CN112941003A

CN112941003A - Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells

Info

Publication number: CN112941003A
Application number: CN202110418599.0A
Authority: CN
Inventors: 周哲敏; 刘中美; 崔睿智; 朱小青; 周丽; 崔文璟; 郭军玲
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2021-06-11

Abstract

The invention discloses a method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells, belonging to the field of bioengineering. The invention obtains the recombinant strain which can catalyze maleic acid to generate L-alanine with high conversion rate by a whole cell through integrally establishing the recombinant strain which is coupled to express the maleate cis-trans isomerase and the L-aspartate beta decarboxylase and carrying out host strain transformation optimization on the recombinant strain. The recombinant strain can generate L-alanine by using maleic acid with relatively low price, almost no intermediate product fumaric acid and L-aspartic acid are accumulated, the conversion rate reaches over 96 percent, the highest production rate reaches 28.43 g/(L.h), and the highest yield reaches 261.2 g/L.

Description

Method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells

Technical Field

The invention relates to a method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cells, belonging to the field of bioengineering.

Background

L-Alanine (L-Alanine, L-Ala) is one of the smallest chiral molecules. It has wide application and great market development potential in the fields of food, medicine, chemical industry and the like. The flavoring agent is mainly used for biochemical research, tissue culture, liver function measurement and flavoring agent, can increase the flavoring effect of the flavoring agent, and can also be used as sour taste correcting agent to improve the sour taste of organic acid.

The current industrial methods for preparing L-alanine are mainly chemical synthesis and biological methods. In the chemical synthesis production, a propionic acid chlorination method is a common idea for synthesizing L-alanine, but the method has the defects of poor product quality, long synthesis route, low yield, high cost, serious environmental pollution and the like, and is not suitable for industrial production.

The biological method for preparing L-alanine becomes a commonly used method in industry; the biological method mainly comprises a catalytic method and a fermentation method, wherein the fermentation method mainly adopts glucose as a raw material for anaerobic fermentation production, and then the L-alanine product is obtained through separation and purification. The process has the advantages of cheap and easily-obtained raw materials, simple flow, difficult extraction, no enzymatic catalysis, strong stereoselectivity and the like. Compared with the prior art, the whole-cell catalytic conversion method has the advantages of strong stereoselectivity, high conversion rate, mild reaction conditions, small environmental pollution and the like, and the one-pot multi-enzyme cascade catalysis does not need the separation and purification of enzyme and the separation and purification of intermediate products, is convenient for the recovery and the reutilization of cells, and greatly reduces the cost, so the one-pot whole-cell catalytic conversion method has better application prospect.

However, in the prior art, the whole cell transformation and synthesis of L-alanine has the technical problems of high cost and complex process, for example, as described in the Chinese patent application publication No. CN109456928A, the recombinant Escherichia coli is constructed to obtain an L-aspartate beta-decarboxylase strain with high enzyme activity, the recombinant strain is subjected to shake flask fermentation, and the L-Asp-Na is used as a substrate to perform whole cell catalytic reaction to prepare the L-alanine. Although the yield of the final product L-alanine reaches more than 94 percent, the method adopts aspartic acid as a substrate, and the aspartic acid is expensive and is not beneficial to large-scale industrial production. Although the fermentation method is low in cost, the process operation is complex and long in time consumption, for example, the Chinese patent application publication No. CN109355242A describes that the yield of L-alanine prepared by adopting the glucose fermentation method reaches 171.0g/L after 55 hours of fermentation, two stages of fermentation are required in the fermentation process, and the fermentation process is relatively complex.

Therefore, finding a low-cost substrate and a genetically engineered bacterium capable of preparing L-alanine by adopting the low-cost substrate reaction become research hotspots and difficulties.

Disclosure of Invention

The technical problem is as follows:

the technical problem to be solved by the invention is as follows: provides a low-cost substrate for preparing L-alanine by reaction, and simultaneously provides a genetically engineered bacterium capable of preparing L-alanine by adopting the low-cost substrate reaction and a method for preparing L-alanine by whole-cell transformation.

The technical scheme is as follows:

in order to solve the technical problems, the invention provides a genetic engineering bacterium for coupling and expressing maleic acid cis-trans isomerase (from serratia marcescens) and L-aspartate beta decarboxylase (from pseudomonas P. dachunhae 21192), and host bacterium modification and optimization are carried out on the recombinant strain to obtain the genetic engineering bacterium capable of catalyzing maleic acid to generate L-alanine at a whole cell high conversion rate (the reaction principle is shown in figure 1).

The invention provides a genetic engineering bacterium, which takes E.coli BL21(DE3) with a knockout of a delta fumAC gene in a genome as an expression host (E.coli BL21(DE3) delta fumAC) and expresses maleic acid cis-trans isomerase and L-aspartic acid beta decarboxylase in a coupling manner.

In one embodiment of the present invention, the method for constructing the expression host e.coli BL21(DE3) Δ fumAC is disclosed in the article: the fumaric acid is efficiently synthesized by transforming the whole cells of the recombinant escherichia coli into the maleic acid [ J ]. the report of food and biotechnology 2016,35(12): 1323-.

In one embodiment of the invention, the genetically engineered bacterium is pRSF_Duet-1Is an expression vector.

In one embodiment of the invention, the amino acid sequence of the maleate cis-trans isomerase is shown in SEQ ID NO. 1.

In one embodiment of the invention, the amino acid sequence of the L-aspartate beta decarboxylase is shown as SEQ ID NO. 3.

In one embodiment of the invention, the nucleic acid sequence of the gene encoding the maleate cis-trans isomerase is shown in SEQ ID NO. 2.

In one embodiment of the invention, the nucleic acid sequence of the gene encoding the L-aspartate beta-decarboxylase is shown in SEQ ID NO. 4.

In one embodiment of the invention, T is also used₇The promoter-RBS sequence overexpresses the L-aspartate lyase AspA on the host genome.

In one embodiment of the present invention, the T is₇The nucleic acid sequence of the promoter-RBS sequence is shown as SEQ ID NO. 5.

In one embodiment of the present invention, the positional relationship between the maleate cis-trans isomerase and the L-aspartate beta decarboxylase on the carrier is as follows: expression vector-L-aspartate beta decarboxylase-maleate cis-trans isomerase.

The invention also provides a method for preparing L-alanine, which comprises the step of preparing L-alanine by using the genetic engineering bacteria or the enzyme generated by the genetic engineering bacteria as a catalyst and maleic acid as a substrate.

In one embodiment of the invention, the final concentration of the substrate maleic acid is 2-3 mol/L.

In one embodiment of the invention, the genetically engineered bacteria are added to the substrate in the form of a cell bacterial suspension, and the volume ratio of the cell bacterial suspension to the substrate solution is 2: 8.

in one embodiment of the present invention, the concentration OD of the genetically engineered bacteria is₆₀₀At least 20.

The invention also provides the application of the genetic engineering bacteria or the method in preparing products containing L-alanine.

Advantageous effects

(1) The invention provides a genetically engineered bacterium capable of efficiently expressing maleic acid cis-trans isomerase and L-aspartate beta decarboxylase, the recombinant strain can generate L-alanine by using relatively cheap maleic acid, and the production method is simple in process, low in cost and beneficial to subsequent large-scale industrial production.

(2) The L-alanine prepared by transforming the maleic acid by the whole cells of the gene engineering bacteria constructed by the invention has high yield and high production efficiency, and almost no intermediate products of fumaric acid and L-aspartic acid are accumulated; when 2M maleic acid is taken as a substrate, the reaction is complete within 6h, the yield reaches 170.6g/L, and the production rate reaches 28.43 g/(L.h); when 2.5M maleic acid is taken as a substrate, the reaction is complete within 10h, the yield reaches 220.02g/L, and the production rate reaches 22.02 g/(L.h); when 3M maleic acid is used as a substrate, the reaction is complete within 15h, the yield reaches 261.2g/L, the production rate reaches 17.41 g/(L.h), and the conversion rate reaches over 96 percent.

Drawings

FIG. 1: schematic diagram of three-enzyme cascade reaction for synthesizing L-alanine by maleic acid.

FIG. 2: different strategies for MaiA coupled with ASD expression.

FIG. 3: enzyme digestion verification results of the recombinant plasmids; wherein, M: marker; 1: verifying the Not I single enzyme digestion of the pAM recombinant plasmid; 2: nde I and Xho I double enzyme digestion verification of the pAM recombinant plasmid; 3: double enzyme digestion verification of Not I and EcoR I of the pAM recombinant plasmid; 4: HindIII single enzyme digestion verification of pMA recombinant plasmid; 5: performing double enzyme digestion verification on BamH I and Hind III of the pMA recombinant plasmid; 6: NdeI and XhoI double enzyme digestion verification of pMA recombinant plasmid.

FIG. 4: SDS-PAGE results of different coupled expressions; wherein, M: marker; 1: pAM was not induced; 2: pAM supernatant expression results; 3: pAM precipitate expression results; 4: pMA was not induced; 5: pMA supernatant expression results; 6: pMA precipitation expression results.

FIG. 5: and comparing the capabilities of two strains expressed in different tandem modes in catalyzing maleic acid under the condition of pH 6-8.

FIG. 6: different cells catalyze 2M maleic acid results; wherein, fig. 6 a: coli BL21(DE3)/pRSF_Duet-l-ASD-MaiA whole cell catalysis 2M maleic acid results; FIG. 6 b: coli BL21(DE3) Δ fumAC/pRSF_Duet-l-ASD-MaiA whole cell catalysis 2M maleic acid results; FIG. 6 c: coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lASD-MaiA whole-cell catalysis 2M maleic acid results.

FIG. 7: coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lASD-MaiA whole cell catalysis 2.5M maleic acid results.

FIG. 8: coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lASD-MaiA whole-cell catalysis 3M maleic acid results.

FIG. 9: comparing the capacities of different ASD enzymes in whole cells to catalyze fumaric acid to generate alanine under the condition of pH 5.0.

Detailed Description

The maleic acid referred to in the examples below was purchased from alatin.

The media involved in the following examples are as follows:

LB liquid medium: 10g/L peptone, 5g/L yeast extract, L0 g/L NaCl.

LB solid medium: 0.2g/L agar was added to LB liquid medium.

2YT liquid medium: 16g/L peptone, 10g/L yeast extract and 5g/L NaCl.

The detection methods referred to in the following examples are as follows:

detection methods for maleic acid, fumaric acid, L-aspartic acid and L-alanine: the concentrations of maleic acid, fumaric acid, L-aspartic acid and L-alanine were determined by High Performance Liquid Chromatography (HPLC).

HPLC detection conditions of maleic acid and fumaric acid: column Spursil 5. mu. m C18 (250X 4.6mM,5 μm), mobile phase pH 2.5, 25mM K₂HPO₄The flow rate of the solution is 1mL/min, the column temperature is 40 ℃, the wavelength of an ultraviolet detector is 210nm, and the sample injection amount is 10 mu L; the detection of L-aspartic acid and L-alanine is carried out by derivatizing with phenyl iso-sulfate (PITC), and connecting with benzene at amino terminalAnd the ring is convenient to separate. The derivation method comprises the following steps: and adding 250 mu L of 1M triethylamine-acetonitrile solution and 250 mu L of 0.1M PITC-acetonitrile solution into 500 mu L of reaction solution, oscillating, uniformly mixing, and reacting for 45min in a dark place. And after derivatization is finished, adding 750 mu L of normal hexane to terminate the reaction, oscillating for 30s to extract out residual derivatization reagent, standing, absorbing lower-layer solution after reaction liquid is obviously layered, and filtering by using a 0.22mm needle head type organic filter membrane. HPLC detection conditions: the column Diamonsil 5. mu. m C18(2) (250X 4.6mm,5 μm) was detected by gradient elution. The mobile phase A is an 80% acetonitrile solution, and the mobile phase B is a 97:3 0.1M sodium acetate-acetonitrile solution; the gradient elution conditions were: reducing the content of the mobile phase B from 95% to 65% in 0-35 min; the time is 35-40 min, and the mobile phase B rises from 65% to 95%; and (4) keeping the concentration of the mobile phase B unchanged for 40-45 min. The detection temperature is 40 ℃, and the detection wavelength is 254 nm.

Example 1: selection of L-aspartate beta-decarboxylase

The L-aspartate beta decarboxylase is adjusted to other 3 sources of L-aspartate beta decarboxylase, which are respectively as follows: ArASD (hereinafter referred to as ASD-1) of AP019740.1 in GenBank, coding L-aspartate beta decarboxylase Pd21192ASD (hereinafter referred to as ASD-2) with a nucleic acid sequence shown as SEQ ID NO.4, pET28a-Pd19121ASD (hereinafter referred to as ASD-3) of WP _016451742.1 in GenBank;

recombinant strains E.coli BL21(DE3)/pET28a-ASD-1, E.coli BL21(DE3)/pET28a-ASD-2 and E.coli BL21(DE3)/pET28a-ASD-3 are synthesized by genes of Jinweizhi company and stored in a storage tube, and the prepared single colony is inoculated in 5mL LB liquid culture medium with 50mg/mL antibiotic concentration and cultured at 37 ℃ and 200rpm for 8 hours to prepare seed liquid; then, the seed solution was transferred to 250mL shake flasks containing 50mL of 2YT medium at an inoculum size of 2% (v/v), respectively, and the antibiotic kanamycin concentration was 50mg/mL, cultured at 37 ℃ and 200rpm to OD₆₀₀0.6-0.8, then adding IPTG with the final concentration of 0.2mmol/L to induce expression for 20 hours at 30 ℃.

The prepared cells inducing expression were collected, resuspended in 50mM phosphate buffer pH 5.0, and diluted to OD₆₀₀10, then 20% (by volume) of resting cells and 80% (by volume) of the substrate fumaric acid, the final concentration of which is the fumaric acidThe alanine production amount is 0.2M, the pH value is 5.0, the reaction system is 25mL, the catalytic reaction is carried out in a shaking table at the temperature of 37 ℃ at 200r/min, the reaction is carried out for 4h, and the sample is taken and the inactivation thin-plate chromatography is carried out to detect the alanine production amount.

The results shown in FIG. 9 indicate that Pd21192ASD catalyzed by fumaric acid at pH 5.0 produced the largest amount of alanine after 4 hours, which was better than the other two ASDs.

Therefore, the gene encoding L-aspartate beta decarboxylase Pd21192ASD, the nucleic acid sequence of which is shown in SEQ ID NO.4, was selected for subsequent studies.

Example 2: construction of recombinant vector for coupled expression of maleate cis-trans isomerase and L-aspartate beta decarboxylase

The method comprises the following specific steps:

(1) selecting enzyme cutting sites according to a gene which has a nucleic acid sequence shown as SEQ ID NO.2 and codes a maleate cis-trans isomerase, a gene which has a nucleic acid sequence shown as SEQ ID NO.4 and codes L-aspartate beta decarboxylase and a carrier, and designing primers shown as a table 1:

table 1: design of enzyme-digested ligation primer

Note that the restriction sites are underlined

(2) Chemically synthesizing a gene MaiA with a nucleic acid sequence shown as SEQ ID NO.2 and encoding maleic acid cis-trans isomerase, and a gene ASD with a nucleic acid sequence shown as SEQ ID NO.4 and encoding L-aspartic acid beta decarboxylase, and carrying out enzyme and plasmid vector pRSF by using Nde I, Xho I, EcoR I, Not I, BamH I and Hind III enzymes_Duet-1Performing double enzyme digestion for 4h, and then recovering and purifying the gel of the enzyme digestion product;

(3) measuring the concentrations of the recovered gene and vector fragment with a nucleic acid quantitative analyzer, mixing the gene fragment and vector fragment at a ratio of 3:1, adding T4DNA ligase, and connecting at 16 deg.C overnight; the ligation products were transformed into competent cells of JM109 and then plated on LB solid medium carrying the corresponding kanamycin antibiotic resistance;

(4) first colony PCR assayThen selecting single colony to 5mL LB liquid medium with antibiotic concentration of 50mg/mL, culturing at 37 deg.C and 200rpm for 8h, performing double enzyme digestion verification on the quality-improved granule (as shown in figure 3), sequencing the correctly verified bacteria, and obtaining the recombinant plasmid pRSF_Duet-l-ASD-MaiA and pRSF_Duet-lMaiA-ASD (specific attachment is shown in fig. 2).

Example 3: construction of recombinant strain for coupled expression of maleate cis-trans isomerase and L-aspartate beta decarboxylase and expression of enzyme

The method comprises the following specific steps:

(1) the recombinant plasmid pRSF prepared in example 1 was used_Duet-l-ASD-MaiA and pRSF_Duet-lE.coli BL21(DE3) Δ fumAC/pRSF are respectively prepared by transforming E.coli BL21(DE3) Δ fumAC competent cells into an expression host E.coli BL21, coating the cells on a LB solid culture medium with kanamycin resistance as a vector, and culturing the cells at 37 ℃ for 12 hours_Duet-lColi BL21(DE3) Δ fumAC/pRSF_Duet-l-MaiA-ASD。

(2) The single colony prepared in the step (1) is selected and inoculated in 5mL LB culture medium with 50mg/mL antibiotic concentration, cultured for 8 hours at 37 ℃ and 200rpm, and then respectively transferred to a 250mL shaking flask filled with 50mL 2YT culture medium by 2% (v/v) inoculum concentration, 50mg/mL antibiotic kana concentration, cultured at 37 ℃ and 200rpm to OD₆₀₀0.6-0.8, then adding IPTG with the final concentration of 0.2mmol/L to induce expression for 20 hours at 25 ℃.

(3) The same amount of induction expression cells are taken, the cells are resuspended, and the crude enzyme solution SDS-PAGE electrophoresis obtained by ultrasonic disruption is used for analyzing the expression condition of the target protein (as shown in figure 4), when the MaiA and the ASD are expressed in series, the expression levels of the two enzymes are different in different series connection modes.

The results show that: pRSF_Duet-lASD-MaiA expresses more ASD and less MaiA; pRSF_Duet-lMaiA is expressed more in MaiA-ASD, while ASD is expressed less.

Example 4: recombinant strain whole cell conversion maleic acid to produce L-alanine

The method comprises the following specific steps:

coli BL21(DE3) Δ fumAC/pRSF was collected from the recombinant cells prepared in example 2_Duet-lColi BL21(DE3) Δ fumAC/pRSF_Duet-l-MaiA-ASD, collecting cell concentrated OD by centrifugation at 6000rpm for 10min₆₀₀For 8, resuspend with maleic acid substrate (final concentration 1mol/L) of different pH, reaction system is 5mL, catalyze the reaction in shaker at 37 ℃ at 200r/min, sample inactivation after reaction for 4h, thin plate chromatography plate assay of aspartic acid and alanine content, two strains of different pH conditions under the catalytic 1M maleic acid results comparison, the results are shown in figure 4 and figure 5.

The results show that E.coli BL21(DE3) Δ fumAC/pRSF_Duet-lColi BL21(DE3) Δ fumAC/pRSF_Duet-lThe MaiA-ASD all have the best catalytic effect at the pH value of 7.5, because the optimal pH value catalyzed by the maleic acid cis-trans isomerase MaiA is 8, the optimal pH value catalyzed by the aspartate lyase AspA is 8.5, and the optimal pH value catalyzed by the aspartate beta decarboxylase ASD is 5, so that the common catalytic efficiency of the three enzymes is the highest under the environment of the pH value of 7.5; coli BL21(DE3) Δ fumAC/pRSF_Duet-lCatalytic effect of ASD-MaiA compared to another tandem form of E.coli BL21(DE3) Δ fumAC/pRSF_Duet-lThe MaiA-ASD has high catalytic effect because the enzyme activity of the ASD is lower under the alkaline condition in the catalytic process, so that the catalytic capability of synthesizing alanine from maleic acid can be improved to a certain extent by enhancing the expression of the ASD.

Example 5: recombinant strain whole cell conversion maleic acid to produce L-alanine

(1) Construction of E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA

T with nucleotide sequence shown as SEQ ID NO.5₇The promoter-RBS sequence was synthesized by Jinzhi corporation before insertion of the L-aspartate lyase AspA gene on the genome of E.coli BL21(DE3) Δ fumAC chromosome, to prepare E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA.

(2) Recombinant plasmid pRSF_Duet-lASD-MaiA is transformed into competent cells of expression hosts E.coli BL21(DE3), E.coli BL21(DE3) delta fumAC and E.coli BL21(DE3) delta fumAC-T7-RBS/AspA respectively to prepare recombinant strains: coli BL21(DE3)/pRSF_Duet-l-ASD-MaiA，E.coli BL21(DE3)△fumAC/pRSF_Duet-l-ASD-MaiA，E.coli BL21(DE3)ΔfumAC-T7-RBS/AspA/pRSF_Duet-l-ASD-MaiA; then spread on the LB solid culture medium of the corresponding kanamycin resistance of the carrier; selecting single bacterium to perform induction expression under OD₆₀₀Growing to 0.6-0.8, adding IPTG with the final concentration of 0.2mol/L, and culturing for 20 hours at the temperature of 25 ℃.

(3) Firstly, preparing a maleic acid solution with the pH of 7.5, and adjusting the pH of the maleic acid to 7.5 by using ammonia water in the process. Collecting the cells induced to express in step (2), resuspending the cells in 50mM phosphate buffer, pH7.5, and diluting to OD ₆₀₀20, mixing resting cells of 20 percent (volume ratio) and maleic acid of 80 percent (volume ratio) as a substrate, wherein the final concentration of the maleic acid is 2M, the reaction system is 50mL, catalyzing the reaction in a shaker at 200r/min and 37 ℃, and sampling every 2h to detect the contents of the maleic acid, the fumaric acid, the L-aspartic acid and the L-alanine in the reaction solution. The results are shown in table 2 and fig. 6a to 6 c:

table 2: after different strains react for 6 hours, the contents of maleic acid, fumaric acid, L-aspartic acid and L-alanine in reaction liquid

Coli BL21(DE3)/pRSF without modification as shown in FIGS. 6 a-6 c and Table 2_Duet-lASD-MaiA and malate pathway knock-out E.coli BL21(DE3) Δ fumAC/pRSF_Duet-lBoth strains of ASD-MaiA had a large amount of fumaric acid as an intermediate product remaining after 6h catalysis, with a large amount of aspartic acid remaining in BL21(DE3) without the malate pathway knocked out, and the unmodified strain failed to completely convert the maleate substrate to the target alanine product due to accumulation of the by-product malate; although the conversion efficiency of the strain for knocking out the malic acid is improved, the fumaric acid is accumulated in large quantity due to insufficient expression only by using the aspartic acid lyase on the escherichia coli genome, and the conversion efficiency is slow; and E.coli BL21(DE3) Δ fumAC-T7-RB enhancing the T7 promoter-RBS sequence before AspAS/AspA/pRSF_Duet-lThe ASD-MaiA strain (figure 6c) can solve the problem of unbalanced expression of the three enzymes in the catalytic process to a certain extent, the maleic acid basic reaction of a 2M substrate is complete at 6h, the accumulation of intermediate products, namely fumaric acid and aspartic acid is almost avoided, the conversion rate reaches over 96 percent, the yield reaches 170.6g/L, and the production rate reaches 28.43 g/(L.h).

Example 6: recombinant strain whole cell conversion maleic acid to produce L-alanine

Coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF cells prepared in example 4 were collected_Duet-lASD-MaiA, resuspension of cells with 50mM phosphate buffer pH7.5, diluted to OD₆₀₀Then 20% (volume ratio) of resting cells and 80% (volume ratio) of substrate maleic acid were mixed, wherein the final concentration of maleic acid was 2.5M, the reaction system was 50mL, the reaction was catalyzed in a shaker at 200r/min and 37 ℃, and the contents of maleic acid, fumaric acid, L-aspartic acid and L-alanine in the reaction solution were measured by sampling every 2 h.

The results are shown in FIG. 7, which shows E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lThe ASD-MaiA strain can be completely converted within 10 hours under the catalysis of 2.5M substrate maleic acid, almost no intermediate products of fumaric acid and aspartic acid remain, the conversion rate reaches over 98 percent, the yield reaches 220.2g/L, and the production rate reaches 22.02 g/(L.h).

Example 7: recombinant strain whole cell conversion maleic acid to produce L-alanine

Coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF cells prepared in example 4 were collected_Duet-lASD-MaiA, resuspension of cells with 50mM phosphate buffer pH7.5, diluted to OD ₆₀₀20, mixing resting cells of 20 percent (volume ratio) and maleic acid of 80 percent (volume ratio) as a substrate, wherein the final concentration of the maleic acid is 3M, the reaction system is 50mL, catalyzing the reaction in a shaking table at 200r/min and 37 ℃, and sampling every 2h to detect the contents of the maleic acid, the fumaric acid, the L-aspartic acid and the L-alanine in the reaction solution.

The results are shown in FIG. 8, E.coli BL21(DE3) Δ fumAC-T7-RBS/AspA/pRSF_Duet-lThe ASD-MaiA strain can be completely converted in 15h under the catalysis of 3M substrate maleic acid, alanine is separated out at the later stage of the reaction process, the conversion rate reaches more than 97%, the yield reaches 261.2g/L, and the production rate reaches 17.41 g/(L.h).

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

<110> university of south of the Yangtze river

<120> method for synthesizing L-alanine by catalyzing maleic acid through double-enzyme coupling whole cell

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Leu Asn Ala Gly Arg Gly Asn Pro Asn Phe Leu Ala Thr Thr Pro Arg

35 40 45

Arg Ala Phe Phe Arg Leu Gly Leu Phe Ala Ala Ala Glu Ser Glu Leu

50 55 60

Ser Tyr Ser Tyr Met Asn Thr Val Gly Val Gly Gly Leu Ala Lys Ile

65 70 75 80

Glu Gly Ile Glu Gly Arg Phe Glu Arg Tyr Ile Ala Glu Asn Arg Asp

85 90 95

Gln Glu Gly Val Arg Phe Leu Gly Lys Ser Leu Ser Tyr Val Arg Asp

100 105 110

Gln Leu Gly Leu Asp Pro Ala Ala Phe Leu His Glu Met Val Asp Gly

115 120 125

Ile Leu Gly Cys Asn Tyr Pro Val Pro Pro Arg Met Leu Asn Ile Ser

130 135 140

Glu Lys Ile Val Arg Gln Tyr Ile Ile Arg Glu Met Gly Ala Asp Ala

145 150 155 160

Ile Pro Ser Glu Ser Val Asn Leu Phe Ala Val Glu Gly Gly Thr Ala

165 170 175

Ala Met Ala Tyr Ile Phe Glu Ser Met Lys Val Asn Gly Leu Leu Lys

180 185 190

Ala Gly Asp Lys Val Ala Ile Gly Met Pro Val Phe Thr Pro Tyr Ile

195 200 205

Glu Ile Pro Glu Leu Ala Gln Tyr Ala Leu Glu Glu Val Ala Ile Asn

210 215 220

Ala Asp Pro Ala Leu Asn Trp Gln Tyr Pro Asp Ser Glu Leu Asp Lys

225 230 235 240

Leu Lys Asp Pro Ala Ile Lys Ile Phe Phe Cys Val Asn Pro Ser Asn

245 250 255

Pro Pro Ser Val Lys Met Asp Glu Arg Ser Leu Glu Arg Val Arg Lys

260 265 270

Ile Val Ala Glu His Arg Pro Asp Leu Met Ile Leu Thr Asp Asp Val

275 280 285

Tyr Gly Thr Phe Ala Asp Gly Phe Gln Ser Leu Phe Ala Ile Cys Pro

290 295 300

Ala Asn Thr Leu Leu Val Tyr Ser Phe Ser Lys Tyr Phe Gly Ala Thr

305 310 315 320

Gly Trp Arg Leu Gly Val Val Ala Ala His Lys Glu Asn Ile Phe Asp

325 330 335

Leu Ala Leu Gly Arg Leu Pro Glu Ser Glu Lys Thr Ala Leu Asp Asp

340 345 350

Arg Tyr Arg Ser Leu Leu Pro Asp Val Arg Ser Leu Lys Phe Leu Asp

355 360 365

Arg Leu Val Ala Asp Ser Arg Ala Val Ala Leu Asn His Thr Ala Gly

370 375 380

Leu Ser Thr Pro Gln Gln Val Gln Met Thr Leu Phe Ser Leu Phe Ala

385 390 395 400

Leu Met Asp Glu Ser Asp Gln Tyr Lys His Thr Leu Lys Gln Leu Ile

405 410 415

Arg Arg Arg Glu Ala Thr Leu Tyr Arg Glu Leu Gly Thr Pro Pro Gln

420 425 430

Arg Asp Glu Asn Ala Val Asp Tyr Tyr Thr Leu Ile Asp Leu Gln Asp

435 440 445

Val Thr Ser Lys Leu Tyr Gly Glu Ala Phe Ser Lys Trp Ala Val Lys

450 455 460

Gln Ser Ser Thr Gly Asp Met Leu Phe Arg Ile Ala Asp Glu Thr Gly

465 470 475 480

Ile Val Leu Leu Pro Gly Arg Gly Phe Gly Ser Asp Arg Pro Ser Gly

485 490 495

Arg Ala Ser Leu Ala Asn Leu Asn Glu Tyr Glu Tyr Ala Ala Ile Gly

500 505 510

Arg Ala Leu Arg Gln Met Ala Asp Glu Leu Tyr Ala Gln Tyr Thr Gln

515 520 525

Gln Gly Asn Lys Arg

530

<210> 4

<211> 1602

<212> DNA

<213> Artificial sequence

<400> 4

atgagcaagg attatcagag tctggcgaac ttgagcccgt ttgagctcaa ggatgagttg 60

atcaagatcg cctcgggcga cggaaaccgc ctcatgctca atgcggggcg gggcaatccc 120

aattttctgg caaccacccc gagaagagca tttttccgtc tgggcttgtt cgcggctgcc 180

gagtcggaac tttcgtattc atatatgaac acggtgggcg tgggaggcct ggcaaagatc 240

gagggcatag aagggcgctt cgagcgctat attgccgaga accgcgatca ggaaggcgtg 300

cgctttctcg gtaaatccct gagttatgta cgcgatcagc tgggcttgga tccggccgcc 360

ttcctgcacg agatggtcga cggtattctg ggctgcaatt accccgttcc ccctcggatg 420

ctgaacatca gcgaaaaaat cgtgcgccag tacatcatcc gtgaaatggg ggccgatgca 480

attcccagcg agtccgtgaa cctgtttgcg gtcgaggggg gaacggccgc catggcatac 540

atcttcgaga gcatgaaggt caacggcctc ctcaaggctg gtgacaaggt agccatcggc 600

atgccggttt tcactccgta catagaaatt ccggaactgg cccagtatgc gttggaggag 660

gtggcaatca atgccgaccc ggccctcaac tggcaatatc ctgattccga actagacaag 720

ctcaaggatc cggccatcaa gatcttcttc tgcgtgaacc ccagcaatcc gccatcggta 780

aagatggacg agcgcagcct ggagcgtgtg cgcaagattg tggcagagca tcgaccggat 840

ctgatgatcc tgaccgatga cgtctatggc acgtttgccg atggctttca gtcgctcttt 900

gcgatttgcc cggccaacac tttgttggtc tattcattct ccaaatactt tggtgccact 960

ggctggcgtc tgggtgtcgt ggccgcccat aaggaaaata tcttcgactt ggcattgggc 1020

aggctgcctg agtccgagaa aacagcgctc gatgatcgct atcgttcact gctacccgat 1080

gtgcgttcat tgaaattcct agatcgtctg gttgccgaca gccgcgctgt tgccttgaac 1140

cacacggccg gtctgtccac gccgcagcag gtccagatga ccttgttctc gttgtttgcg 1200

ctcatggacg agagcgacca gtacaagcac acgctcaagc aactgatacg acgtcgtgaa 1260

gcaacgctct atcgcgagtt gggaacgcct ccgcaaagag atgaaaatgc ggtcgattac 1320

tacaccttga ttgacctgca ggacgtgacg tcgaagcttt atggcgaagc gttctcgaaa 1380

tgggcagtca agcagtcctc gaccggcgac atgctgttcc ggattgccga cgaaacaggg 1440

atcgtgctcc tgccgggacg tggctttgga tcggaccgtc catcgggacg cgcctccttg 1500

gccaatctca acgagtatga gtacgcggcc ataggtcgtg cgctgcgaca aatggctgac 1560

gagctgtacg cgcaatacac ccagcaaggg aacaagcgct ga 1602

<210> 5

<211> 334

<212> DNA

<213> Artificial sequence

<400> 5

gtttctcctg taatagcagc cggttaaccc cggctacctg aatgggttgc gaatcgcgtt 60

tagcttatat tgtggtcatt agcaaaattt caagatgttt gcgcaactat ttttggtagt 120

aatcccaaag cggtgatcta tttcacaaat taataattaa ggggtaaaaa ccgacactta 180

aagtgatcca gattacggta gaaatcctca agcagcatat gatctcgggt attcggtcga 240

tgcaggggat aatcgtcggt cgaaaaacat tcgaaaccac atatattctg tgtgtttaaa 300

gcaaatcatt ggcagcttga aaaagaaggt tcac 334

Claims

1. A genetically engineered bacterium is characterized in that E.coli BL21(DE3) with a gene delta fumAC knocked out in a genome is taken as an expression host, and maleic acid cis-trans isomerase and L-aspartate beta decarboxylase are expressed in a coupling mode.

2. The genetically engineered bacterium of claim 1, wherein the pRSF is pRSF_Duet-1Is an expression vector.

3. The genetically engineered bacterium of claim 1 or 2, wherein the amino acid sequence of the maleate cis-trans isomerase is shown in SEQ ID No.1, and the amino acid sequence of the L-aspartate beta decarboxylase is shown in SEQ ID No. 3.

4. The genetically engineered bacterium of any one of claims 1 to 3, further comprising T having a nucleotide sequence shown as SEQ ID No.5₇promoter-RBS sequence, overexpressing L-aspartate lyase AspA on the host genome.

5. The genetically engineered bacterium of any one of claims 1 to 4, wherein the positional relationship between the maleate cis-trans isomerase and the L-aspartate beta decarboxylase on the vector is as follows: expression vector-L-aspartate beta decarboxylase-maleate cis-trans isomerase.

6. A method for producing L-alanine, characterized in that the genetically engineered bacterium according to any one of claims 1 to 5 or an enzyme produced by the genetically engineered bacterium is used as a catalyst, and maleic acid is used as a substrate to produce L-alanine.

7. The method of claim 6, wherein the substrate maleic acid has a final concentration of 2 to 3 mol/L.

8. The method of claim 6 or 7, wherein the genetically engineered bacteria are added to the substrate in the form of a cell suspension, and the volume ratio of the cell suspension to the substrate solution is 2: 8.

9. the method according to any one of claims 6 to 8, wherein the bacterial concentration OD of the genetically engineered bacteria₆₀₀At least 20.

10. Use of the genetically engineered bacterium of any one of claims 1 to 5, or the method of any one of claims 6 to 9, for the preparation of a product containing L-alanine.