Recombinant Escherichia coli for highly efficient production of D-salvianic acid and its construction method and application

Abstract

The present invention discloses a recombinant Escherichia coli for efficiently producing D-salvianic acid and its construction method and application. By changing the plasmid copy number and the strength of RBS, the present invention finely regulates the expression levels of the pathway enzymes L-amino acid deaminase LAAD, phenylpyruvate reductase LaPPR and glucose dehydrogenase GDH of D-salvianic acid, achieving the balance of the cascade pathway reaction. The optimal recombinant strain E. coli 13 (containing plasmids pRSF-LAAD-LaPPR, PCDF-GDH) adopts a fed-batch strategy (a total of 60 g / L L-DOPA is fed) in a 5 L fermenter and is transformed for 16 h, and the yield of D-salvianic acid reaches 58.86 g / L, and the conversion rate is 97.6%. The method of the present invention has great potential and wide value for increasing the yield of D-salvianic acid industrially.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli for efficiently producing D-salvianic acid and a construction method and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Salvianic acid, scientific name R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid, D-(+)-β-(3,4-dihydroxyphenyl) lactic acid, English names are Danshensu, D-DSS, (R)-(+)-3-(3,4-Dihydroxyphenyl)-lacticacid, molecular formula is C9H 10 O5, molecular weight is 198.17, is a dextrorotatory phenolic acid compound, and there is no natural L-salvianic acid at present. Salvianic acid is an important active ingredient in the water extract of Salvia miltiorrhiza, and has a wide range of pharmacological activities. It is mainly used to treat cardiovascular and cerebrovascular related diseases. A variety of studies have shown that salvianic acid can also play roles such as antioxidant, anti-inflammatory, anti-hepatic fibrosis, anti-atherosclerosis, and inhibition of thrombus formation.

[0003] At present, the synthesis methods of salvianic acid mainly include chemical synthesis method and biosynthesis method. Among them, the chemical synthesis method mainly starts with 3,4-dihydroxybenzaldehyde, and through six-step synthesis reactions of benzyl protection, Darzens synthesis reaction, selective ring-opening of Lewis acid, reduction by NaBH4, hydrolysis and hydrogenation, the total yield of D-salvianic acid is 48.4%. The obtained products are mostly racemates with low optical purity. In terms of the biosynthesis method, in recent years, domestic and foreign researchers have explored and studied the production of salvianic acid by microorganisms from two aspects of biocatalysis and metabolic engineering. So far, there are mainly the following several methods: Findrik et al. from the University of Zagreb first used snake venom oxidase to convert L-DOPA into 3,4-dihydroxyphenylpyruvic acid, and then used lactate dehydrogenase to convert 3,4-dihydroxyphenylpyruvic acid into salvianic acid. Zhao Guangrong et al. from Tianjin University constructed an engineered Escherichia coli to ferment and produce salvianic acid using glucose as a substrate; in this pathway, lactate dehydrogenase and hydroxylase complex convert 4-hydroxyphenylpyruvic acid into salvianic acid, and then the salvianic acid synthesis pathway is improved by modular optimization strategy and the strategy of knocking out genes regulating the production pathway, and finally the metabolically engineered Escherichia coli obtains a salvianic acid yield of 7.1 g / L. Wang Jian et al. from Tongji University used phenylpyruvic acid as a substrate, and tandem D-mandelic acid dehydrogenase, phenylalanine-4-hydroxylase, and hydroxyphenylacetic acid-3-hydroxylase to produce salvianic acid by whole-cell catalysis method, and finally the salvianic acid yield is 8 g / L. The yield of D-salvianic acid obtained by the above methods is too low and the reaction time is too long. Further increasing the yield of D-salvianic acid has become a problem to be solved and is also one of the focuses of scientific research personnel in various countries in the world. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides an engineered Escherichia coli strain with high yield of D-salvianic acid, as well as a method for producing D-salvianic acid using this engineered strain. By overexpressing the pathway enzymes L-amino acid deaminase, phenylpyruvate reductase, and glucose dehydrogenase in Escherichia coli cells, and then optimizing the plasmid copy number to obtain the optimal expression vector and gene combination, and regulating the expression levels of the three enzymes at different levels of ribosome binding site (RBS) to further optimize the enzyme activity ratio of the three enzymes in vivo, the efficient production of D-salvianic acid is achieved, laying a foundation for the industrial production of D-salvianic acid.

[0005] The first object of the present invention is to provide a recombinant Escherichia coli for the efficient production of D-salvianic acid, which overexpresses L-amino acid deaminase LAAD H295S,V437S , phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH.

[0006] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the L-amino acid deaminase is as shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the phenylpyruvate reductase is as shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding the glucose dehydrogenase is as shown in SEQ ID NO.3.

[0007] In one embodiment of the present invention, the L-amino acid deaminase and the phenylpyruvate reductase are expressed using plasmid pRSF-Duet-1, and the glucose dehydrogenase is expressed using plasmid pCDF-Duet-1.

[0008] In one embodiment of the present invention, the recombinant expression vector is that all the pathway enzyme genes are sequentially linked to the expression vector in the order of independent open reading frames, and the order of the open reading frames is: promoter, ribosome binding site RBS, pathway enzyme encoding gene, terminator.

[0009] In one embodiment of the present invention, the expression of the phenylpyruvate reductase is regulated using an RBS with a nucleotide sequence as shown in SEQ ID NO.10.

[0010] In one embodiment of the present invention, the host of the recombinant Escherichia coli is Escherichia coli E.coli BL21.

[0011] The second object of the present invention is to provide a method for constructing the recombinant Escherichia coli, and the steps of the construction method are as follows:

[0012] (1) Obtain single-gene expression vectors of L-amino acid deaminase, phenylpyruvate reductase, and glucose dehydrogenase;

[0013] (2) Amplify the genes encoding the three enzymes respectively, and ligate the coding genes of L-amino acid deaminase and phenylpyruvate reductase to the pRSF-Duet-1 expression vector according to the order of independent reading frames, and ligate the coding gene of glucose dehydrogenase to the pCDF-Duet-1 expression vector to obtain the recombinant vectors pRSF-LAAD H295S,V437S -LaPPR and pCDF-GDH;

[0014] (3) Transform the recombinant vectors obtained in step (2) into Escherichia coli E. coli BL21 to obtain recombinant E. coli strains.

[0015] The third object of the present invention is to provide the application of the recombinant Escherichia coli in the production of D-salvianolic acid. The application is to ferment and collect the recombinant Escherichia coli cells in a fermentation medium, and use the recombinant Escherichia coli cells as whole-cell catalysts to catalyze the production of L-DOPA and glucose to generate D-salvianolic acid in a whole-cell transformation production system.

[0016] In one embodiment of the present invention, the fermentation is inoculated into a fermentation medium at an inoculum size of 1-10%, cultured for 2-3 h, induced by adding 0.1-0.4 mmol / L IPTG, the induction temperature is 25-37 °C, and the recombinant Escherichia coli cells are collected by centrifugation after fermentation.

[0017] In one embodiment of the present invention, the fermentation medium is: glucose 25 g / L, yeast powder 20 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 3 g / L, VB1 1 mg / L, VB2 1.5 mg / L, VB6 1.5 mg / L.

[0018] In one embodiment of the present invention, in the whole-cell transformation production system, the recombinant Escherichia coli cells are used as cell catalysts, 5-30 g / L L-DOPA and 6-40 g / L glucose are used as substrates, and 4-6 g / L Na2SO3 is used as an antioxidant for the transformation reaction.

[0019] In one embodiment of the present invention, in the whole-cell transformation production system, after reacting for 1-3 h, 1-20 g / L DOPA and 1.2-36 g / L glucose are added to the whole-cell transformation production system, and the pH of the transformation reaction system is adjusted to 6.5-7.0.

[0020] The beneficial effects of the present invention are:

[0021] The present invention realizes the balance of the cascade pathway reaction by finely regulating the expression levels of the pathway enzymes L - amino acid deaminase LAAD, phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH by changing the plasmid copy number and the strength of the RBS. The optimal recombinant strain E. coli 13 (containing plasmids pRSF - LAAD - LaPPR and PCDF - GDH) was fermented in a 5 L fermenter using a fed - batch strategy (a total of 60 g / L L - dopa was fed). After 16 h of conversion, the D - danshensu yield reached 58.86 g / L, and the conversion rate was 97.6%. The method of the present invention has great potential and wide value for increasing the production of D - danshensu industrially. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows a schematic diagram of the D - danshensu cascade pathway;

[0023] Figure 2 The figure shows a schematic diagram of the construction of gene expression vectors with different copy numbers;

[0024] Figure 3 The figure shows the comparison of D - danshensu yields and conversion rates of 6 recombinant strains with different copy numbers in shake - flask conversions

[0025] Figure 4 The figure shows a schematic diagram of the construction of gene expression vectors with different RBS levels;

[0026] Figure 5 The figure shows the comparison of D - danshensu yields and conversion rates of 10 recombinant strains with different RBS levels in shake - flask conversions;

[0027] Figure 6 The figure shows the D - danshensu yield of the optimal recombinant strain E. coli 13 in fed - batch conversion on a 5 L fermenter. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0029] The present invention first provides a recombinant Escherichia coli for efficiently producing D - danshensu, and the recombinant Escherichia coli overexpresses L - amino acid deaminase LAAD H295S,V437S , phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH.

[0030] Encoding the L - amino acid deaminase LAAD H295S,V437SThe nucleotide sequence of the gene is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the phenylpyruvate reductase LaPPR is shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding the glucose dehydrogenase GDH is shown in SEQ ID NO.3.

[0031] In the present invention, genes encoding three enzymes, L-amino acid deaminase LAAD H295S,V437S , phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH, were amplified respectively, and were connected to the expression vectors pACYC-Duet-1, pCDF-Duet-1, pET-Duet-1, and pRSF-Duet-1 according to the order of independent reading frames. The connection types and orders of the three genes on each vector were different, and the pathway enzymes were assembled to obtain various combination methods. Finally, it was screened that L-amino acid deaminase and phenylpyruvate reductase were expressed using plasmid pRSF-Duet-1, and glucose dehydrogenase was expressed using plasmid pCDF-Duet-1, that is, the expression vectors pCDF-GDH and pRSF-LAAD H295S,V437S -LaPPR were introduced into the Escherichia coli host, and a high yield and conversion rate of D-salvianolic acid B could be obtained.

[0032] In the present invention, the recombinant expression vector is that all the pathway enzyme genes are sequentially connected to the expression vector according to the order of independent open reading frames. The order of the open reading frames is: promoter, ribosome binding site RBS, pathway enzyme coding gene, terminator.

[0033] In the present invention, different levels of RBS were also tried to regulate the enzyme expression level, so as to further optimize the enzyme activity ratio of the enzymes in vivo. Finally, it was found that when the RBS with the nucleotide sequence shown in SEQ ID NO.10 was used to regulate the expression of phenylpyruvate reductase, the obtained recombinant bacteria could obtain a high yield and conversion rate of D-salvianolic acid B.

[0034] In the present invention, the host of the recombinant Escherichia coli is Escherichia coli BL21, but this host is not used to limit the present invention, and other hosts that can achieve the purpose of the present invention also belong to the protection scope of the present invention.

[0035] The present invention also provides a method for constructing the recombinant Escherichia coli, and the steps of the construction method are:

[0036] (1) Obtain single-gene expression vectors of L-amino acid deaminase, phenylpyruvate reductase, and glucose dehydrogenase;

[0037] (2) Amplify the genes encoding the three enzymes respectively, and ligate the encoding genes of L-amino acid deaminase and phenylpyruvate reductase to the pRSF-Duet-1 expression vector in the order of independent reading frames, and ligate the encoding gene of glucose dehydrogenase to the pCDF-Duet-1 expression vector to obtain the recombinant vectors pRSF-LAAD H295S,V437S -LaPPR and pCDF-GDH;

[0038] (3) Transform the recombinant vectors obtained in step (2) into Escherichia coli E. coli BL21 to obtain recombinant E. coli strains.

[0039] The present invention also provides the application of the recombinant Escherichia coli in the production of D-salvianic acid. The application is to ferment and collect the recombinant Escherichia coli cells in a fermentation medium, and use the recombinant Escherichia coli cells as a whole-cell catalyst to catalyze the production of D-salvianic acid from L-dopa and glucose in a whole-cell transformation production system.

[0040] In the present invention, the fermentation is inoculated into the fermentation medium at an inoculum size of 1-10%, cultured for 2-3 h, induced by adding 0.1-0.4 mmol / L of IPTG, the induction temperature is 25-37 °C, and after the fermentation is completed, centrifuged at 6000 rpm for 10 min, and the recombinant Escherichia coli cells are stored at -20 °C for later use.

[0041] In the present invention, the fermentation medium is: glucose 25 g / L, yeast powder 20 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 3 g / L, VB1 1 mg / L, VB2 1.5 mg / L, VB6 1.5 mg / L.

[0042] In the present invention, during the whole fermentation stage, a glucose mother liquor is also supplemented to an appropriate concentration, and the composition of the feeding solution is: glucose mother liquor 800 g / L.

[0043] In the present invention, in the whole-cell transformation production system, using the recombinant Escherichia coli cells as a cell catalyst, using 5-30 g / L of L-dopa and 6-40 g / L of glucose as substrates, and using 4-6 g / L of Na2SO3 as an antioxidant for the transformation reaction.

[0044] In the present invention, in the whole-cell transformation production system, after reacting for 1-3 h, 1-20 g / L of dopa and 1.2-36 g / L of glucose are added to the whole-cell transformation production system, and the pH of the transformation reaction system is adjusted to 6.5-7.0.

[0045] The present invention will be further illustrated with specific examples as follows:

[0046] Example 1: Construction of a single-gene expression vector related to pathway enzymes

[0047] The L-amino acid deaminase used in the present invention is the mutant LAAD already available in the laboratory H295S,V437S , the phenylpyruvate reductase LaPPR gene is derived from Lactobacillus sp. CGMCC 9967, and the glucose dehydrogenase GDH gene is derived from Bacillus sp. G3. Lactobacillus sp. CGMCC 9967 and Bacillus sp. G3 were inoculated into 25 mL of LB liquid medium and cultured at 37 °C and 200 rpm for 10 h. The cells were collected, and the genomic DNA of Lactobacillus sp. CGMCC 9967 and Bacillus sp. G3 was extracted using a bacterial genomic DNA extraction kit.

[0048] According to the published genomic information sequences, primers corresponding to each pathway enzyme were designed respectively. Using the above-extracted genomic DNA and the plasmids already available in the laboratory as templates, and using a standard PCR amplification system and program, the corresponding LAAD H295S,V437S , LaPPR, and GDH gene fragments were amplified. After plasmid pET-28a was double-digested with BamHI and XhoI, agarose nucleic acid electrophoresis was used for gel recovery, and the linearized plasmid pET-28a was obtained. The gene fragments obtained by the above PCR amplification were respectively ligated to the double-digested plasmid using a one-step homologous recombinase. The system was 20 μL, and the condition was 37 °C for 30 min. The ligation product was transformed into JM109 competent cells, and single colonies were picked for PCR verification. If the sequencing results were consistent with the theoretical sequence, it was proved that the single-gene expression vector was successfully constructed. Thus, 3 expression vectors were obtained, namely: pET28a-LAAD H295S,V437S , pET28a-LaPPR, and pET28a-GDH. These three vectors are required for subsequent replacement of different expression vectors as amplification templates.

[0049] Example 2: Construction of vectors with different gene copy numbers of pathway enzymes

[0050] Primers with homologous arms were designed according to the restriction enzyme sites, and the corresponding LAADH was amplified 295S,V437S, Gene fragments of LaPPR and GDH, plasmids pRSF-Duet-1, pCDF-Duet-1, and pET-Duet-1 were double-digested with BamHI and HindIII, and then subjected to agarose nucleic acid electrophoresis for gel extraction to obtain linearized plasmids pRSF-Duet-1, pCDF-Duet-1, and pET-Duet-1. The gene fragments obtained by the above PCR amplification were respectively ligated to the double-digested plasmids using one-step homologous recombinase. The reaction system was 20 μL, and the condition was 37 °C for 30 min. The ligation products were transformed into JM109 competent cells, and single colonies were picked for PCR verification. Positive transformants were sequenced. If the sequencing results were consistent with the theoretical sequences, it proved that the single-gene expression vectors were successfully constructed. Thus, 7 expression vectors were obtained, namely: pRSF-LAAD H295S,V437S , pRSF-LaPPR, pET-LAAD H295S,V437S , pET-LaPPR, pET-GDH, pCDF-LaPPR, pCDF-GDH.

[0051] Primers with homologous arms were designed according to the restriction enzyme sites, and the corresponding LaPPR and GDH gene fragments were amplified. Plasmids pCDF-LaPPR, pRSF-LaPPR, pRSF-LAAD H295S,V437S , pET-LaPPR were double-digested with KpnI and XhoI, and then subjected to agarose nucleic acid electrophoresis for gel extraction to obtain linearized plasmids pCDF-LaPPR, pRSF-LaPPR, pRSF-LAAD H295S,V437S , pET-LaPPR. The gene fragments obtained by the above PCR amplification were respectively ligated to the double-digested plasmids using one-step homologous recombinase. The reaction system was 20 μL, and the condition was 37 °C for 30 min. The ligation products were transformed into JM109 competent cells, and single colonies were picked for PCR verification. Positive transformants were sequenced. If the sequencing results were consistent with the theoretical sequences, it proved that the single-gene expression vectors were successfully constructed. Thus, 5 expression vectors were obtained, namely: pCDF-LaPPR-GDH, pET-LaPPR-GDH, pRSF-LaPPR-GDH, pRSF-LAAD H295S,V437S -GDH, pRSF-LAAD H295S,V437S -LaPPR.

[0052] The successfully constructed expression vectors above were combined pairwise according to the requirements of path enzyme expression, and the double plasmids were simultaneously transformed into BL21 competent cells. Thus, 6 recombinant bacteria were obtained, namely E.coli1 (pRSF-LAAD H295S,V437S / pCDF-LaPPR-GDH), E.coli2 (pRSF-LAAD H295S,V437S / pET-LaPPR-GDH), E. coli 3 (pET-LAAD H295S,V437S / pRSF-LaPPR-GDH), E. coli 4 (pET-LaPPR / pRSF-LAAD H295S,V437S -GDH), E. coli 5 (pET-GDH / pRSF-LAAD H295S,V437S -LaPPR), E. coli 6 (pCDF-GDH / pRSF-LAAD H295S,V437S -LaPPR).

[0053] Example 3: Shake-flask fermentation of recombinant bacteria E. coli 1 - E. coli 6

[0054] Cultivation of recombinant bacteria: A monoclonal colony was inoculated into 50 mL (250 mL shake flask) of LB medium as the seed liquid for shake-flask fermentation and cultured at 37 °C and 200 rpm for 10 h. The inoculation amount of the seed liquid was 2%, and it was inoculated into 150 mL of fermentation medium and cultured at 37 °C until the OD 600 reached 0.8. IPTG was added to a final concentration of 0.4 mM, and induction was carried out at 25 °C for 14 h. After completion, the cells were collected by centrifugation at 6000 rpm for 10 min and stored at -40 °C as the whole-cell catalyst required for biotransformation. Composition of the fermentation medium: glucose 25 g / L, yeast powder 20 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 3 g / L, VB1 1 mg / L, VB2 1.5 mg / L, VB6 1.5 mg / L.

[0055] Shake-flask transformation system: The recombinant bacteria were suspended in 20 mM Tris-HCl buffer, with L-DOPA 30 g / L, glucose 36 g / L, wet cells 20 g / L, sodium sulfite 5 g / L, and reacted at 30 °C for 16 h. During the reaction process, 2M NaOH was used to maintain the pH around 7.0.

[0056] According to the determination of the yield of D-salvianic acid, the results are as Figure 3 shown. After 16 h of transformation, the optimal recombinant strain E. coli 6 could produce 25.3 g / L of D-salvianic acid, and the conversion rate was 83.9% (30 g / L L-DOPA).

[0057] Example 4: Construction of gene expression vectors with different RBS levels and comparison of shake-flask transformation

[0058] Based on the successfully constructed expression vectors with different gene copy numbers in Example 2, after evaluating the biotransformation using L-DOPA as the substrate, the vector with the optimal triple-enzyme gene combination was obtained. A total of 10 different strengths of RBS replacement primers were set to replace the RBS of the vector itself in front of the LaPPR gene. First, whole plasmid PCR was performed, and the PCR products were purified. The original template was removed using DPNI, and the digested products were transformed into BL21 competent cells. The colonies with correct bands verified by colony PCR were sent for sequencing. Correct sequencing indicated the successful construction of gene expression vectors with different RBS levels. Finally, a total of 10 recombinant bacteria were obtained, namely E.coli7, E.coli8, E.coli9, E.coli10, E.coli11, E.coli12, E.coli13, E.coli14, E.coli15, and E.coli16.

[0059] The RBS sequences used in Table 1

[0060]

[0061] The culture conditions and transformation conditions of the recombinant bacteria were the same as those in Example 4. According to the determination of the yield of D-salvianic acid, the results were as Figure 5 shown. After 16 h of transformation, the optimal recombinant strain E.coli13 could produce 29.71 g / L of D-salvianic acid, and the conversion rate was 98.53% (30 g / L L-DOPA).

[0062] Example 5: Fed-batch transformation of the optimal recombinant strain E.coli13 in a 5 L fermenter

[0063] Cultivation of recombinant bacteria: Pick the E.coli13 recombinant strain for on-fermenter fermentation. Inoculate a single colony into 50 mL (250 mL shake flask) of LB medium as the seed liquid for the fermenter, and culture it at 37 °C and 200 rpm for 10 h. The liquid volume in the fermenter is 2 L, and the fermentation medium is: glucose 25 g / L, yeast powder 20 g / L, disodium hydrogen phosphate 6 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate 3 g / L, VB1 1 mg / L, VB2 1.5 mg / L, VB6 1.5 mg / L. The pH during the whole fermentation process is controlled at about 7.0. Inoculate into the fermenter at an inoculation amount of 2%, and the inoculation amount of the seed liquid is 2% into the fermentation medium. Maintain the dissolved oxygen level at 25 ± 1.5%, the air supply rate at 6 L / min, the stirring speed at 100 - 500 rpm, and culture at 37 °C until the OD 600 reaches 15, add IPTG to a final concentration of 0.4 mM, induce at 25 °C for 14 h, and after completion, centrifuge at 6000 rpm for 10 min to collect the cells and store them at -40 °C as the whole-cell catalyst required for biotransformation.

[0064] 5L fermenter conversion (1L reaction system): Suspend the recombinant bacterial cells in 20 mM Tris-HCl buffer, initially feed 20 g / L L-DOPA, 24 g / L glucose, and 4 g / L sodium sulfite, and then feed 10 g / L L-DOPA and 12 g / L glucose every 2 h, for a total of four feedings. The total feed is 60 g / L L-DOPA, 72 g / L glucose, and 4 g / L sodium sulfite. Carry out the conversion at 30 °C, use 2 M NaOH to maintain the pH around 7.0 during the reaction process, and the stirring speed is 400 rpm. React for a total of 16 h, and take samples every 2 h to detect the contents of L-DOPA, 3,4-dihydroxyphenylpyruvic acid, and D-salvianic acid in the samples.

[0065] According to the determination of the yield of D-salvianic acid, the results are as Figure 6 shown. After 16 h of conversion, the yield of D-salvianic acid reaches 58.86 g / L, and the conversion rate is 97.6% (a total of 60 g / L L-DOPA is fed in).

[0066] The above results show that the technology of the present invention uses genetic engineering technology to change the strength of the RBS and the plasmid copy number to finely regulate the expression levels of the enzymes L-amino acid deaminase (LAAD H295S,V437S ), phenylpyruvate reductase (LaPPR), and glucose dehydrogenase (GDH) in the D-salvianic acid cascade pathway, which can effectively improve the yield of D-salvianic acid.

[0067] The above-described embodiments are only preferred embodiments given to fully illustrate the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.

Claims

1. A recombinant Escherichia coli for producing D-salvianic acid, characterized in that, The recombinant Escherichia coli overexpresses L-amino acid deaminase LAAD H295S,V437S , phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH; Encoding the L-amino acid deaminase LAAD H295S,V437S The nucleotide sequence of the gene is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the phenylpyruvate reductase LaPPR is shown in SEQ ID NO.2; the nucleotide sequence of the gene encoding the glucose dehydrogenase GDH is shown in SEQ ID NO.

3.

2. The recombinant Escherichia coli according to claim 1, characterized in that, L-amino acid deaminase LAAD H295S,V437S L-amino acid deaminase LAAD and phenylpyruvate reductase LaPPR were expressed using plasmid pRSF-Duet-1, and glucose dehydrogenase GDH was expressed using plasmid pCDF-Duet-1.

3. The recombinant Escherichia coli according to claim 1, characterized in that, The expression of phenylpyruvate reductase LaPPR is regulated by an RBS with a nucleotide sequence as shown in SEQ ID NO.

10.

4. The recombinant Escherichia coli according to claim 1, wherein The host of the recombinant Escherichia coli is Escherichia coli E.coli BL21.

5. A method for constructing the recombinant Escherichia coli according to any one of claims 1 to 4, characterized in that, The steps of the construction method are as follows: (1) Obtain the single-gene expression vectors of L-amino acid deaminase LAAD H295S,V437S , phenylpyruvate reductase LaPPR, and glucose dehydrogenase GDH; (2) Amplify the genes encoding the three enzymes respectively, and ligate the encoding genes of L-amino acid deaminase LAAD H295S,V437S and phenylpyruvate reductase LaPPR to the pRSF-Duet-1 expression vector in the order of independent reading frames, and ligate the encoding gene of glucose dehydrogenase GDH to the pCDF-Duet-1 expression vector to obtain recombinant vectors pRSF-LAAD H295S ,V437S -LaPPR and pCDF-GDH; (3) Transform the recombinant vector obtained in step (2) into Escherichia coli E.coli BL21 to obtain a recombinant Escherichia coli strain.

6. Use of the recombinant Escherichia coli according to any one of claims 1 to 4 in the production of D-salvianic acid, characterized in that, The application is to ferment and collect the recombinant E. coli cells in a fermentation medium, and use the recombinant E. coli cells as a whole-cell catalyst to catalyze the production of D-salvianic acid from L-DOPA and glucose in a whole-cell transformation production system.

7. The application according to claim 6, wherein The fermentation is carried out by inoculating into a fermentation medium at an inoculum size of 1-10%, culturing for 2-3 h, adding 0.1-0.4 mmol / L of IPTG for induction, with an induction temperature of 25-37°C, and centrifuging to collect the recombinant E. coli cells after fermentation.

8. The application according to claim 6, characterized in that, In the whole-cell transformation production system, using the recombinant E. coli cells as a cell catalyst, with 5-30 g / L of L-DOPA and 6-40 g / L of glucose as substrates, and 4-6 g / L of Na2SO3 as an antioxidant for the transformation reaction.

9. The application according to claim 6, wherein In the whole-cell transformation production system, after reacting for 1-3 h, 1-20 g / L of DOPA and 1.2-36 g / L of glucose are added to the whole-cell transformation production system, and the pH of the transformation reaction system is adjusted to 6.5-7.0.

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