CN109593702B

CN109593702B - Method for synthesizing L-phenyllactic acid by whole cell transformation of genetic engineering strain

Info

Publication number: CN109593702B
Application number: CN201910044513.5A
Authority: CN
Inventors: 夏雨; 茅菁菁; 王周平; 刘祖河; 刘琦
Original assignee: Jiangnan University
Current assignee: Xinghua Green Biological Preparation Co ltd
Priority date: 2019-01-17
Filing date: 2019-01-17
Publication date: 2020-10-09
Anticipated expiration: 2039-01-17
Also published as: CN109593702A

Abstract

The invention discloses a method for synthesizing L-phenyllactic acid by whole cell transformation of a genetic engineering strain, and particularly relates to a method capable of realizing cofactor NAD⁺A gene engineering strain for coexpressing phenylalanine dehydrogenase and L-hydroxyisocaproate reductase with NADH self-circulation, a method for synthesizing L-phenyllactic acid by converting a substrate L-phenylalanine through whole cells, belonging to the technical field of biological engineering. The L-phenyllactic acid is synthesized by the whole cell transformation of a recombinant strain E.coli BL21(DE3)/pET28a-pdh-ldh, and the whole cell transformation rate can reach 88.9-95.6% under the action of adding a surfactant. The method realizes cofactor NAD⁺And NADH self-circulation, reduces the addition of the cofactor, reduces the production cost, and has wide application prospect in the field of industrial synthesis of L-phenyllactic acid.

Description

Method for synthesizing L-phenyllactic acid by whole cell transformation of genetic engineering strain

Technical Field

The invention relates to a method for synthesizing L-phenyllactic acid by whole cell transformation of a genetic engineering strain, in particular to a method capable of realizing cofactor NAD⁺A gene engineering strain for coexpressing phenylalanine dehydrogenase and L-hydroxyisocaproate reductase with NADH self-circulation, a method for synthesizing L-phenyllactic acid by converting a substrate L-phenylalanine through whole cells, belonging to the technical field of biological engineering.

Background

L-phenyllactic acid (L-PLA), namely L-2-hydroxy-3-phenylpropionic acid, is one of two chiral enantiomers of PLA, naturally coexists with the chiral enantiomer D-PLA in lactic acid bacteria fermentation products and honey, has special biological activity, and can be widely applied to the fields of chemical industry, medicines, pesticides, biosynthesis and the like as a chiral intermediate.

The L-PLA not only has an inhibiting effect on various food-borne pathogenic bacteria, but also can inhibit the growth of most gram-positive bacteria and gram-negative bacteria, and prolongs the shelf life of food. L-PLA is a small molecular organic acid, has high stability and strong hydrophilicity, is easy to diffuse in food and feed, is a metabolite of L-phenylalanine, has no toxicity to human and animal cells, and has great development potential in food preservation.

The synthesis of the L-phenyllactic acid mainly adopts a chemical method and a biological method, the chemical method has complex synthetic steps, needs organic chemical reagents and causes certain pollution to the environment. In comparison, the biological preparation method of the L-phenyllactic acid has the advantages of mild reaction conditions, simple process and no need of chiral resolution. Biological methods are also classified into microbial fermentation, enzymatic catalysis, and whole-cell transformation. The microbial fermentation method has long fermentation time, needs product separation and has complex operation. The enzyme catalysis method needs to consume coenzyme I, has high cost and complex enzyme purification operation, and is not suitable for industrial production. The whole-cell conversion method avoids the separation and purification of enzyme, provides a stable cell environment for enzyme catalytic reaction, has shorter conversion time than a fermentation method, and has great potential in the field of industrial synthesis of L-phenyllactic acid.

At present, phenylalanine is used as a substrate to convert and synthesize phenyllactic acid and realize cofactor NAD⁺And NADH circulation, at least three enzymes are required, such as amino acid deaminase and lactate dehydrogenase for converting phenylalanine to synthesize phenyllactic acid, glucose dehydrogenase is added into the system, and the cofactor NAD is realized under the action of the lactate dehydrogenase and the glucose dehydrogenase⁺And the circulation of NADH. Therefore, the synthesis method of the L-phenyllactic acid is simple and convenient to operate, low in cost, high in conversion rate and suitable for industrial production, and has important application value for industrially preparing the L-phenyllactic acid.

Disclosure of Invention

The first purpose of the invention is to provide a genetic engineering bacterium, which co-expresses phenylalanine dehydrogenase (PheDH) and L-hydroxyisocaproate reductase (L-HicDH), wherein the phenylalanine dehydrogenase contains an amino acid sequence shown in SEQ ID NO.1, and the L-hydroxyisocaproate reductase contains an amino acid sequence shown in SEQ ID NO. 2.

In one embodiment of the present invention, e.coli BL21(DE3) is used as a host.

In one embodiment of the present invention, a pET series vector is used as an expression vector.

The second purpose of the invention is to provide a method for constructing the gene engineering bacteria, the gene coding phenylalanine dehydrogenase, the gene coding L-hydroxyisocaproate reductase and the vector are connected by enzyme digestion to obtain recombinant plasmids, and the recombinant plasmids are transferred into host cells.

In one embodiment of the invention, the genetically engineered bacterium is escherichia coli e.coli BL21(DE3)/pET28a-pdh-ldh, and is characterized in that a coexpression plasmid pET28a-pdh-ldh of phenylalanine dehydrogenase gene (pdh) from Bacillus badius and L-hydroxyisocaproate reductase gene (ldh) from Lactobacillus paracasei (Lactobacillus paracasei) is constructed by taking pET28a as a vector, and is transformed into e.coli BL21(DE3) to obtain a coexpression strain e.coli BL21(DE3)/pET28a-pdh-ldh, and the coexpression of the two enzymes in escherichia coli is realized.

In one embodiment of the present invention, the gene encoding phenylalanine dehydrogenase is represented by SEQ ID NO. 3.

In one embodiment of the present invention, the gene encoding L-hydroxyisocaproate reductase is set forth in SEQ ID NO. 3.

The third purpose of the invention is to provide a method for expressing the genetic engineering bacteria, which comprises the steps of inoculating the genetic engineering bacteria into an LB liquid culture medium, culturing at 35-38 ℃ and 150-170 rpm for 10-14 h to serve as seed liquid; inoculating the seed liquid into LB culture medium with the inoculation amount of 1.0-5.0%, and performing shaking culture until OD is reached₆₀₀0.4-0.6, adding an inducer IPTG, and culturing for 10-20 h at 20-25 ℃.

The fourth purpose of the invention is to provide a method for producing phenyllactic acid, which takes L-phenylalanine as a substrate and the genetically engineered bacterium as a biocatalyst.

In one embodiment of the present invention, the reaction is carried out at 22-26 ℃ and 200-220 rpm for 7-15 hours.

In one embodiment of the present invention, the mass ratio of the wet cells to the substrate is (3-24): 1.

In one embodiment of the invention, triton-X-100 with the final concentration of 0.005-0.2% (V/V), Tween-20 with the final concentration of 0.005-0.2% (V/V) or CTAB with the final concentration of 0.005-0.2% (W/V) is added into the reaction system.

The fifth purpose of the invention is to provide the application of the method for producing phenyllactic acid in the fields of chemical industry, pharmacy or biosynthesis.

The invention constructs a plasmid pET28a-pdh-ldh for coexpression of phenylalanine dehydrogenase and L-hydroxy acid reductase, and transfers the plasmid into escherichia coli to obtain a recombinant strain E.coli BL21(DE3)/pET28 a-pdh-ldh. The L-phenyllactic acid is synthesized by whole cell transformation of a recombinant strain E.coli BL21(DE3)/pET28a-pdh-ldh, the conversion rate of the L-phenyllactic acid synthesized by the whole cell transformation of 20.0 g/L-160.0 g/L is 30.0-70.0%, and the conversion rate can be improved to 89.6-95.6% under the condition of adding a surfactant. The invention only needs two enzymes to solve the problem of cofactor NAD⁺Compared with the circulation problem of NADH, the method reduces the types of enzymes required by the circulation of the cofactor, reduces the addition amount of the cofactor, reduces the production cost and has wide application prospect in the field of industrial synthesis of L-phenyllactic acid.

Drawings

FIG. 1: the procedure for constructing the co-expression plasmid pET28 a-pdh-ldh.

FIG. 2: SDS-PAGE picture of the whole bacterial liquid after the induction expression of the recombinant gene engineering bacteria.

FIG. 3: the reaction process of converting and synthesizing L-phenyl lactic acid with L-phenylalanine as substrate.

FIG. 4: conversion rate of L-phenyllactic acid under different whole cell concentration conditions.

FIG. 5: conversion rate of L-phenyllactic acid under different surfactant conditions.

Detailed Description

(I) culture Medium

LB culture medium: 10.0g/L of peptone, 10.0g/L of sodium chloride and 5.0g/L of yeast powder.

(II) HPLC determination of L-phenyllactic acid content

Column: sunfire C18; flow rate: 1.0 mL/min; temperature: 30 ℃; the detection wavelength was 254 nm; mobile phase: acetonitrile (buffer A, containing 0.1% trifluoroacetic acid) and H₂O (buffer B, containing 0.1% trifluoroacetic acid); gradient: gradient wash from 95.0% water in 22 minTo 100.0% acetonitrile.

EXAMPLE 1 construction of the Co-expression plasmid pET28a-pdh-ldh

A phenylalanine dehydrogenase gene pdh and an L-hydroxyisocaproate reductase gene ldh were artificially synthesized. PCR amplification reaction (50.0. mu.L):_ddH₂35.5 μ L of O, 10.0 μ L of 5 × Phusion HF Buffer, 1.0 μ L of dNTPs, 1.0 μ L of pdh-up/ldh-up, 1.0 μ L of pdh-down/ldh-down, 1.0 μ L of template plasmid and 0.5 μ L of Phusion enzyme, reaction conditions of 1) pre-denaturation at 98 ℃ for 30s, 2) denaturation at 98 ℃ for 10s, 3) primer annealing at 68 ℃ (pdh)/58.5 ℃ (ldh for 30s, 4) primer extension at 72 ℃ for 35s (pdh)/50s (ldh) (repeating steps 2-4, cycle 30 times), and 5) extension at 72 ℃ for 7min, PCR products are verified by agarose gel electrophoresis, impurities are removed by using an agarose gel recovery kit, and the purified target gene is stored at 4 ℃ for later use.

TABLE 1 primers for amplifying genes pdh and ldh

Primer name	Primer sequences
		pdh-up	5’-GGGCCCCATATGAGCTTAGTAGAAAAAACATCC-3’
pdh-down	5’-GGGCCCGAATTCTTAGTTGCGAATATCCCATT-3’
		ldh-up	5’-GGGCCCGAATTCAAGGAGATATAATGGCACGTAAGATTGGAATTATCGG-3’
ldh-down	5’-GATCCCCTCGAGGAGTGTATCCACAATTTCGTCGA-3’

(2) pET28a-pdh-ldh Co-expression plasmid construction (FIG. 1) plasmid pET28a was used as an expression vector to construct a recombinant plasmid pET28a-pdh 1. plasmid pET28a and gene pdh were digested simultaneously with NdeI and EcoRI restriction enzymes, digestion system (30.0. mu.L) 10 × Buffer 3.0. mu.L, pET28a-pdh 5.0. mu.L, NdeI 1.0. mu.L, EcoRI 1.0. mu.L,_ddH₂and supplementing O to 30.0 mu L, carrying out water bath at 37 ℃ for 15min, and carrying out water bath at 80 ℃ for 5 min.

The product after enzyme digestion is treated with T₄The DNA was ligated with a ligase to obtain a recombinant plasmid pET28 a-pdh1. the ligation system (20.0. mu.L) was pET28a 3.0.0. mu.L, pdh gene fragment 9.0. mu.L, 5 × Buffer 4.0. mu.L, and T₄DNA ligase 1.0 μ L, sterile water make up to 20.0 μ L, water bath at 22 deg.C for 15min, ice bath at 4 deg.C overnight ligation.

The ligation product was transferred to E.coli DH 5. alpha. competent cells, and the resulting recombinant strain was named E.coli DH 5. alpha./pET 28a-pdh 1. A single colony of a transformant is picked from a plate, inoculated into an LB (containing 1.0mmol/L kanamycin) liquid culture medium for overnight culture, subjected to plasmid extraction and enzyme digestion, subjected to 1.0% agarose gel electrophoresis verification, and subjected to sequencing, so that the constructed recombinant plasmid is named as pET28a-pdh 1.

The recombinant plasmid pET28a-pdh1 and the gene ldh fragment were digested simultaneously with EcoRI and XhoI restriction enzymes, 10 × Buffer 3.0. mu.L, pET28a-pdh-ldh 5.0. mu.L, XhoI 1.0. mu.L, EcoRI 1.0. mu.L,_ddH₂and supplementing O to 30.0 mu L, carrying out water bath at 37 ℃ for 15min, and carrying out water bath at 80 ℃ for 5 min.

The product after enzyme digestion is treated with T₄The DNA was ligated with a ligase to obtain a recombinant plasmid pET28 a-pdh-ldh. ligation scheme (20.0. mu.L), pET28a-pdh 14.0. mu.L, ldh gene fragment 5.0. mu.L, 5 × Buffer 4.0. mu.L, T₄DNA ligase 1.0 μ L, sterile water make up to 20.0 μ L, water bath at 22 deg.C for 15min, ice bath at 4 deg.C overnight ligation.

The ligation product was transferred to E.coli DH 5. alpha. competent cells, and the resulting recombinant strain was named E.coli DH 5. alpha. (pET28 a-pdh-ldh). Single colony of transformant is picked from the plate and inoculated into LB (containing 1.0mmol/L kanamycin) liquid culture medium for overnight culture, after plasmid extraction and enzyme digestion, 1.0 percent agarose gel electrophoresis verification is carried out, bacterial liquid after successful verification is sequenced, and the constructed co-expression plasmid is named as pET28 a-pdh-ldh.

Example 2 construction and inducible expression of the Co-expression Strain E.coli BL21(DE3)/pET28a-pdh-ldh

After activating and culturing the recombinant strain E.coli DH5 alpha/pET 28a-pdh-ldh, extracting a plasmid, transferring a co-expression plasmid pET28a-pdh-ldh into E.coli BL21(DE3) competent cells, and naming the obtained co-expression strain E.coli BL21(DE3)/pET28 a-pdh-ldh.

The co-expression strain E.coli BL21(DE3)/pET28a-pdh-ldh was inoculated into LB liquid medium (containing 1.0mmol/L kanamycin) and cultured overnight at 37 ℃ and 160rpm as a seed solution. The seed solution was inoculated at an inoculum size of 1.0% into 100.0mL of LB (containing 1.0mmol/L kanamycin) liquid medium, and shake-cultured to OD₆₀₀0.6, adding 0.8mmol/L isopropyl- β -D-thiogalactoside (IPTG), culturing at 22 ℃ for 14h, centrifuging at 4 ℃ and 8000rpm for 10min to remove supernatant, collecting thalli, washing the thalli twice with 0.85% physiological saline for later use, and identifying the thalli by SDS-PAGE electrophoresis (figure 2), wherein lane 1 and lane 2 are non-induced whole bacterial liquid, lane 3 is IPTG induced whole bacterial liquid, and the sizes of two bands in lane 3 respectively accord with the sizes of target proteins of phenylalanine dehydrogenase (PheDH) and L-hydroxyisocaproate reductase (L-HicDH), which indicates that the two target proteins are successfully co-expressed in E.coli BL21(DE 3).

EXAMPLE 3 Synthesis of L-phenyllactic acid by transformation of the Co-expression Strain E.coli BL21(DE3)/pET28a-pdh-ldh Whole cells

The conversion and synthesis of L-phenyllactic acid by taking L-phenylalanine as a substrate comprises two steps of reactions: deamination and reduction (figure 3). And (3) deamination reaction: phenylalanine dehydrogenase deaminates L-phenylalanine to phenylpyruvic acid with concomitant NAD⁺Conversion to NADH. Reduction reaction: l-hydroxyisocaproate reductase reduces phenylpyruvic acid to L-phenyllactic acid accompanied by NADH to NAD⁺Is performed. Therefore, the conversion synthesis of L-phenyllactic acid by the tandem reaction of phenylalanine dehydrogenase and L-hydroxyisocaproate reductase can realize the cofactor NAD⁺And self-circulation of NADH.

Whole cell transformation System (1.0 mL): 40.0mmol/L L-phenyllactic acid, 10.0mmol/L NAD⁺20.0-160.0 g/L whole cell, 150.0mmol/L ammonium formate buffer (pH 7.0). The system was reacted at 200rpm for 12h at 25 ℃. The sample was then centrifuged at 4000rpm, the supernatant was removed, and the supernatant was passed through a 0.22 μm membrane and subjected to HPLC.

The results show (FIG. 4) that the abscissa is the concentration of the coexpression strain E.coli BL21(DE3)/pET28a-pdh-ldh whole cells and the ordinate is the conversion of L-phenyllactic acid. The coexpression strain E.coli BL21(DE3)/pET28a-pdh-ldh is transformed into L-phenyllactic acid by whole cells, and the transformation rate of the L-phenyllactic acid synthesized by 20.0g/L to 160.0g/L of the whole cells is 30.0 percent to 70.0 percent.

Different surfactants were added to the conversion system: Triton-X-100 (Triton-X-100) (the final concentration of the reaction system is 0.005-0.2% V/V), Tween-20 (Tween-20) (the final concentration of the reaction system is 0.005-0.2% V/V) or Cetyl Trimethyl Ammonium Bromide (CTAB) (the final concentration of the reaction system is 0.005-0.2% W/V), the co-expression strain E.coli BL21(DE3)/pET28a-pdh-ldh is transformed into L-phenyllactic acid by whole cells under the same conditions, and the transformation rate of synthesizing the L-phenyllactic acid by the transformation of the whole cells of 20.0-160.0 g/L can reach 88.9-95.6% (figure 5). The two enzymes selected by the invention can solve the problem of cofactor NAD in the whole cell transformation process⁺And the recycling problem of NADH, reduces the addition amount of the cofactor, reduces the production cost of the L-phenyllactic acid, and has great potential in the field of industrial production of the L-phenyllactic acid.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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Claims

1. A genetically engineered bacterium is characterized in that phenylalanine dehydrogenase and L-hydroxyisocaproate reductase are co-expressed, the amino acid sequence of the phenylalanine dehydrogenase is shown as SEQ ID NO.1, and the amino acid sequence of the L-hydroxyisocaproate reductase is shown as SEQ ID NO. 2; said baseDue to engineering bacteria, theE.coliBL21(DE3) is the host.

2. The genetically engineered bacterium of claim 1, wherein the pET-series vector is an expression vector.

3. A method for constructing the genetically engineered bacteria of any one of claims 1-2, comprising the steps of carrying out enzyme digestion on a gene coding phenylalanine dehydrogenase, a gene coding L-hydroxyisocaproate reductase and a vector to obtain a recombinant plasmid, and transferring the recombinant plasmid into a host cell.

4. A method for expressing the genetically engineered bacteria of any one of claims 1-2, characterized in that the genetically engineered bacteria are inoculated into LB liquid culture medium, cultured at 35-38 ℃ and 150-170 rpm for 10-14 h, and used as seed liquid; inoculating the seed liquid into LB culture medium with the inoculation amount of 1.0-5.0%, and performing shaking culture until OD is reached₆₀₀0.4-0.6, adding inducer IPTG, and culturing at 20-25 ℃ for 10-20 h.

5. A method for producing L-phenyllactic acid, which is characterized in that L-phenylalanine is used as a substrate, and the genetically engineered bacterium as claimed in any one of claims 1 to 3 is used as a biocatalyst.

6. The method of claim 5, wherein the reaction is carried out at a temperature of 22-26 ℃ and a speed of 200-220 rpm for 7-15 hours.

7. The method according to claim 5, wherein the mass ratio of the wet cells to the substrate is (3-24): 1.

8. the method according to claim 6, wherein the mass ratio of the wet cells to the substrate is (3-24): 1.

9. the method according to any one of claims 5 to 8, wherein triton-X-100 is added to the reaction system at a final concentration of 0.005% to 0.2% (V/V), Tween-20 is added to the reaction system at a final concentration of 0.005% to 0.2% (V/V), or CTAB is added to the reaction system at a final concentration of 0.005% to 0.2% (W/V).

10. Use of the method of any one of claims 5-9 for the preparation of L-phenyllactic acid.