CN116064348A - Recombinant escherichia coli for efficiently producing D-salvianic acid, and construction method and application thereof - Google Patents

Recombinant escherichia coli for efficiently producing D-salvianic acid, and construction method and application thereof Download PDF

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CN116064348A
CN116064348A CN202211116758.2A CN202211116758A CN116064348A CN 116064348 A CN116064348 A CN 116064348A CN 202211116758 A CN202211116758 A CN 202211116758A CN 116064348 A CN116064348 A CN 116064348A
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吴静
杨家辉
宋伟
周怡雯
陈修来
高聪
刘佳
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Abstract

The invention discloses a recombinant escherichia coli for efficiently producing D-salvianic acid A, a construction method and application thereof, wherein the expression levels of a pathway enzyme L-amino acid deaminase LAAD, a phenylpyruvate reductase LaPPR and a glucose dehydrogenase GDH of the D-salvianic acid A are finely regulated and controlled by changing the copy number of plasmids and the strength of RBS, so that the cascade pathway reaction balance is realized; the optimal recombinant strain E.coli13 (containing plasmid pRSF-LAAD-LaPPR, PCDF-GDH) is transformed for 16h by adopting a fed-batch strategy (60 g/L L-dopa co-fed) under a 5L fermentation tank, the yield of D-salvianic acid A reaches 58.86g/L, and the transformation rate is 97.6%. The method of the invention has great potential and wide value in industry for improving the yield of D-salvianic acid A.

Description

Recombinant escherichia coli for efficiently producing D-salvianic acid, and construction method and application thereof
Technical Field
The invention relates to recombinant escherichia coli for efficiently producing D-salvianic acid A, and a construction method and application thereof, and belongs to the technical field of bioengineering.
Background
Salvianic acid, R- (+) -3- (3, 4-Dihydroxyphenyl) -2-hydroxy propionic acid, D- (+) -beta- (3, 4-Dihydroxyphenyl) lactic acid, english name of Danshensu, D-DSS, (R) - (+) -3- (3, 4-Dihydroxyphenyl) -lactic acid, molecular formula of C 9 H 10 O 5 The molecular weight is 198.17, which is a dextro-phenolic acid compound, and no natural levo-salvianic acid exists at present. The salvianic acid A is an important effective component of the water extract of the red sage root, has wide pharmacological activity, is mainly used for treating cardiovascular and cerebrovascular related diseases, and various researches indicate that the salvianic acid A can also play roles in resisting oxidation, inflammation, liver fibrosis, atherosclerosis, thrombosis and the like.
At present, the synthesis mode of tanshinol mainly comprises a chemical synthesis method and a biological synthesis method, wherein the chemical synthesis method mainly takes 3, 4-dihydroxybenzaldehyde as an initial part, and adopts benzyl protection, darzens synthesis reaction, selective ring opening of Lewis acid and NaBH 4 The total yield of the final D-salvianic acid A is 48.4% by six synthesis reactions of reduction, hydrolysis and hydrogenation, and the obtained product is mainly raceme and has lower optical purity. In the aspect of biosynthesis, in recent years, domestic and foreign personnel have explored and studied microbial production of salvianic acid from two aspects of biocatalysis and metabolic engineering, so far, the following are mainly available: findrik et al, university of Sagleibu, utilized a snake venom oxidase to convert L-dopa to 3, 4-dihydroxyphenylpyruvate, and then a lactate dehydrogenase to convert 3, 4-dihydroxyphenylpyruvate to salvianic acid. Construction of Tianjin university Zhao Anrong and the likeEngineering escherichia coli, and fermenting and producing tanshinol by taking glucose as a substrate; in the way, the lactate dehydrogenase and hydroxylase complex converts 4-hydroxyphenylpyruvate into salvianic acid A, then the salvianic acid A synthesis way improves the production of the salvianic acid A through a modularized optimization strategy and a knockout regulation production way gene strategy, and finally the yield of the salvianic acid A obtained by metabolic engineering escherichia coli is 7.1g/L. Wang Jian from the university of the same university and the like uses phenylpyruvic acid as a substrate, and connects D-mandelic acid dehydrogenase, phenylalanine-4-hydroxylase and hydroxyphenylacetic acid-3-hydroxylase in series, and adopts a whole cell catalysis method to produce the salvianic acid with the final yield of 8g/L. The method has the advantages that the yield of the D-salvianic acid A is too low, the reaction time is too long, the further improvement of the yield of the D-salvianic acid A becomes a problem to be solved, and meanwhile, the method is one of focuses of current scientific researchers around the world.
Disclosure of Invention
In order to solve the technical problems, the invention provides an escherichia coli engineering bacterium for high-yield D-salvianic acid A and a method for producing the D-salvianic acid A by using the engineering bacterium. The invention synthesizes the path L-amino acid deaminase, phenylpyruvate reductase and glucose dehydrogenase of the D-salvianic acid in the escherichia coli cells by over-expression, then obtains an optimal expression vector and a gene combination mode by optimizing the plasmid copy number, and regulates and controls the expression level of the three enzymes by different levels RBS so as to further optimize the enzyme activity proportion of the three enzymes in the body, thereby realizing the high-efficiency production of the D-salvianic acid and laying a foundation for the industrialized production of the D-salvianic acid.
The first object of the present invention is to provide a recombinant E.coli which efficiently produces D-tanshinol and overexpresses L-amino acid deaminase LAAD H295S,V437S Phenylpyruvate reductase LaPPR and glucose dehydrogenase GDH.
In one embodiment of the invention, the nucleotide sequence of the gene encoding the L-amino acid deaminase is shown as SEQ ID NO. 1; the nucleotide sequence of the gene for encoding the phenylpyruvate reductase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for encoding the glucose dehydrogenase is shown as SEQ ID NO. 3.
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.
In one embodiment of the present invention, the recombinant expression vector is formed by sequentially connecting all the pathase genes to the expression vector in the sequence of independent open reading frames, wherein the sequence of the open reading frames is as follows: a promoter, a ribosome binding site RBS, a gene encoding a pathway enzyme, and a terminator.
In one embodiment of the invention, RBS with nucleotide sequence shown as SEQ ID NO.10 is used for regulating and controlling the expression of the phenylpyruvate reductase.
In one embodiment of the invention, the host of the recombinant escherichia coli is escherichia coli E.coli BL21.
The second object of the present invention is to provide a construction method of the recombinant escherichia coli, which comprises the following steps:
(1) Obtaining single gene expression vectors of L-amino acid deaminase, phenylpyruvate reductase and glucose dehydrogenase;
(2) Amplifying the genes encoding the three enzymes respectively, connecting the genes encoding the L-amino acid deaminase and the phenylpyruvate reductase to pRSF-Duet-1 expression vector according to the independent reading frame sequence, and connecting the gene encoding the glucose dehydrogenase to pCDF-Duet-1 expression vector to obtain recombinant vector pRSF-LAAD H295S,V437S -LaPPR and pCDF-GDH;
(3) And (3) converting the recombinant vector obtained in the step (2) into E.coli BL21 to obtain the E.coli recombinant strain.
The third object of the invention is to provide the application of the recombinant escherichia coli in the production of D-salvianic acid A, wherein the application is to collect the recombinant escherichia coli thalli by fermentation in a fermentation medium, and the recombinant escherichia coli thalli is used as a whole-cell catalyst to catalyze L-dopa and glucose to produce D-salvianic acid A in a whole-cell conversion production system.
In one embodiment of the invention, the fermentation is carried out by inoculating 1-10% of the inoculum size into a fermentation culture medium, culturing for 2-3h, adding 0.1-0.4mmol/L IPTG for induction, and centrifuging to collect recombinant escherichia coli thalli after the fermentation is finished, wherein the induction temperature is 25-37 ℃.
In one embodiment of the invention, the fermentation medium is: 25g/L glucose, 20g/L yeast powder, 6g/L disodium hydrogen phosphate, 2g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate and VB 1 1mg/L,VB 2 1.5 mg/L，VB 6 1.5mg/L。
In one embodiment of the invention, in the whole cell transformation production system, the recombinant escherichia coli thalli are used as cell catalysts, 5-30g/L L-dopa and 6-40g/L glucose are used as substrates, and Na is used as a substrate 2 SO 3 4-6g/L as antioxidant.
In one embodiment of the invention, 1-20g/L dopa and 1.2-36g/L glucose are added into the whole cell transformation production system after the whole cell transformation production system reacts for 1-3 hours, and the pH value of the transformation reaction system is regulated to 6.5-7.0.
The beneficial effects of the invention are as follows:
the invention realizes the cascade path reaction balance by changing the plasmid copy number and RBS strength to finely regulate and control the expression levels of the pathway enzyme L-amino acid deaminase LAAD, the phenylpyruvate reductase LaPPR and the glucose dehydrogenase GDH of D-salvianic acid A; the optimal recombinant strain E.coli13 (containing plasmid pRSF-LAAD-LaPPR, PCDF-GDH) is transformed for 16h by adopting a fed-batch strategy (60 g/L L-dopa co-fed) under a 5L fermentation tank, the yield of D-salvianic acid A reaches 58.86g/L, and the transformation rate is 97.6%. The method of the invention has great potential and wide value in industry for improving the yield of D-salvianic acid A.
Drawings
FIG. 1 is a schematic diagram of a cascade path of D-tanshinol;
FIG. 2 is a schematic diagram showing construction of gene expression vectors with different copy numbers;
FIG. 3 shows comparison of D-tanshinol yield and conversion rate of 6 recombinant strains with different copy numbers in shake flask transformation
FIG. 4 is a schematic diagram showing construction of gene expression vectors at different RBS levels;
FIG. 5 shows a comparison of D-tanshinol yield and conversion rate of 10 different RBS level recombinant strains in shake flask transformation;
FIG. 6 shows the yield of D-danshensu obtained by fed-batch conversion of the optimal recombinant strain E.coli13 on a 5L fermenter.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
The invention firstly provides a recombinant escherichia coli for efficiently producing D-salvianic acid A, which overexpresses L-amino acid deaminase LAAD H295S,V437S Phenylpyruvate reductase LaPPR and glucose dehydrogenase GDH.
Encoding said L-amino acid deaminase LAAD H295S,V437S The nucleotide sequence of the gene of (2) is shown as SEQ ID NO. 1; the nucleotide sequence of the gene for encoding the phenylpyruvate reductase LaPPR is shown as SEQ ID NO. 2; the nucleotide sequence of the gene encoding the glucose dehydrogenase GDH is shown as SEQ ID NO. 3.
The invention respectively amplifies and codes three enzymes L-amino acid deaminase LAAD H295S,V437S Genes of phenylpyruvate reductase LaPPR and glucose dehydrogenase GDH are respectively connected to pACYC-Duet-1, pCDF-Duet-1, pET-Duet-1 and pRSF-Duet-1 expression vectors according to independent reading frame sequences, the three genes are respectively different in connection type and sequence of each vector, the pathases are assembled to obtain various combination modes, finally, the L-amino acid deaminase and the phenylpyruvate reductase are obtained by screening, the plasmid pRSF-Duet-1 is used for expression, and the glucose dehydrogenase is expressed by the plasmid pCDF-Duet-1, so that the expression vector pCDF-GDH and the expression vector pRSF-LAAD are obtained H295S,V437S The LaPPR is introduced into an escherichia coli host, so that higher D-salvianic acid A yield and conversion rate can be obtained.
In the invention, the recombinant expression vector is formed by sequentially connecting all path enzyme genes with the expression vector according to the sequence of independent open reading frames, wherein the sequence of the open reading frames is as follows: a promoter, a ribosome binding site RBS, a gene encoding a pathway enzyme, and a terminator.
In the invention, RBS with different levels is adopted to regulate and control the enzyme expression level, so that the enzyme activity proportion of in-vivo enzyme is further optimized, and finally, RBS with a nucleotide sequence shown as SEQ ID NO.10 is adopted to regulate and control the expression of phenylpyruvate reductase, and the obtained recombinant bacterium can obtain higher D-salvianic acid A yield and conversion rate.
In the present invention, the host of the recombinant escherichia coli is escherichia coli e.coli BL21, but the host is not limited to the present invention, and other hosts capable of achieving the object of the present invention are also included in the scope of the present invention.
The invention also provides a construction method of the recombinant escherichia coli, which comprises the following steps:
(1) Obtaining single gene expression vectors of L-amino acid deaminase, phenylpyruvate reductase and glucose dehydrogenase;
(2) Amplifying the genes encoding the three enzymes respectively, connecting the genes encoding the L-amino acid deaminase and the phenylpyruvate reductase to pRSF-Duet-1 expression vector according to the independent reading frame sequence, and connecting the gene encoding the glucose dehydrogenase to pCDF-Duet-1 expression vector to obtain recombinant vector pRSF-LAAD H295S,V437S -LaPPR and pCDF-GDH;
(3) And (3) converting the recombinant vector obtained in the step (2) into E.coli BL21 to obtain the E.coli recombinant strain.
The invention also provides an application of the recombinant escherichia coli in the production of the D-salvianic acid A, wherein the application is to collect the recombinant escherichia coli thalli by fermentation in a fermentation medium, take the recombinant escherichia coli thalli as a whole-cell catalyst and catalyze L-dopa and glucose in a whole-cell conversion production system to generate the D-salvianic acid A.
In the invention, the fermentation is carried out by inoculating 1-10% of inoculum size into a fermentation culture medium, culturing for 2-3h, adding 0.1-0.4mmol/L IPTG for induction, centrifuging at 6000rpm for 10min after fermentation is finished at the induction temperature of 25-37 ℃, and preserving recombinant escherichia coli thallus at-20 ℃ for standby.
In the present invention, the fermentation medium is: 25g/L glucose, 20g/L yeast powder, 6g/L disodium hydrogen phosphate, 2g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate and VB 1 1mg/L,VB 2 1.5 mg/L，VB 6 1.5mg/L。
In the invention, the whole fermentation stage also comprises the step of feeding glucose mother liquor to a proper concentration, wherein the ingredients of the feeding liquor are as follows: glucose mother liquor 800g/L.
In the whole cell transformation production system, the recombinant escherichia coli thalli are used as cell catalysts, 5-30g/L L-dopa and 6-40g/L glucose are used as substrates, and Na is used as a substrate 2 SO 3 4-6g/L as antioxidant.
In the whole cell transformation production system, 1-20g/L dopa and 1.2-36g/L glucose are added into the whole cell transformation production system after the whole cell transformation production system reacts for 1-3 hours, and the pH value of the transformation reaction system is regulated to 6.5-7.0.
The invention is further illustrated below in connection with specific examples:
example 1: construction of Patase-associated monogenic expression vectors
The L-amino acid deaminase used in the invention is a laboratory existing mutant LAAD 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. Inoculating Lactobacillus sp.CGMCC 9967 and Bacillus sp.G3 into 25mL LB liquid culture medium, culturing at 37deg.C and 200rpm for 10h, collecting thallus, and extracting Lactobacillus sp.CGMCC 9967 and Bacillus sp.G3 genome DNA by using bacterial genome extraction kit.
Respectively designing primers corresponding to each path enzyme according to the published genome information sequence, and amplifying to obtain corresponding LAAD by using the extracted genome DNA and the existing plasmids in the laboratory as templates and using a standard PCR amplification system and a standard PCR amplification program H295S,V437S LaPPR, GDH gene fragment. After the plasmid pET-28a is digested by BamHI and XhoI, agarose nucleic acid electrophoresis is adopted to carry out gel recovery, and linearized plasmid p is obtained by recoveryET-28a. The gene fragments obtained by PCR amplification are respectively connected with plasmids after double enzyme digestion by adopting one-step homologous recombinase, the system is 20 mu L, and the temperature is 37 ℃ for 30min. The ligation product was transformed into JM109 competent cells, single colonies were picked for PCR verification, positive transformants were sequenced, and the sequencing result was consistent with the theoretical sequence, which demonstrated successful construction of single gene expression vectors, thereby obtaining 3 expression vectors, respectively: pET28a-LAAD H295S,V437S pET28a-LaPPR, pET28a-GDH. The three vectors are used for later replacement of different expression vectors as amplification templates.
Example 2: construction of Path enzyme copy number vectors of different genes
Designing a primer with a homology arm according to the enzyme cutting site, and amplifying to obtain a corresponding LAADH 295S,V437S The LaPPR and GDH gene fragments, and the plasmids pRSF-Duet-1, pCDF-Duet-1 and pET-Duet-1 are subjected to double digestion by BamHI and HindIII, and then gel recovery is carried out by agarose nucleic acid electrophoresis, so that linearized plasmids pRSF-Duet-1, pCDF-Duet-1 and pET-Duet-1 are obtained. The gene fragments obtained by PCR amplification are respectively connected with plasmids after double enzyme digestion by adopting one-step homologous recombinase, the system is 20 mu L, and the temperature is 37 ℃ for 30min. The ligation product was transformed into JM109 competent cells, single colonies were picked for PCR verification, positive transformants were sequenced, and the sequencing result was consistent with the theoretical sequence, which demonstrated successful construction of single gene expression vectors, thereby obtaining 7 expression vectors, each: pRSF-LAAD H295S,V437S 、pRSF-LaPPR、pET-LAAD H295S,V437S 、pET-LaPPR、pET-GDH、pCDF-LaPPR、pCDF-GDH。
Designing primers with homology arms according to enzyme cutting sites, and amplifying to obtain corresponding LaPPR and GDH gene fragments, and obtaining plasmids pCDF-LaPPR, pRSF-LaPPR and pRSF-LAAD H295S,V437S After being subjected to KpnI and XhoI double digestion, pET-LaPPR is subjected to agarose nucleic acid electrophoresis to carry out gel recovery, and linearized plasmids pCDF-LaPPR, pRSF-LaPPR and pRSF-LAAD are obtained by recovery H295S,V437S pET-LaPPR. The gene fragments obtained by PCR amplification are respectively connected with plasmids after double enzyme digestion by adopting one-step homologous recombinase, the system is 20 mu L, and the temperature is 37 ℃ for 30min. Connection productTransforming the substance into JM109 competent cells, selecting single colony for PCR verification, sequencing positive transformant, and confirming that the single gene expression vector is constructed successfully if the sequencing result is consistent with the theoretical sequence, thereby obtaining 5 expression vectors, namely: pCDF-LaPPR-GDH, pET-LaPPR-GDH, pRSF-LAAD H295S,V437S -GDH、pRSF-LAAD H295S,V437S -LaPPR。
The successfully constructed expression vectors are combined two by two according to the expression requirement of the pathase, and the double plasmids are simultaneously transformed into BL21 competent cells, thereby obtaining 6 recombinant bacteria, namely E.coli1 (pRSF-LAAD) H295S,V437S /pCDF-LaPPR-GDH)、E.coli2(pRSF-LAAD H295S,V437S /pET-LaPPR-GDH)、E.coli3(pE T-LAAD H295S,V437S /pRSF-LaPPR-GDH)、E.coli4(pET-LaPPR/pRSF-LAAD H295S,V437S -GDH)、E.coli5(pET-GDH/pRSF-LAAD H295S,V437S -LaPPR)、E.coli6(pCDF-GDH/pRSF-LAAD H295S,V437S -LaPPR)。
Example 3: recombinant bacterium E.coli1-E.coli6 shake flask fermentation
Culturing recombinant bacteria: the monoclonal was inoculated into 50mL (250 mL shaking flask) LB medium as seed liquid for shaking flask fermentation, and cultured at 37℃and 200rpm for 10 hours. The seed liquid inoculation amount is 2%, the seed liquid is inoculated to 150mL of fermentation culture medium, and is cultured at 37 ℃ until OD 600 IPTG was added to a final concentration of 0.4mM at 25℃for 14 hours, and after completion, the cells were collected by centrifugation at 6000rpm for 10 minutes and stored at-40℃to obtain the whole cell catalyst required for bioconversion. Fermentation medium components: 25g/L glucose, 20g/L yeast powder, 6g/L disodium hydrogen phosphate, 2g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate and VB 1 1mg/L,VB 2 1.5 mg/L，VB 6 1.5mg/L。
Shake flask transformation system: the recombinant bacteria were suspended in 20mM Tris-HCl buffer, L-dopa 30g/L,36g/L glucose, 20g/L wet cells, 5g/L sodium sulfite, and reacted at 30℃for 16 hours, while maintaining the pH around 7.0 using 2M NaOH.
According to the yield measurement of D-salvianic acid, the result is shown in FIG. 3, the optimal recombinant strain E.coli6 can produce 25.3g/L D-salvianic acid with a conversion rate of 83.9% (30 g/L L-dopa) after 16h of transformation.
Example 4: gene expression vector construction and shake flask transformation comparison of different RBS levels
On the basis of successfully constructing expression vectors with different gene copy numbers in example 2, after biological transformation is evaluated by taking L-dopa as a substrate, the vector with the optimal three-enzyme gene combination mode is obtained, 10 RBSs with different intensities are arranged as replacement primers, RBSs of the vector in front of the LaPPR gene are replaced, firstly, full-plasmid PCR is carried out, PCR products are purified, DPNI is utilized to remove an original template, the digested products are transformed into BL21 competent cells, correct sequencing is carried out through colony PCR verification strips, and the correct sequencing shows that the gene expression vectors with different RBS levels are successfully constructed, so that 10 recombinant bacteria, namely E.coli7, E.coli8, E.coli9, E.coli10, E.coli11, E.coli12, E.coli13, E.coli14, E.coli15 and E.coli16 are finally obtained.
RBS sequence employed in Table 1
Figure BDA0003845908710000071
The recombinant culture conditions and transformation conditions were the same as in example 4. According to the yield measurement of D-salvianic acid A, the result is shown in FIG. 5, the optimal recombinant strain E.coli13 can produce 29.71g/L D-salvianic acid A with a conversion rate of 98.53% (30 g/LL-dopa) after 16h of transformation.
Example 5: optimal recombinant strain E.coli13 fed-batch conversion in 5L fermentors
Culturing recombinant bacteria: e.coli13 recombinant strain was picked up and subjected to tank fermentation. The single clone was inoculated in 50mL (250 mL shaking flask) LB medium as seed solution for the upper tank, and cultured at 37℃and 200rpm for 10 hours. The liquid amount of the fermentation tank is 2L, and the fermentation culture medium is: 25g/L glucose, 20g/L yeast powder, 6g/L disodium hydrogen phosphate, 2g/L potassium dihydrogen phosphate, 3g/L magnesium sulfate and VB 1 1mg/L,VB 2 1.5 mg/L，VB 6 1.5mg/L. The pH in the whole fermentation process is controlled to be about 7.0. Inoculating into fermentation tank according to 2% inoculum size, inoculating into fermentation medium with seed solution inoculum size of 2%, maintaining dissolved oxygen level of 25+ -1.5%, air supply rate of 6L/min, stirring at 100-500rpm, and culturing at 37deg.CTo OD 600 15, IPTG was added to a final concentration of 0.4mM, induction was performed at 25℃for 14 hours, and after completion, the cells were collected by centrifugation at 6000rpm for 10 minutes and stored at-40℃to obtain a whole cell catalyst required for bioconversion.
5L fermenter conversion (1L reaction System): the recombinant bacterial cells are suspended in 20mM Tris-HCl buffer solution, 20g/L L-dopa, 24g/L glucose and 4g/L sodium sulfite are initially fed, then 10g/L L-dopa and 12g/L glucose are respectively fed every 2 hours, the total feeding is four times, and the total feeding is 60g/L L-dopa, 72g/L glucose and 4g/L sodium sulfite. The reaction is carried out at 30 ℃, 2M NaOH is used for maintaining the pH near 7.0 in the reaction process, the stirring speed is 400rpm, the total reaction is carried out for 16 hours, and the contents of L-dopa, 3,4-dihydroxyphenyl pyruvic acid and D-salvianic acid in the sample are detected by sampling every 2 hours.
According to the measurement of the yield of D-salvianic acid A, the result is shown in FIG. 6, the yield of D-salvianic acid A reaches 58.86g/L and the conversion rate is 97.6% (60 g/L L-dopa co-fed).
The results show that the technology adopts the genetic engineering technology to change the intensity of RBS and the copy number of plasmid to finely regulate and control the enzyme L-amino acid deaminase (LAAD) in the D-salvianic acid A cascade path H295S,V437S ) The expression level of phenylpyruvate reductase (LaPPR) and Glucose Dehydrogenase (GDH) can effectively improve the yield of D-tanshinol.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A recombinant escherichia coli for efficiently producing D-salvianic acid, which is characterized in that the recombinant escherichia coli overexpresses L-amino acid deaminase LAAD H295S,V437S Phenylpyruvate reductase LaPPR and glucose dehydrogenase GDH.
2. The recombinant escherichia coli according to claim 1, wherein the nucleotide sequence of the gene encoding the L-amino acid deaminase is shown as SEQ ID No. 1; the nucleotide sequence of the gene for encoding the phenylpyruvate reductase is shown as SEQ ID NO. 2; the nucleotide sequence of the gene for encoding the glucose dehydrogenase is shown in SEQ ID NO. 3.
3. The recombinant E.coli according to claim 1, wherein the L-amino acid deaminase and phenylpyruvate reductase are expressed using plasmid pRSF-Duet-1 and the glucose dehydrogenase is expressed using plasmid pCDF-Duet-1.
4. The recombinant E.coli according to claim 1, wherein RBS having a nucleotide sequence shown in SEQ ID NO.10 is used to regulate the expression of phenylpyruvate reductase.
5. The recombinant escherichia coli according to claim 1, wherein the host of the recombinant escherichia coli is escherichia coli e.coli BL21.
6. A method for constructing recombinant E.coli according to any one of claims 1 to 5, wherein the method comprises the steps of:
(1) Obtaining single gene expression vectors of L-amino acid deaminase, phenylpyruvate reductase and glucose dehydrogenase;
(2) Amplifying the genes encoding the three enzymes respectively, connecting the genes encoding the L-amino acid deaminase and the phenylpyruvate reductase to pRSF-Duet-1 expression vector according to the independent reading frame sequence, and connecting the gene encoding the glucose dehydrogenase to pCDF-Duet-1 expression vector to obtain recombinant vector pRSF-LAAD H295S,V437S -LaPPR and pCDF-GDH;
(3) And (3) converting the recombinant vector obtained in the step (2) into E.coli BL21 to obtain the E.coli recombinant strain.
7. The use of the recombinant escherichia coli according to any one of claims 1 to 5 for producing D-salvianic acid, wherein the use is to ferment and collect the recombinant escherichia coli thalli in a fermentation medium, and catalyze L-dopa and glucose in a whole cell transformation production system by taking the recombinant escherichia coli thalli as a whole cell catalyst.
8. The use according to claim 7, wherein the fermentation is carried out by inoculating 1-10% of the inoculum size into a fermentation medium, culturing for 2-3h, adding 0.1-0.4mmol/L IPTG for induction, and centrifuging to collect recombinant E.coli cells after fermentation is completed at 25-37 ℃.
9. The use according to claim 7, wherein in the whole cell transformation production system, the recombinant E.coli cells are used as cell catalyst, 5-30g/L L-dopa, 6-40g/L glucose are used as substrate, na is used as substrate 2 SO 3 4-6g/L as antioxidant.
10. The use according to claim 7, wherein 1-20g/L dopa and 1.2-36g/L glucose are added to the whole cell conversion production system after 1-3 hours of reaction, and the pH of the conversion reaction system is adjusted to 6.5-7.0.
CN202211116758.2A 2022-09-14 2022-09-14 Recombinant escherichia coli for efficiently producing D-salvianic acid, and construction method and application thereof Pending CN116064348A (en)

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