purity of 91 percent, the research on the enzymatic separation of D-lysine is less at present, Liu Yitai and the like firstly adopt a chemical lysine racemization method to convert L-D64-furcellarase into D by a lysine method, and then adopt Hawai bacterium to prepare Hawai by the melissa with the property difference of D-lysineHafniaalvei) The D-lysine decarboxylase in the method is used for stereoselective biotransformation of D L-lysine to obtain 8.5 g D-lysine from 30 g D L-lysine with the yield of 28%, Takahashi and the like utilize a chemical biological coupling method, L-lysine hydrochloride is used as a raw material, D L-lysine reaction liquid with the mass concentration of 100 g/L is obtained in a chemical racemization mode, L-lysine is directly degraded by oxygenase and deaminase, finally, 47 g/L of D-lysine remains in the mixed reaction liquid, and the yield of D-lysine after crystallization and separation is 38%.

So far, no D-lysine production method has been reported, which is characterized in that DapA and L YR are separately expressed in two cells, a mixed fermentation mode is utilized, so that the accumulation of intermediate products is reduced, the yield of D L-lysine is improved, and a final product is obtained by an enzymatic resolution method.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for producing D-lysine by mixed fermentation based on an enzymatic resolution method, the method realizes that L-lysine does not compete for glucose with D L-lysine synthesis bacteria, and the D-lysine prepared by combining the enzymatic resolution method has mild conditions and few intermediate products, and is suitable for large-scale industrial production.

A mixed bacteria fermentation production method of D-lysine based on an enzymatic resolution method comprises the following steps:

step 1, constructing a recombinant strain E.coli B L-pTrc 99 a-DapA;

step 2, constructing a glucose metabolism defective strain of E.coli B L-pET 28 a-L YR;

step 3, selecting recombinant strains E.coli B L-pTrc 99a-DapA and E.coli B L-pET 28 a-L YR, adding the recombinant strains into a fermentation culture medium, using glucose and glycerol as carbon sources, culturing until OD is about 0.6, inducing by IPTG, and continuously culturing to produce D L-lysine;

and 4, converting L-lysine in the D L-lysine into 1, 5-pentanediamine by using crude enzyme liquid containing lysine decarboxylase, thereby obtaining the D-lysine.

The improvement is that in the step 1, the E.coli B L-pTrc 99a-DapA construction method comprises the steps of selecting a primer, copying dihydropicolinic acid synthase on Escherichia coli MG1655 through PCR, connecting with a vector pTrc99a, transferring into a cloning vector Trans1-T1, primarily screening through a L B plate, selecting a point, carrying out colony PCR verification, and sequencing.

The improvement is that the E.coli B L-pET 28 a-L YR glucose metabolism deficient strain in the step 2 is constructed by the method that the strain is derived fromP. mirabilisThe gene L YR of BCRC10725 was codon-optimized and then transferred to pET28a synthesized by Jinwei Zhi CoNcoI /XhoTransferring a cloning vector Trans1-T1 between the I enzyme cutting sites, primarily screening by a L B plate, selecting the sites, carrying out colony PCR verification and sequencing.

As an improvement, the E-coli B L-pET 28 a-L YR glucose metabolism deficient strain construction method in the step 2

The method comprises knocking out phosphotransferase system (PTS) of Escherichia coli B L21 (DE3) by Crispr-Cas9 editing technologyptsG、ptsH、ptsI、crrAndglkthe gene coded protein is obtained by transforming the constructed plasmid pET28 a-L YR into the competence of B L21 (DE3) of glucose metabolism defect, thus obtaining the E.coli B L-pET 28 a-L YR glucose metabolism defect strain.

As a modification, the recombinant strains e.coli B L-pTrc 99a-DapA and e.coli B L-pET 28 a-L YR in step 3 had a cell OD ratio of 3: 1.

As a refinement, the molar ratio of the addition of glucose and glycerol in step 3 is 6: 1.

as a refinement, the induction temperature in step 3 is 35 ℃.

In the improvement, the final concentration of the crude enzyme solution containing lysine decarboxylase in the step 4 is 1mg/m L.

Has the advantages that:

compared with the prior art, the method for producing D-lysine by mixed fermentation has the advantages that:

1. knockdown of the phosphotransferase System (PTS) Using the Crispr-Cas9 techniqueptsG、ptsH、ptsI、crrAndglkthe gene codes protein, so that L-lysine does not compete with D L-lysine synthetic bacteria for glucose, thereby improving the yield of D L-lysine and reducing the generation of byproducts.

2. The enzymatic resolution method is used for resolving D L-lysine to prepare high-concentration D-lysine, namely the advantages of mild reaction conditions, strong stereoselectivity, high catalytic efficiency and the like in the resolution process are achieved.

3. Compared with the method of singly utilizing E.coli B L-pET 28 a-L YR to ferment and produce by L-lysine, the method reduces the production cost and improves the molar yield of the D-lysine by 35 percent.

Drawings

FIG. 1 shows the optimized OD inoculation ratio at OD600 for E.coli B L-pTrc 99a-DapA and E.coli B L-pET 28 a-L YR 1;

FIG. 2 shows the optimization of molar concentration ratio of glucose and glycerol in the initial culture of mixed bacteria of E.coli B L-pTrc 99a-DapA and E.coli B L-pET 28 a-L YR 1;

FIG. 3 shows the optimized induction temperature for mixed culture of E.coli B L-pTrc 99a-DapA and E.coli B L-pET 28 a-L YR 1;

FIG. 4 shows the effect of the concentration of crude lysine decarboxylase on the yield of high-purity D-lysine.

Detailed Description

The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.

In the experiment, Escherichia coli MG1655, Escherichia coli Trans1-T1, Escherichia coli B L21 (DE3), plasmid pTrc99a and plasmid pET28a are all commercial products and can be purchased conventionally, and the construction method of engineering bacteria Escherichia coli AST3 is disclosed in application No. 2020101559422 and is named as a construction method of glucose metabolism defective strains and a patent of producing 1, 5-pentanediamine by mixed fermentation of the strains.