CN116769687A

CN116769687A - Recombinant escherichia coli for producing L-phenyllactic acid as well as construction method and application thereof

Info

Publication number: CN116769687A
Application number: CN202310618583.3A
Authority: CN
Inventors: 李辉; 邓志强; 何响; 罗亮; 宁雅芳; 胥龙奇; 彭滢
Original assignee: Dabang Hunan Bio Pharmacy Co ltd
Current assignee: Dabang Hunan Bio Pharmacy Co ltd
Priority date: 2023-05-29
Filing date: 2023-05-29
Publication date: 2023-09-19

Abstract

The invention belongs to the technical field of synthetic biology, and particularly relates to recombinant escherichia coli for producing L-phenyllactic acid, and a construction method and application thereof. The recombinant escherichia coli is constructed by taking coding genes of non-membrane-bound L-amino acid deaminase, catalase, L-lactate dehydrogenase and glucose dehydrogenase as target genes, taking double expression plasmids pRSFDuet-1 and pETDuet-1 as vectors and taking escherichia coli BL21 (DE 3) as a host. The engineering bacteria is used as a catalyst for homogenizing and crushing the expressed engineering bacteria to prepare crude enzyme liquid, 65g/L of L-phenylalanine can be completely converted into L-phenyllactic acid within 15h, the ee value of the product can reach 99.9%, and the chemical selectivity and the enantioselectivity are high. The method for synthesizing the L-phenyllactic acid by converting the L-phenylalanine by the one-bacterium multienzyme provided by the invention has the advantages of high substrate concentration, high product yield, mild reaction conditions and short reaction time, and is suitable for industrial production.

Description

Recombinant escherichia coli for producing L-phenyllactic acid as well as construction method and application thereof

Technical Field

The invention belongs to the technical field of synthetic biology, and particularly relates to recombinant escherichia coli for producing L-phenyllactic acid, and a construction method and application thereof.

Background

Phenyllactic acid (Phenyllactic acid), also called beta-phenyllactic acid, has a chemical name of 2-Hydroxy-3-phenylpropionic acid (2-Hydroxy-3-phenylpropionic acid), is a novel natural broad-spectrum antibacterial substance discovered in recent years, and has wide popularization and application space in the fields of food, feed, cosmetics, medicines, biological materials and the like. The phenyllactic acid can be used as a preservative in the food field, has a strong inhibition effect on most food-borne pathogenic bacteria, and can play a role in preserving and fresh keeping; the compound feed can be used as a feed antibiotic substitute in the field of feed, has good inhibition effect on common animal intestinal pathogenic bacteria, and can play roles of enhancing immunity and promoting growth; can be used as raw material with effects of resolving macula, removing wrinkle and preventing corrosion in cosmetic field, and can promote growth of epidermal cells and prolong shelf life of cosmetics. The phenyllactic acid structure contains a chiral carbon atom and has a pair of enantiomers, namely D-phenyllactic acid and L-phenyllactic acid, and the two single enantiomers can be used as medical intermediates, thus being an important building block for synthesizing various chiral medicaments such as PF1022A, YM-254890, englitazone and the like; can also be used as chiral monomer for synthesizing novel biological material polylactic acid to synthesize D-polylactic acid or L-polylactic acid with high optical activity.

The phenyllactic acid can be divided into natural phenyllactic acid and synthetic phenyllactic acid according to the production process, wherein the natural phenyllactic acid is mainly obtained by two ways of extracting natural fermented food and synthesizing natural raw materials by a biological method. With the increasing importance and demand of "green" and "natural" products, natural phenyllactic acid will be more acceptable to consumers in the future. The biological method for synthesizing the natural phenyllactic acid comprises a biological fermentation method and a biological catalysis method, wherein the biological fermentation method firstly screens probiotic strains for producing the phenyllactic acid in high yield from fermented foods, and then carries out strain improvement or fermentation process optimization on the probiotic strains, so that the phenylalanine is converted into the phenyllactic acid under certain conditions, but the yield is lower and the separation and purification difficulty is higher. The biocatalysis method is a method for leading the synthetic route of converting phenylalanine into phenyllactic acid into a proper host, converting and synthesizing phenyllactic acid by using intact cells or enzyme produced by the intact cells as a catalyst, and has the advantages of simple reaction system, high chiral selectivity, few byproducts, simple separation and purification and the like, and becomes the most promising method in the phenyllactic acid synthesis process.

At present, many patent reports on directly synthesizing phenyllactic acid by using natural L-phenylalanine as a substrate by a biocatalysis method are reported, wherein the L-phenylalanine is catalyzed by L-amino acid deaminase (LAAD) to generate phenylpyruvic acid, and then D-phenyllactic acid or L-phenyllactic acid is catalyzed by D-lactate dehydrogenase (DLDH) or L-lactate dehydrogenase (LLDH). The patent CN201810048337.8 constructs recombinant escherichia coli producing D-phenyllactic acid by coexpression of L-amino acid deaminase (LAAD) derived from general bacillus, D-lactic acid dehydrogenase (DLDH) derived from Lactobacillus sp.CGMCC 9967 and Formate Dehydrogenase (FDH) derived from Proteus mirabilis, and can produce 30g/L of D-phenyllactic acid by whole-cell transformation of L-phenylalanine. Patent CN202110253638.6 constructs recombinant E.coli producing L-phenyllactic acid by coexpression of L-amino acid deaminase (LAAD) derived from Proteus vulgaris, phenylpyruvate reductase (LaPPR) derived from Lactobacillus sp.CGMCC 9967 and Glucose Dehydrogenase (GDH) derived from Bacillus subtilis, and L-phenyllactic acid producing 21.4g/L can be produced by whole cell transformation of L-phenylalanine. Patent CN202210657451.7 constructed recombinant E.coli producing L-phenyllactic acid and D-phenyllactic acid, respectively, by coexpression of L-amino acid deaminase (LAAD) derived from Rhizoctonia cerealis, L-lactate dehydrogenase (LLDH) derived from Lactobacillus paracasei or D-lactate dehydrogenase (DLDH) derived from Lactobacillus Nasalidis, phosphite dehydrogenase (PtxD) derived from Ralstonia, 29.4g/L of L-phenyllactic acid or 27.3g/L of D-phenyllactic acid could be produced by whole cell transformation of L-phenylalanine.

The L-amino acid deaminase LAAD reported in the patent is formed by membrane-bound water, is positioned on a cell membrane when being expressed in escherichia coli in a recombination mode, and needs an electron transfer chain on the cell membrane to participate in the process of catalyzing the deamination of the L-phenylalanine, so that oxygen is consumed and water is generated. The membrane-bound characteristics of the aquatic formed LAAD reported in the above patent are beneficial to the construction of whole cell catalysts because the activity and stability of the membrane-bound protein are significantly reduced after the recombinant cells are damaged or broken. However, due to mass transfer obstruction of cell membranes, the whole cell catalysis efficiency is far lower than that of free enzyme catalysis, so that the product concentration of the synthesized phenyllactic acid by the whole cell catalysis reported by the patent is lower, and a certain distance is left from industrial application. Therefore, the screening activity is high, the non-membrane-combined L-amino acid deaminase catalyzed by free enzyme can be efficiently carried out, and the L-lactic dehydrogenase capable of efficiently and selectively reducing the phenylpyruvic acid to generate the L-phenyllactic acid is further excavated, so that the method has important application value for biocatalytic synthesis of the L-phenyllactic acid.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides recombinant escherichia coli for producing L-phenyllactic acid, and a construction method and application thereof.

In a first aspect of the present invention, a recombinant engineering bacterium for synthesizing L-phenyllactic acid by converting L-phenylalanine is provided, wherein the recombinant engineering bacterium is obtained by co-expressing non-membrane-bound L-amino acid deaminase LAAO and catalase KatE in a high copy number double expression plasmid pRSFDuet-1 and co-expressing L-lactate dehydrogenase LLDH and glucose dehydrogenase GDH in a high copy number double expression plasmid pETDuet-1 in an E.coli host.

According to the specific embodiment of the recombinant escherichia coli, the non-membrane-bound L-amino acid deaminase LAAO is non-membrane-bound L-amino acid deaminase derived from streptomyces coronensis (streptomyces bottropensis), and is used for catalyzing the oxidative deamination of phenylalanine and generating hydrogen peroxide, wherein the amino acid sequence of the non-membrane-bound L-amino acid deaminase is shown as SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: shown at 5.

According to the specific embodiment of the recombinant escherichia coli disclosed by the invention, the catalase KatE is catalase derived from escherichia coli K-12 (E.coli K-12), and catalyzes the decomposition of hydrogen peroxide into oxygen and water so as to timely remove the hydrogen peroxide, thereby avoiding the toxicity of the hydrogen peroxide to cells and catalysts, and the amino acid sequence of the catalase KatE is shown as SEQ ID No:2, the nucleotide sequence is shown as SEQ ID NO: shown at 6.

According to the specific embodiment of the recombinant escherichia coli, the L-lactic dehydrogenase LLDH is L-lactic dehydrogenase derived from lactobacillus plantarum DBPLA2021 (Lactobacillus plantarum DBPLA 2021), and is used for selectively reducing phenylpyruvic acid to generate L-phenyllactic acid, wherein the amino acid sequence of the L-lactic acid is shown as SEQ ID No:3, the nucleotide sequence is shown as SEQ ID No: shown at 7. The lactobacillus plantarum is lactobacillus plantarum DBPLA2021 reported by the inventor in China patent application No. 202310403498.5, and is preserved in China Center for Type Culture Collection (CCTCC) in the 2 nd month of 2023, wherein the preservation number is M2023206.

According to the specific embodiment of the recombinant escherichia coli, the glucose dehydrogenase GDH is glucose dehydrogenase derived from bacillus megatherium (Bacillus megaterium), and is used for catalyzing glucose to generate gluconic acid and generating NADH, so that cofactor regeneration circulation is realized, and the amino acid sequence of the cofactor regeneration circulation is shown as SEQ ID No:4, the nucleotide sequence is shown as SEQ ID NO: shown at 8.

According to a specific embodiment of the recombinant E.coli, the E.coli host is E.coli BL21 (DE 3).

In a second aspect of the present invention, there is provided a method for constructing the aforementioned recombinant E.coli, specifically comprising:

s1, codon optimization and total gene synthesis: respectively carrying out escherichia coli codon optimization and total gene synthesis on a coding gene of non-membrane-combined L-amino acid deaminase, a coding gene of catalase, a coding gene of L-lactate dehydrogenase and a coding gene of glucose dehydrogenase to obtain a first synthetic sequence, a second synthetic sequence, a third synthetic sequence and a fourth synthetic sequence;

s2, constructing a recombinant expression vector: inserting a first synthetic sequence between the cleavage sites EcoR I and Hind III on the pRSFDuet-1 plasmid, and inserting a second synthetic sequence between the cleavage sites Nde I and Kpn I on the pRSFDuet-1 plasmid to obtain a first recombinant expression vector; inserting the third synthetic sequence between the cleavage sites Nco I and Pst I on the pETDuet-1 plasmid, and inserting the fourth synthetic sequence between the cleavage sites NdeI and Xho I on the pETDuet-1 plasmid to obtain a second recombinant expression vector;

s3, transforming a recombinant expression vector: and transforming the first recombinant expression vector and the second recombinant expression vector into competent cells of the escherichia coli to obtain the recombinant escherichia coli.

In a third aspect of the invention, the application of the recombinant escherichia coli in the synthesis of L-phenyllactic acid by converting L-phenylalanine is provided, and the principle of the biocatalysis of L-phenyllactic acid by synthesizing L-phenyllactic acid by catalyzing is shown in figure 1, wherein the biocatalysis of L-phenyllactic acid is simple, convenient, efficient and easy for industrial production. Specifically, the application includes:

fermenting and culturing the recombinant escherichia coli, and adding an inducer to induce expression;

ii) re-suspending the thalli by using phosphate buffer solution, and then carrying out homogenate wall breaking to obtain crude enzyme solution;

iii) converting the L-phenylalanine into L-phenyllactic acid by using the crude enzyme solution as a catalyst.

According to a specific embodiment of the application of the invention, in the step i), a single colony of recombinant escherichia coli is selected and inoculated into a TB medium, and the culture is carried out at 37 ℃ until the OD600 reaches 4.0-6.0, so as to obtain seed liquid; inoculating the seed liquid into a fermentation culture medium containing glycerol, ventilating, stirring and culturing, regulating the air quantity, the rotating speed and the tank pressure to be 30-40%, controlling the pH value to be 7.0 by adding ammonia water, and continuing until the fermentation is finished; and (3) fermenting until the glycerol is exhausted for 10-12 h, wherein DO is rapidly increased by more than 70%, and then adding glycerol at a time according to the proportion of 2% of the initial volume of the fermentation, and adding glycerol of 2% of the initial volume of the fermentation when the DO is suddenly increased to more than 70% after the glycerol is completely consumed, wherein the adding time is 6-8, and the fermentation is continued until the fermentation is finished. When the OD600 value reaches more than 80, adding IPTG with the final concentration of 0.2-0.6 mM for induction, and inducing at 20-28 ℃ for 10-16 h; preferably, IPTG was added at a final concentration of 0.4mM and induced at 24℃for 12h; preferably, the fermentation medium comprises the following concentrations of the components: 30-50 g/L of glycerin, 3-7 g/L of corn steep liquor dry powder, 4-8 g/L of ammonium sulfate, 8-12 g/L of monopotassium phosphate, 0.8-1.2 g/L of sodium citrate, 1-3 g/L of magnesium sulfate heptahydrate, 0.1-0.3 g/L of thiamine, 0.08-0.12 g/L of calcium chloride dihydrate and 0.1-0.2 g/L of ferric chloride hexahydrate; the pH is 6.8-7.2; more preferably, the fermentation medium comprises the following concentrations of the components: 40g/L of glycerin, 5g/L of corn steep liquor dry powder, 6g/L of ammonium sulfate, 10g/L of monopotassium phosphate, 1g/L of sodium citrate, 2g/L of magnesium sulfate heptahydrate, 0.2g/L of thiamine, 0.1g/L of calcium chloride dihydrate and 0.15g/L of ferric chloride hexahydrate; pH 7.0.

According to a specific embodiment of the application of the invention, in step ii), the recombinant bacterium fermentation broth is resuspended in a phosphate buffer solution with a pH of 7.0 in a ratio of 1:1, and the low-temperature and ultra-high-pressure continuous flow cell disruption instrument is used for homogenizing the wall-broken fermentation product to obtain a crude enzyme solution, preferably, the disruption condition of high-pressure homogenization is that the temperature is 2-6 ℃, the pressure is 600-1200 bar, and more preferably, the temperature is 4 ℃, and the pressure is 800-1000 bar.

According to a specific embodiment of the application of the invention, in the step iii), a reaction system is formed by taking crude enzyme solution as a catalyst, L-phenylalanine as a substrate, glucose as an auxiliary substrate and NAD+ as coenzyme in phosphate buffer solution with pH equal to 6.5, the temperature is controlled at 30 ℃ in the reaction process, the pH is controlled at 6.5, and ventilation and stirring are continuously carried out, so that the reaction solution containing L-phenyllactic acid is obtained after the reaction is completed. And (3) detecting the contents of the product L-phenyllactic acid and the intermediate phenylpyruvate and the residual quantity of the substrate L-phenylalanine in the reaction system by HPLC tracking. And after the reaction is finished, regulating the pH to 1.5-2.0, heating at 80 ℃ for 10min, filtering to remove broken cells and other solids in the reaction liquid, extracting with ethyl acetate for three times, combining the extracting solutions, distilling under reduced pressure to obtain a crude L-phenyllactic acid product, and measuring the optical purity of the L-phenyllactic acid in the sample by chiral chromatography.

Preferably, the final concentration of the crude enzyme solution is 100-300 ml/L, the final concentration of the L-phenylalanine is 60-80 g/L, the final concentration of the glucose is 150-200 g/L, the final concentration of the NAD+ is 0.2-0.6 mM, and the reaction time is 12-20 h. More preferably, the final concentration of the crude enzyme solution is 200ml/L, the final concentration of L-phenylalanine is 65g/L, the final concentration of glucose is 160g/L, the final concentration of NAD+ is 0.4mM, and the reaction time is 15 hours.

The beneficial effects of the invention are as follows:

1. the recombinant escherichia coli provided by the invention firstly co-expresses coding genes of four key enzymes including non-membrane-combined L-amino acid deaminase, catalase, L-lactic dehydrogenase and glucose dehydrogenase for synthesizing the L-phenyllactic acid by a biocatalysis method, and can realize the synthesis of the L-phenyllactic acid by converting the L-phenylalanine by one bacterium and multiple enzymes.

2. The invention provides an application of recombinant escherichia coli: 1) The non-membrane-combined L-amino acid deaminase which can be expressed in a cell in a soluble way is used for the first time to catalyze the oxidative deamination reaction in the process of synthesizing L-phenyllactic acid from L-phenylalanine, so that the problem that the catalytic efficiency is low due to the fact that the activity and stability of the L-amino acid deaminase are obviously reduced after cell damage or wall breaking caused by the co-expression strain of the traditional membrane-combined aquatic forming L-amino acid deaminase is solved; 2) On the basis of the catalytic modules of the non-membrane-combined L-amino acid deaminase and the L-lactic dehydrogenase, a catalase and glucose dehydrogenase auxiliary module is also introduced, so that hydrogen peroxide byproducts generated in a reaction system are rapidly cleared and coenzyme NAD+ is circularly regenerated, cell/catalyst poisoning caused by hydrogen peroxide aggregation and large-scale use of coenzyme NAD+ are avoided, and the high-speed smooth performance of enzyme catalytic reaction is facilitated; 3) The method realizes the efficient synthesis of L-phenyllactic acid by catalyzing the L-phenylalanine by using the cell disruption crude enzyme liquid, and particularly, 65g/L of L-phenylalanine can be completely converted into L-phenyllactic acid within 15h by taking the 4 enzyme co-expression strain crude enzyme liquid as a catalyst. Compared with whole cell catalysis, the crude enzyme liquid catalysis overcomes mass transfer obstruction of cell membranes, the catalysis efficiency and the L-phenyllactic acid yield are obviously improved, and the method has mild reaction conditions and short reaction time and has important industrial application value.

Drawings

FIG. 1 is a schematic diagram of the present invention for catalyzing L-phenylalanine to synthesize L-phenyllactic acid using a co-expression strain;

FIG. 2 is a diagram showing the co-expression plasmid pRSFDuet-LAAO-KatE of L-amino acid deaminase and catalase of the present invention;

FIG. 3 is a diagram of the co-expression plasmid pETDuet-LLDH-GDH of L-lactate dehydrogenase and glucose dehydrogenase of the present invention;

FIG. 4 is an HPLC chart of mixed sample injection of substrate L-phenylalanine, intermediate phenylpyruvic acid and product L-phenyllactic acid standard in equal proportion, wherein the peak outlet time of the mixed sample injection is 4.557min, 8.537min and 9.352min respectively;

FIG. 5 is a diagram showing the results of HPLC detection of substrate L-phenylalanine, intermediate phenylpyruvate and product L-phenyllactic acid at the reaction time of 5 hours in the crude enzyme solution reaction system;

FIG. 6 is a forward HPLC chart of mixed sample injection of an L-phenyllactic acid standard substance and a D-phenyllactic acid standard substance in equal proportion, wherein the peak outlet time of the forward HPLC chart is 17.018min and 21.623min respectively;

FIG. 7 is a forward HPLC chromatogram of crude L-phenyllactic acid product prepared in example 4 of the present invention.

Detailed Description

The technical scheme of the present invention will be further described with reference to specific examples, and it should be understood that the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. It is therefore to be understood that other equivalents and modifications may be resorted to without departing from the spirit and scope of the invention. The experimental techniques used in the examples below are conventional in the art unless otherwise specified; materials, reagents and the like used, unless otherwise specified, are commercially available and the methods of use are referred to in the commodity specifications.

The embodiment of the invention adopts a reversed-phase high performance liquid chromatography to quantitatively detect a substrate L-phenylalanine, an intermediate phenylpyruvic acid and a product L-phenyllactic acid, and the specific conditions are as follows:

chromatographic column: inertSustatin C18,5 μm,4.6 mm. Times.250 mm; mobile phase: methanol-water-trifluoroacetic acid (volume ratio 40:60:0.1); detection wavelength: 210nm; flow rate: 1ml/min; column temperature: 25 ℃; sample injection amount: 20 μl. L-phenylalanine (Phe) retention time 4.557min; phenylpyruvic Acid (PPA) retention time was 8.537min; the retention time of L-phenyllactic acid (LPLA) was 9.352min.

The embodiment of the invention adopts chiral chromatographic column to detect the optical purity of L-phenyllactic acid by HPLC, and the specific conditions are as follows:

chromatographic column:AD-H,5 μm,4.6 mm. Times.250 mm; mobile phase: n-hexane-isopropanol-trifluoroacetic acid (volume ratio 95:5:0.1); detection wavelength: 210nm; flow rate: 1ml/min; column temperature: 30 ℃; sample injection amount: 20 μl; the retention time of D-phenyllactic acid was 17.018min and the retention time of L-phenyllactic acid was 21.623min.

The protein and the coding gene thereof according to the embodiment of the invention:

the non-membrane-bound L-amino acid deaminase LAAO is non-membrane-bound L-amino acid deaminase derived from streptomyces coronensis (streptomyces bottropensis), and has an amino acid sequence shown in SEQ ID NO:1, the nucleotide sequence of the escherichia coli after codon optimization is shown as SEQ ID NO:5 is shown in the figure; the catalase KatE is catalase derived from escherichia coli K-12 (Escherichia coli K-12), and the amino acid sequence of the catalase KatE is shown in SEQ ID NO:2, the nucleotide sequence of the escherichia coli after codon optimization is shown as SEQ ID NO:6 is shown in the figure; the L-lactic dehydrogenase LLDH is L-lactic dehydrogenase derived from lactobacillus plantarum DBPLA2021 (Lactobacillus plantarum DBPLA 2021), and the amino acid sequence of the L-lactic dehydrogenase is shown as SEQ ID NO:3, the nucleotide sequence of the escherichia coli after codon optimization is shown as SEQ ID NO: shown in figure 7; the glucose dehydrogenase GDH is glucose dehydrogenase derived from bacillus megaterium (Bacillus megaterium), and the amino acid sequence of the glucose dehydrogenase GDH is shown as SEQ ID NO:4, the nucleotide sequence of the escherichia coli after codon optimization is shown as SEQ ID NO: shown at 8.

Example 1: construction of codon optimization and Co-expression Strain

1.1 coding liberalization and Gene Synthesis

In order to improve the expression levels of non-membrane-bound L-amino acid deaminase LAAO, catalase KatE, L-lactate dehydrogenase LLDH and glucose dehydrogenase GDH in escherichia coli, the gene optimization sequence is obtained by performing complete gene codon optimization on the genes by utilizing Synthetic Gene Designer software according to the codon preference of the escherichia coli on the premise of not changing the amino acid sequence. Adding an (EcoR I) enzyme cutting site at the 5 'end of the L-amino acid deaminase LAAO gene optimization sequence, and adding an (Hind III) enzyme cutting site at the 3' end to obtain a LAAO synthesis sequence; adding an (Nde I) enzyme cutting site at the 5 'end and an (Kpn I) enzyme cutting site at the 3' end of a catalase KatE gene optimization sequence to obtain a KatE synthesis sequence; adding an (Nco I) enzyme cutting site at the 5 'end of the optimized sequence of the LLDH gene of the L-lactate dehydrogenase, and adding an (Pst I) enzyme cutting site at the 3' end to obtain a LLDH synthetic sequence; and adding an (NdeI) enzyme cutting site at the 5 'end and an (Xho I) enzyme cutting site at the 3' end of the optimized sequence of the GDH gene of the glucose dehydrogenase to obtain a GDH synthetic sequence. Then, complete gene synthesis was performed on LAAO synthetic sequence, katE synthetic sequence, LLDH synthetic sequence and GDH synthetic sequence, and the gene synthesis was completed by Beijing qing department biotechnology Co., ltd and was ligated to pMD18-T vector.

1.2 construction and transformation of Co-expression plasmids

The L-amino acid deaminase LAAO gene synthesis sequence was inserted between the (EcoR I) and (Hind III) cleavage sites of the first multiple cloning site of pRSFDuet-1, and the catalase KatE gene synthesis sequence was inserted between the (Nde I) and (Kpn I) cleavage sites of the second multiple cloning site of pRSFDuet-1, to obtain a recombinant plasmid pRSFDuet-LAAO-KatE coexpression of LAAD and KatE, as shown in FIG. 2. The L-lactate dehydrogenase gene LLDH synthetic sequence was inserted between the (Nco I) and (Pst I) cleavage sites of the first multiple cloning site of pETDuet-1, and the glucose dehydrogenase gene GDH synthetic sequence was inserted between the (NdeI) and (Xho I) cleavage sites of the second multiple cloning site of pRSFDuet-1, to obtain a recombinant plasmid pETDuet-LLDH-GDH coexpressing LLDH and GDH, as shown in FIG. 3. Recombinant plasmids pRSFDuet-LAAO-KatE and pETDuet-LLDH-GDH were transformed into E.coli BL21 (DE 3), and screening and verification were performed in LB plates containing ampicillin and kanamycin resistance to obtain co-expression strains simultaneously expressing recombinant plasmids pRSFDuet-LAAO-KatE and pETDuet-LLDH-GDH.

1.3 shake flask induced expression of Co-expressed Strain and enzyme catalytic verification

Co-expression strains grown on ampicillin and kanamycin-containing plates were selected as single colonies and inoculated into LB liquid medium, cultured at 37℃and 220rpm, and when OB600 reached 0.6, 1.0mM IPTG was added for induction of expression, at 28℃for 12h. And (3) centrifuging at a low temperature of 12000rpm for 10min to collect thalli, respectively performing whole cell catalysis and cell disruption crude enzyme liquid catalysis, wherein the catalytic system is 200mL, the concentration of substrate L-phenylalanine is 5g/L, the concentration of auxiliary substrate glucose is 15g/L, the concentration of coenzyme NAD+ is 1.0mM, the reaction temperature is 30 ℃, and the pH is controlled to be 7.0. The reverse phase high performance liquid chromatography is used for detecting the product L-phenyllactic acid and the intermediate phenylpyruvic acid in the reaction liquid, and the whole cell catalytic reaction liquid and the crude enzyme liquid catalytic reaction liquid both detect the L-phenyllactic acid and the phenylpyruvic acid, which indicates that the recombinant co-expression strain for converting the L-phenylalanine into the L-phenyllactic acid is successfully constructed.

Example 2: optimization of enzyme production conditions for co-expressed strains

2.1 Effect of inducer IPTG concentration on conversion Rate

The single colony of the co-expression recombinant escherichia coli constructed in the step 1.2 is selected and cultured in a TB shake flask culture medium containing ampicillin and kanamycin at 37 ℃ and 220rpm, when the OD600 of the thallus reaches 0.6, IPTG (0 mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0 mM) with different concentrations is respectively added, the mixture is induced for 16 hours at 28 ℃, and the thallus is collected by centrifugation. After ultrasonic wall breaking, crude enzyme liquid is obtained, in vitro catalytic reaction verification is carried out respectively, the catalytic system is 200ml, the concentration of substrate L-phenylalanine is 15g/L, the concentration of auxiliary substrate glucose is 40g/L, the concentration of coenzyme NAD+ is 1.0mM, the reaction temperature is 30 ℃, and the pH is controlled at 7.0 by automatic titration of NaOH. And (3) carrying out HPLC detection on the product L-phenyllactic acid after 5h of crude enzyme catalysis. The results showed that the highest conversion rate was achieved when induced with IPTG at a final concentration of 0.4mM and the concentration of the product L-phenyllactic acid was 10.4g/L.

2.2 Effect of Induction temperature on conversion Rate

Selecting the recombinant escherichia coli single colony constructed in the step 1.2, culturing the recombinant escherichia coli single colony in a TB shake flask culture medium containing ampicillin and kanamycin at 37 ℃ and 220rpm, adding IPTG with the medium-to-final concentration of 0.4mM when the OD600 of the thalli reaches 0.6, respectively carrying out induced expression at different temperatures (18 ℃,20 ℃, 22 ℃,24 ℃, 26 ℃,28 ℃ and 30 ℃) for 16 hours, centrifuging, collecting thalli, and carrying out ultrasonic wall breaking to obtain crude enzyme liquid. In vitro catalytic reaction verification is carried out by referring to the reaction system, and the result shows that the conversion rate reaches the highest when the induction is carried out at 24 ℃, and the concentration of the product L-phenyllactic acid is 12.7g/L.

2.3 Effect of duration of Induction on conversion Rate

Selecting the single colony of the co-expression recombinant escherichia coli obtained in the step 1 into a TB shake flask culture medium containing ampicillin and kanamycin, culturing at 37 ℃ and 220rpm, adding IPTG with the medium-to-final concentration of 0.4mM when the OD600 of the thalli reaches 0.6, respectively inducing for 8h, 10h, 12h, 14h, 16h and 18h at 24 ℃, centrifugally collecting the thalli, and performing ultrasonic wall breaking to obtain crude enzyme liquid. In-vitro catalytic reaction verification is carried out by referring to the reaction system, and the result shows that the induction rate reaches the highest 12h conversion rate, and the concentration of the product L-phenyllactic acid is 14.6g/L.

Example 3: high density fermentation expression of co-expressed strains

The coexpression strain obtained in example 1 was inoculated into a plurality of 1L flasks containing 200mL of TB medium, ampicillin and kanamycin were added thereto, and shake culture was performed at 37℃and 220rpm for 12 hours, to prepare a shake flask seed culture solution. The above-mentioned cultured seed liquor is inoculated into 50L fermentation tank containing 30L high-density fermentation culture medium according to 2% inoculation quantity, and ampicillin and kanamycin are added to start fermentation culture, and its initial air quantity is 50L/min, initial rotating speed is 300rpm, initial tank pressure is 0.05Mpa, initial temperature is 37 deg.C and initial pH is 7.0. The dissolved oxygen is controlled to be more than 30% by increasing the air quantity, the rotating speed and the tank pressure, the highest air quantity is 150L/min, the highest rotating speed is 750rpm, the highest tank pressure is 0.08Mpa, and the pH is controlled to be 7.0 continuously to the end by automatically feeding 25% ammonia water. And (3) when the fermentation is completed for 10 to 12 hours, glycerol is exhausted, DO is rapidly increased by more than 70 percent, glycerol is fed in a proportion of 2 percent of the initial volume of the fermentation at one time, and then when the DO is suddenly increased to more than 70 percent after the glycerol is completely consumed, the glycerol accounting for 2 percent of the initial volume of the fermentation is fed in by adopting the same fed-batch strategy, and the feeding times are 6 to 8 times and are continued until the fermentation is completed. When the OD600 value reaches 80 or above, cooling to 24 ℃, adding IPTG with the final concentration of 0.4mM, and after the fermentation is finished after the induction culture is completed for about 12 hours, sampling and monitoring the OD600 every hour, and taking the time point when the OD600 value is reduced by 5-10 units from the highest value as the fermentation end point. OD600 at the end of fermentation was 156, and the wet weight of the cells was 248g/L. And (3) after the fermentation is finished, re-suspending the recombinant bacteria fermentation by adopting a phosphate buffer solution with the pH value of 7.0 according to the proportion of 1:1, and homogenizing and crushing the fermentation liquor by utilizing a low-temperature ultrahigh-pressure continuous flow cell crusher to obtain crude enzyme liquid, wherein the crushing condition of high-pressure homogenization is that the temperature is 4 ℃ and the pressure is 800-1000 bar.

The escherichia coli high-density fermentation medium comprises the following components: 40g/L glycerin, 5g/L corn steep liquor dry powder, 6g/L ammonium sulfate, 10g/L monopotassium phosphate, 1g/L sodium citrate, 2g/L magnesium sulfate heptahydrate, 0.2g/L thiamine, 0.1g/L calcium chloride dihydrate and 0.15g/L, pH 7.0.0 ferric chloride hexahydrate.

Example 4: optimization of enzymatic reaction conditions for co-expressed strains

The crude enzyme solution prepared in the example 3 is used as a catalyst, and the reaction condition of a reaction system for catalyzing L-phenylalanine to synthesize L-phenyllactic acid by one-bacterium multienzyme is optimized.

4.1 optimum temperature selection for catalytic systems

200ml of the reaction system was added with 20g/L of L-phenylalanine, 50g/L of glucose, 1.0mM of coenzyme NAD+,100ml/L of crude enzyme solution, and the pH was controlled at 7.0 by automatic titration of sodium hydroxide. The enzyme catalytic reaction is carried out at different temperatures (20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃) respectively, HPLC detection is carried out on the product L-phenyllactic acid after the reaction for 10 hours, and the result shows that the conversion rate reaches the highest when the reaction is carried out at 30 ℃, and the concentration of the product L-phenyllactic acid is 16.3g/L.

4.2 optimum pH selection for catalytic systems

200ml of a reaction system, L-phenylalanine with the medium concentration of 20g/L, glucose with the medium concentration of 50g/L, coenzyme NAD+ with the medium concentration of 1.0mM and crude enzyme solution with the medium concentration of 100ml/L are added, the reaction temperature is 30 ℃, the pH is controlled to be 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 respectively by automatic titration of sodium hydroxide for enzyme catalytic reaction, HPLC detection is carried out on the product L-phenyllactic acid after the reaction for 10 hours, and the result shows that the reaction conversion rate reaches the highest when the pH is 6.5, and the concentration of the product L-phenyllactic acid is 18.7g/L.

4.3 optimal substrate concentration and optimal reaction time selection for catalytic System

Directly adding coenzyme NAD+ with the final concentration of 1.0mM into 200ml of crude enzyme solution, controlling the reaction temperature to 30 ℃, automatically titrating sodium hydroxide to control the pH to 6.5, and respectively carrying out enzyme catalytic reaction under different substrate concentrations (10 g/L phenylalanine+250 g/L glucose, 20g/L phenylalanine+50 g/L glucose, 30g/L phenylalanine+75 g/L glucose, 40g/L phenylalanine+100 g/L glucose, 50g/L phenylalanine+125 g/L glucose, 60g/L phenylalanine+150 g/L glucose, 70g/L phenylalanine+175 g/L glucose, 80g/L phenylalanine+200 g/L glucose). The product L-phenyllactic acid was tested by HPLC at 2h intervals and stopped at 30 h. The result shows that when the concentration of the substrate L-phenylalanine is 70g/L, the conversion rate can reach more than 98% after 15 hours of reaction, and the concentration of the product L-phenyllactic acid is 68g/L. Continuing to increase the substrate concentration and extending the reaction time, the conversion rate gradually decreases and the product concentration increases extremely slowly, so that from the economical point of view of biocatalysis, a substrate concentration of 70g/L is preferred, and a reaction time of 15 hours is preferred.

4.4 selection of the optimum amount of crude enzyme solution of the catalytic System

Based on the optimal reaction temperature, the optimal reaction pH, the optimal substrate concentration and the optimal reaction time, the influence of different addition amounts (25 ml/L,50ml/L, 100ml/L, 200ml/L, 400ml/L, 800ml/L and 1000 ml/L) of the crude enzyme solution on the conversion is studied, and the result shows that when the dosage of the crude enzyme solution is in the range of 200-1000 ml/L, the conversion efficiency is not obviously increased along with the increase of the dosage of the crude enzyme solution, and the optimal dosage of the crude enzyme solution is 200ml/L from the economical point of biocatalysis.

4.5 selection of the most suitable amount of the catalytic System coenzyme NAD+

Based on the optimal reaction temperature, the optimal reaction pH, the optimal substrate concentration, the optimal crude enzyme solution dosage and the optimal reaction time, the influence of different NAD+ coenzyme concentrations (0 mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0 mM) on the crude enzyme solution conversion is studied, and the result shows that the L-phenylalanine conversion efficiency is not remarkably increased along with the increase of the NAD+ concentration when the NAD+ coenzyme concentration is in the range of 0.4-1.0 mM, and the optimal addition amount of the NAD+ coenzyme is 0.4mM from the economical aspect of biocatalysis.

4.6 selection of the most suitable amount of catalytic System coenzyme FMN

Based on the optimum reaction temperature, the optimum reaction pH, the optimum substrate concentration, the optimum crude enzyme solution amount, the optimum reaction time and the optimum NAD+ addition amount, the influence of different FMN coenzyme concentrations (0 mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM and 1.0 mM) on the conversion of the crude enzyme solution was studied, and as a result, it was revealed that the L-phenylalanine conversion efficiency did not significantly improve with the increase of the FMN concentration in the range of 0 to 1.0mM, and therefore, from the viewpoint of biocatalysis economy, the coenzyme FMN was not additionally added to the reaction system.

Example 5: co-expression strain crude enzyme liquid catalysis L-phenylalanine synthesis L-phenyllactic acid

The crude enzyme solution prepared in example 3 is used as a catalyst, and the optimal reaction conditions screened in example 4 are used for converting L-phenylalanine in a 200L biocatalysis reactor to synthesize L-phenyllactic acid. 150L of reaction system, controlling the temperature to 30 ℃, adding 10kg of substrate L-phenylalanine, 25kg of auxiliary substrate glucose, 40g of NAD+ coenzyme and 75g of polyethylene glycol defoamer into 100L of phosphate buffer (pH=6.5), stirring and mixing uniformly, adjusting the pH to 6.5 by using 2.5M NaOH solution, adding 30L of crude enzyme solution prepared in example 2 according to the proportion of 200ml/L, adding phosphate buffer to fix the volume to 150L, and starting the reaction by ventilation. Continuously supplying oxygen in the reaction process, introducing air at 60L/min, stirring at 250rpm, and automatically feeding 2.5M NaOH solution to control the pH to be 6.5. HPLC analysis was performed by sampling every 1 hour from the beginning of the reaction, and the contents of the substrate L-phenylalanine, intermediate phenylpyruvate and product L-phenyllactic acid in the reaction solution were detected, and the sampling detection results at the time of 5 hours of the reaction are shown in FIG. 5. The reaction is completed after 15 hours, the substrate conversion rate is over 99.5 percent, and the yield is more than 95 percent.

And adding concentrated sulfuric acid into the completely converted reaction solution, regulating the pH value to 1.5-2.0, heating to 80 ℃ for 10min, cooling to room temperature, centrifuging or filtering to remove precipitate, adding an equal volume of ethyl acetate for extraction, repeating for 3 times, merging the extracting solutions, and distilling under reduced pressure to obtain the crude L-phenyllactic acid product. The crude L-phenyllactic acid product was dissolved in isopropanol to a concentration of 2g/L, and the ee value of the sample was 99.9% by chiral chromatography, and the detection result was shown in FIG. 7.

Claims

1. A recombinant escherichia coli, which comprises a gene encoding a non-membrane-bound L-amino acid deaminase, a gene encoding a catalase, a gene encoding an L-lactate dehydrogenase, and a gene encoding a glucose dehydrogenase.

2. The recombinant escherichia coli according to claim 1, wherein the non-membrane-bound L-amino acid deaminase is a hydrogen peroxide-producing L-amino acid deaminase derived from streptomyces bojojoi, and has an amino acid sequence as shown in SEQ ID No: 1.

3. The recombinant escherichia coli according to claim 1, wherein the catalase is derived from escherichia coli K-12, and the amino acid sequence of the catalase is shown as SEQ ID No: 2.

4. The recombinant escherichia coli of claim 1, wherein the L-lactate dehydrogenase is derived from a nucleic acid sequence having a accession number of cctccc NO: m2023206 Lactobacillus plantarum DBPLA2021, the amino acid sequence of which is shown in SEQ ID No: 3.

5. The recombinant escherichia coli according to claim 1, wherein the glucose dehydrogenase is derived from bacillus megaterium and has an amino acid sequence as shown in SEQ ID No: 4.

6. The recombinant E.coli according to claim 1, wherein the E.coli host is E.coli BL21 (DE 3).

7. The method for constructing recombinant E.coli according to any one of claims 1 to 6, comprising the steps of:

8. Use of a recombinant e.coli according to any one of claims 1 to 6 for the synthesis of L-phenyllactic acid by conversion of L-phenylalanine, characterized in that said use comprises:

9. The use according to claim 8, wherein in step i) the recombinant E.coli single colony is selected and inoculated into TB medium, and the culture is carried out at 37 ℃ until the OD600 reaches 4.0-6.0, thus obtaining seed liquid; inoculating the seed liquid into a fermentation culture medium containing glycerol, ventilating, stirring and culturing, regulating the air quantity, the rotating speed and the tank pressure to be 30-40%, controlling the pH value to be 7.0 by adding ammonia water, and continuing until the fermentation is finished; fermenting until the glycerol is exhausted for 10-12 h, wherein DO is rapidly increased by more than 70%, adding glycerol at a time according to the proportion of 2% of the initial volume of fermentation, adding glycerol of 2% of the initial volume of fermentation when DO is suddenly increased to more than 70% after the glycerol is completely consumed, adding 6-8 times, and continuing until the fermentation is finished; when the OD600 value reaches 80 or above, adding IPTG with the final concentration of 0.2-0.6 mM for induction, and inducing at 20-28 ℃ for 10-16 h;

preferably, the fermentation medium comprises the following concentrations of the components: 30-50 g/L of glycerin, 3-7 g/L of corn steep liquor dry powder, 4-8 g/L of ammonium sulfate, 8-12 g/L of monopotassium phosphate, 0.8-1.2 g/L of sodium citrate, 1-3 g/L of magnesium sulfate heptahydrate, 0.1-0.3 g/L of thiamine, 0.08-0.12 g/L of calcium chloride dihydrate and 0.1-0.2 g/L of ferric chloride hexahydrate; the pH is 6.8-7.2.

10. The use according to claim 8, characterized in that in step iii) the crude enzyme solution is used as catalyst, L-phenylalanine is used as substrate, glucose is used as auxiliary substrate, NAD+ is used as coenzyme, the reaction system is formed in phosphate buffer solution with pH equal to 6.5, the temperature is controlled to 30 ℃ during the reaction, the pH is controlled to 6.5, and aeration and stirring are continued;

preferably, the final concentration of the crude enzyme solution is 100-300 ml/L, the final concentration of the L-phenylalanine is 60-80 g/L, the final concentration of the glucose is 150-200 g/L, the final concentration of the NAD+ is 0.2-0.6 mM, and the reaction time is 12-20 h.