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    • 专利摘要:
    Provided are a recombinant E. coli and the use thereof in the production of danshensu. The recombinant E. coli simultaneously expresses α-hydroxycarboxylic acid dehydrogenase, L-α-amino acid aminotransferase, and any one of glucose dehydrogenase, L-lactic dehydrogenase and L-glutamate dehydrogenase, and genes thereof involved in the decomposition of phenolic compounds are knocked out on the basis of host E. coli.
    公开号:ES2825205A2
    申请号:ES202090050
    申请日:2018-10-25
    公开日:2021-05-14
    发明作者:Yujie Cai；Tianzhen Xiong；Jinbin Liu；Yanrui Ding；Yajun Bai；Xiaohui Zheng
    申请人:Jiangnan University；
    IPC主号:
    专利说明:

    [0002] Strain modified by genetic engineering and its application in the production of danshensu
    [0004] Technical field
    [0006] The present invention relates to a genetically engineered strain and its application in the production of danshensu, and belongs to the technical field of bioengineering.
    [0008] Background of the technique
    [0010] The danshensu extracted from Salvia miltiorrhiza, has the scientific names of R - (+) - 3- (3,4-dihydroxyphenyl) -2-hydroxypropanoic acid and D - (+) - p- (3,4-dihydroxyphenyl) lactic acid , has the common names danshensu, D-DSS, R-DSS, (R) - (+) - 3- (3,4-dihydroxyphenyl) -lactic acid and (R) - (+) - 3- (3 , 4-dihydroxyphenyl) -2-hydroxypropanoic, and is a dextrophenolic acid compound. Currently, there is no natural L-danshensu.
    [0012] Danshensu is an important active principle in an aqueous extract of Salvia miltiorrhiza, and the structure in the aqueous extract of Salvia miltiorrhiza was obtained and identified in China in 1980 (Study on Water-soluble Active Ingredients of Salvia miltiorrhiza, Structure of ND (+ ) P (3,4-Dihydroxyphenyl) lactic acid, Shanghai First Medical College Journal, 1980, 05 (7), 384-385). Various studies have shown that danshensu has important pharmacological effects and has unique therapeutic effects in the treatment of cardiovascular and cerebrovascular diseases.
    [0014] At present, danshensu is extracted mainly from Salvia miltiorrhiza (patent CN200810038853.9). The danshensu content in Salvia miltiorrhiza is low, the cost to plant Salvia miltiorrhiza is high, and the yield is limited. Therefore, danshensu is not only expensive but also far from meeting market demand. The patent CN201310559498.0 proposes a method to construct a strain modified by genetic engineering of Escherichia coli to produce danshensu by means of glucose fermentation. Since the anabolic pathway involves the use of a hydroxylase, the enzyme is likely to oxidize a product of the metabolic process and affect the performance of danshensu. At the same time, since E. coli fermentation is an oxygen-intensive process, it also oxidizes danshensu. Therefore, the current method has a lower yield, and the cost will be higher than that of a plant extraction process. Patent CN201210190171.6 proposes a method to produce danshensu by hydrolyzing salvianolic acid B. It is necessary to extract salvianolic acid B from Salvia miltiorrhiza, and the Chemical hydrolysis process has a large number of side reactions, which is also not suitable for large-scale production. A catalyst for the chiral synthesis of danshensu (patent CN201210420488.4) is extremely expensive and is currently only maintained at the laboratory level.
    [0016] As early as 1988, Roth et al. proposed a method to treat levodopa by a chemical method to obtain the corresponding 3,4-dihydroxyphenylpyruvic acid, and then synthesize S - (+) - 3- (3,4-dihydroxyphenyl) -2-hydroxypropanoic acid (S-DSS, L -DSS) by an enzymatic method (Enzymatic Synthesis of (S) - (-) - 3- (3,4-Dihydroxyphenyl) lactic Acid, Arch. Pharm. (Weinheim) 321, 179-180 (1988)). Z. Findrik, et al. They used snake venom amino acid oxidase to convert levodopa to 3,4-dihydroxyphenylpyruvic acid, then used D-lactate dehydrogenase for reduction to produce D- (3,4-dihydroxyphenyl) lactic acid (Modeling and Optimization of the (R) - ( +) - 3,4-dihydroxyphenyllactic Acid Production Catalyzed with D-lactate dehydrogenase from Lactobacillus leishmannii Using Genetic Algorithm, Chem. Biochem. Eng. Q. 19 (4) 351-358 (2005)). The preparation of the 3,4-dihydroxyphenylpyruvic acid intermediate by the two methods is expensive and complicated to operate.
    [0018] Summary of the invention
    [0020] Building on the shortcomings of various current methods, the present invention provides an optically pure transaminase-based danshensu production method, and constructs a multi-enzyme co-expression engineered strain to achieve efficient danshensu production. The technical problem to be solved by the present invention is to provide a recombinant strain that can produce danshensu at low cost. At the same time, the present invention has to solve the technical problems of construction and application of the strain.
    [0022] The present invention is primarily concerned with a recombinant strain that can produce optically pure danshensu at low cost. The recombinant strain expressed simultaneously -hidroxicarboxilato dehydrogenase and the -amino acid transaminase, and any one of the following: glucose dehydrogenase, L-lactate dehydrogenase and L-glutamate dehydrogenase, and deactivates a gene related to the decomposition of phenolic compounds based on a host E. coli.
    [0024] In one example, the -hidroxicarboxilato a dehydrogenase is a dehydrogenase D- -hidroxicarboxilato, and is plantarnm ATCC 14917 Lactobacillus, Enterococcus faecalis ATCC 35038 or Lactobacillus fermentum ATCC 14931.
    [0025] In one example, the dehydrogenase is a L- -hidroxicarboxilato to -hidroxicarboxilato dehydrogenase, and Bacillus coagulans DSM 1 strain DSM 20196 Weissella confused fermentum ATCC 14931 or Lactobacillus.
    [0027] In one example, the dehydrogenase is D to -hidroxicarboxilato to -hidroxicarboxilato dehydrogenase, whose amino acid sequence has a registration No. WP_003643296.1, WP_002335374.1 or EEI22188.1 in NCBI.; the dehydrogenase is a L- -hidroxicarboxilato to -hidroxicarboxilato dehydrogenase, whose amino acid sequence has a no. WP_013858488.1 registration, WP_003607654.1 or WP_035430779.1 in NCBI.
    [0029] In one example, the nucleotide sequence of D- a -hydroxycarboxylate dehydrogenase has a registration number of NZ_GL379761 REGION: COMPLEMENT (533562..534560), NZ_KB944641 REGION: 161892..162830, or ACGI01000078 REGION: 20793..21791 in NCBI; The nucleotide sequence of L- a -hydroxycarboxylate dehydrogenase has a NCBI Accession No. of NZ_ATUM01000014 REGION: 39316..40254, NZ_JQAY01000006 REGION: 69708..70640, or NZ_GG669901 REGION: 45517..46470.
    [0031] In one example, the L- a- amino acid transaminase is from E. coli BL21, Lactobacillus plantarum ATCC 14917 or Lactobacillus paracasei ATCC 334.
    [0033] In one example, the amino acid sequence of L - amino acid transaminase to have a no. WP_000462687.1 registration, WP_000486988.1, WP_003643296.1 or YP_806114.1 in NCBI.
    [0035] In one example, the nucleotide sequence of L- a -amino acid transaminase has a registration number of NC_012892 REGION: COMPLEMENT (989603..990793), NC_012892 REGION: 4174517..4175710, NZ_GL379768 REGION: complement (121900 .. 123087), or NC_008526 REGION: complement (840419..841594) in NCBI.
    [0037] In one example, glucose dehydrogenase is from Bacillus subtilis ATCC 13952.
    [0039] In one example, the amino acid sequence of glucose dehydrogenase has an NCBI accession # of WP_013351020.1.
    [0041] In one example, the nucleotide sequence of glucose dehydrogenase has an NCBI Accession # of NZ_CP009748 REGION: 386154..38693.
    [0043] In one example, the L-lactate dehydrogenase is from Lactococcus lactis ATCC 19257.
    [0045] In one example, the amino acid sequence of L-lactate dehydrogenase has a # WP_003131075.1 registration in NCBI.
    [0047] In one example, the nucleotide sequence of L-lactate dehydrogenase has NCBI accession # of NZ_JXJZ01000017 REGION: 18532..19509.
    [0049] In one example, L-glutamate dehydrogenase is from E. coli BL21, Rhodobacter sphaeroides ATCC BAA-808, Clostridium symbiosum ATCC 14940, or Bacillus subtilis 168.
    [0050] In one example, the amino acid sequence of L-glutamate dehydrogenase has an NCBI accession number of WP_000373021.1, WP_011338202.1, WP_003497202.1, or WP_010886557.1.
    [0052] In one example, the nucleotide sequence of L-glutamate dehydrogenase has an accession # of NC_012892 REGION: 1786741..1788084, NC_007493 REGION: complement (2129131..2130558), NZ_KE992901 REGION: complement (17603..18955) o NC_000964 REGION: complement (2402067..2403350) in NCBI.
    [0054] In one example, the recombinant strain is a genetically engineered recombinant strain obtained by ligation of genes encoding L- a- amino acid transaminase, a -hydroxycarboxylate dehydrogenase, and any one of glucose dehydrogenase, L-lactate dehydrogenase, and L-glutamate dehydrogenase. to a plasmid to construct a recombinant three gene co-expression plasmid, and then transformation of the recombinant plasmid into a corresponding strain.
    [0056] In one example, the recombinant strain is constructed using E. coli BL21 (DE3) as the host.
    [0058] In one example, the gene related to the breakdown of phenolic compounds is any one or a combination of hpaD and mhpB.
    [0060] In one example, the nucleotide sequence of the phenolic breakdown gene has a registration number of NC_012892 REGION: complement (4505585..4506436) and NC_012892 REGION: 339806..340750 in NCBI.
    [0062] In one example, the recombinant strain further enhances the expression of one or more pyruvic acid transporter genes, an L-lactic acid transporter gene, a glutamic acid transporter gene, a NAD synthesis gene, and a gene of pyridoxal phosphate synthesis, in which the pyruvic acid transporter genes, the L-lactic acid transporter gene and the glutamic acid transporter gene are not expressed at the same time.
    [0063] In one example, boosted expression adds a constitutive promoter in front of the gene whose expression is to be boosted in an E. coli BL21 (DE3) genome.
    [0065] In one example, the gene whose expression is to be enhanced is any one or more of the pyruvic acid transport related genes, lldP (lactic acid transporter gene), gltS (glutamic acid transporter gene), nadA (gene synthesis gene) and pdxJ (pyridoxal phosphate synthesis gene), in which genes related to pyruvic acid transport include btsT and ybdD (gene for transporting pyruvic acid into cells).
    [0067] In one example, the NCBI registration numbers for btsT and ybdD are: for nadA, NC_012892 REGION: 740487..741530; for pdxJ, NC_012892 REGION: complement (2567591..2568322).
    [0069] In one example, the lldP registration # at NCBI is: NC_012892 REGION: 3646638..3648293; for nadA, NC_012892 REGION: 740487..741530; for pdxJ, NC_012892 REGION: complement (2567591..2568322).
    [0071] In one example, the NCBI gltS registration # is: NC_012892 REGION: plugin (3694931..3696136); for nadA, NC_012892 REGION: 740487..741530; for pdxJ, NC_012892 REGION: complement (2567591..2568322).
    [0073] The present invention further relates to a method for producing danshensu using the recombinant strain of the present invention.
    [0075] In one example, danshensu production is carried out by whole cell transformation production.
    [0077] In one example, in a whole cell transformation production system, the wet weight of cells is 1-200 g / L and the levodopa concentration is 1-200 g / L.
    [0079] When the recombinant strain simultaneously expressed -hidroxicarboxilato dehydrogenase, L- -amino acid transaminase to glucose dehydrogenase and in the production system for processing whole cell, the concentration of pyruvic acid is 1-200 g / l and the glucose concentration is from 1-200 g / l.
    [0081] When the recombinant strain simultaneously expressed -hidroxicarboxilato dehydrogenase, transaminase and -amino acid to L-lactate dehydrogenase L, the production system for processing whole cell includes L-lactic acid of 1-200 g / l.
    [0082] When the recombinant strain expresses simultaneously -hidroxicarboxilato dehydrogenase, L- a- amino acid transaminase and L-glutamate dehydrogenase, the whole cell transformation production system includes 1200 g / L L-glutamic acid.
    [0084] The whole cell transformation production system has a pH of 6.0-9.0, and reacts at 15-40 ° C for 1-48 hours.
    [0086] Beneficial effects of the present invention:
    [0088] The present invention constructs a novel multi-enzyme coexpression engineered strain, and performs low-cost production of danshensu. Furthermore, the transport of a substrate is promoted and the breakdown of products is reduced by the inactivation or enhanced expression of related genes in the E. coli genome. The (D / L) - to -hidroxicarboxilato dehydrogenase selected by the present invention has the characteristics of low substrate specificity and strong optical specificity, and can produce D-and L-danshensu danshensu optically pure. The production process is simple, raw materials are readily available, impurities are few and good prospects for industrial application are achieved.
    [0090] Detailed description of the invention
    [0092] The functional core of the genetically engineered strain provided by the present invention is that three enzymes, respectively L- a- amino acid transaminase, a -hydroxycarboxylate dehydrogenase, and any one of glucose dehydrogenase, L-lactate dehydrogenase and L-glutamate dehydrogenase, they can be expressed simultaneously. The principle is: in the whole cell of the genetically engineered strain, glucose dehydrogenase or L-lactate dehydrogenase or L-glutamate dehydrogenase uses NAD in the cell as a coenzyme to dehydrogenize the corresponding glucose, or L-lactate dehydrogenase or L glutamate dehydrogenase generates NADH and pyruvic acid or gluconic acid or corresponding -cetoglutárico acid; levodopa is deaminated by L - α-amino acid transaminase to generate 3,4-dihydroxyphenylpyruvic acid, and pyruvic acid is converted to alanine by ammonia; -hidroxicarboxilato the dehydrogenase used to NADH produced by the dehydrogenation process of glucose to reduce 3,4-dihidroxifenilpirúvico acid danshensu and simultaneously regenerating the coenzyme NAD. Furthermore, the transport of a substrate is promoted and the breakdown of products is reduced by the inactivation or enhanced expression of related genes in an E. coli genome.
    [0093] To solve the above technical problems, the technical solution adopted by the present invention is as follows:
    [0095] 1. Strains and plasmids of the present invention
    [0097] Lactobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038, Lactobacillus fermentum ATCC 14931, Lactobacillus paracasei ATCC 334, Bacillus subtilis ATCC 13952, E. coli BL21 (DE3) and Lactococcus lactis ATCC 19257 (American Type Collection ATCC 19257). Bacillus coagulans DSM 1 and Weissella confusa strain DSM 20196 acquired from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, German Collection of Microorganisms and Cell Cultures). The plasmids PETDuet-1, pACYCDue-1, pCOLADuet-1 and pRSFDuet-1 and E. coli BL21 (DE3) were purchased from Novagen.
    [0099] 2. Inactivation and enhanced constitutive expression of related genes in E. coli
    [0100] (1) Deactivation of genes related to the breakdown of phenolic compounds in E. coli
    [0102] The phenolic substances in the present invention are highly susceptible to decomposition by enzymes in E. coli. According to the bibliography (Biodegradation of Aromatic Compounds by E. coli, Microbiol Mol Biol Rev.2001,65 (4): 523-569.), Related genes are deactivated to avoid the decomposition of products and substrates. The selected genes are hpaD and mhpB, whose NCBI registration numbers are NC_012892 REGION: complement (4505585..4506436) and NC_012892 REGION: 339806..340750.
    [0104] (2) Enhanced constitutive expression of pyruvic acid transporter gene from E. coli
    [0105] In the whole cell transformation process, it is necessary to transport the substrate into the cell to carry out the reaction. Enhancing the pyruvic acid transporter protein is useful for the rapid and long-term maintenance of a high concentration of the intracellular substrate, and is favorable for the advancement of the reaction. The selected pyruvic acid transport related genes are btsT and ybdD, whose NCBI record numbers are NC_012892 REGION: complement (4496239..4498389) and NC_012892 REGION: 592652..592849. Dopa is similar to aromatic amino acids, and it is necessary to absorb amino acids and the like during cell culture. Therefore, the cells themselves express a large amount of the amino acid transporter protein, and it is not necessary to enhance the expression.
    [0107] (3) Enhanced constitutive expression of important genes related to the synthesis of E. coli coenzyme
    [0109] In the process of reduction to -hidroxicarboxilato dehydrogenase it is necessary to use NADH as a coenzyme. Enhanced expression of key enzymes in an E. coli NAD synthesis pathway can increase the level of NAD in cells, thereby being beneficial for danshensu production. The selected gene is nadA. The NCBI registration number is: NC_012892 REGION: 740487..741530.
    [0111] Pyridoxal phosphate (amine) is a coenzyme of L- a- amino acid transaminase, and overexpression of the core pdxJ gene in the coenzyme pathway is beneficial for levodopa synthesis. The registration number in NCBI is: NC_012892 REGION: complement (2567591..2568322).
    [0113] 3. Selection of enzymes
    [0115] (1) Selection of L- a -amino acid transaminase
    [0117] -Amino acid transaminase La is present widely in bacteria, fungi and mammalian cells. Usually, a transaminase with -cetoglutárico acid or oxaloacetic acid as acid receptor ammonia is the most active. In this process, the acid or oxaloacetic acid -cetoglutárico is expensive, while the value of the glutamic acid or aspartic acid produced accordingly is much smaller than that of the corresponding precursor. A comprehensive review of the corresponding ketoacids to 20 L-amino acids reveals that natural prices pyruvic acid and alanine are comparable. Thus, the present invention selects pyruvic acid as the ammonia receptor for co-production of alanine and danshensu. The genes for L- a -amino acid transaminase lpt and lct are cloned from Lactobacillus plantarum ATCC 14917 and Lactobacillus paracasei ATCC 334, respectively, and their amino acid sequences have the accession numbers of WP_003643296.1 and YP_806114 .1 in NCBI. Two L- a- amino acid transaminase genes ectl and ect2 are cloned from E. coli BL1 (DE3), and their amino acid sequences have NCBI accession numbers WP_000462687.1 and WP_000486988.1 .
    [0119] (2) Selection of a dehydrogenase -hidroxicarboxilato
    [0121] According to the optimal substrate, it contains lactate dehydrogenase to -hidroxicarboxilato dehydrogenase, a hydroxyacid dehydrogenase isohexanoato, mandelic acid dehydrogenase, glyoxylate reductase, etc. These enzymes can act extensively on a variety of substrates to generate hydroxycarboxylic acids, usually named according to their substrates of optimal function. The present invention selects enzymes that have high optical properties and have strong activity against 3,4-dihydroxyphenylpyruvic acid, to produce D- or L-danshensu. The D- a -hydroxycarboxylate dehydrogenase genes lpldhd, efmdhd and lfldhd are cloned, respectively, from Lactobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038 and Lactobacillus fermentum ATCC 14931, and the same amino acid sequences have the same number of amino acids. registration of WP_003643296.1, WP_002335374.1 and EEI22188.1 in NCBI. The genes for L- a -hydroxycarboxylate dehydrogenase bcldhl, wcldhl and lfldhl are obtained, respectively, from Bacillus coagulans DSM 1, Weissella confusa strain DSM 20196 and Lactobacillus fermentum ATCC 14931, and their amino acid sequences have the no. Of Registration of WP_013858488.1, WP_003607654.1 and WP_035430779.1 in NCBI.
    [0123] (3) Selection of glucose dehydrogenase
    [0125] In biotransformation reactions, the NADH required to -hidroxicarboxilato dehydrogenase and / or NADPH as a coenzyme, usually formate dehydrogenase, glucose dehydrogenase, phosphite, etc. Glucose dehydrogenase is the most active compared to other enzymes and therefore the present invention obtains the glucose dehydrogenase gene bsgdh (whose amino acid sequence is WP_013351020.1) from Bacillus subtilis ATCC 13952.
    [0127] (4) Selection of L-lactate dehydrogenase
    [0129] L-lactic acid is the cheapest organic acid, and alanine produced by transamination of pyruvic acid after dehydrogenation has a high added value. L-lactate dehydrogenase is widely present in a variety of microorganisms. Using L-lactic acid as a substrate, the hydrogen generated in L-lactic acid is transferred to the coenzyme NAD or NADP to produce NADH or NADPH. NADH or NADPH may be used as the hydrogen donor dehydrogenase hydroxycarboxylic acid mentioned above. Generally, lactate dehydrogenase with NAD (NADP) as a coenzyme tends to synthesize lactic acid with pyruvic acid as a substrate. However, when lactic acid is excessive, and the like, lactate dehydrogenase will remove hydrogen from lactic acid to produce pyruvic acid. The present invention obtains the gene for L-lactate dehydrogenase llldh (whose amino acid sequence is WP_003131075.1) from Lactococcus lactis ATCC 19257.
    [0131] (5) Selection of L-glutamate dehydrogenase
    [0132] The L-glutamic acid is the most economical amino acid, and the acid -cetoglutárico produced after the dehydrogenation can be used as receiver for ammonia transamination and deamination of levodopa. L-glutamate dehydrogenase is widely found in almost all organisms. Using L-glutamic acid as a substrate, the hydrogen produced in L-glutamic acid is transferred to the coenzyme NAD or NADP to produce NADH or NADPH. NADH or NADPH can be used as the hydrogen donor of the above-mentioned hydroxycarboxylic acid dehydrogenase. The present invention obtains the genes for L-glutamic acid ecgdh (whose amino acid sequence is WP_000373021.1), rsgdh (whose amino acid sequence is WP_011338202.1), csgdh (whose amino acid sequence is WP_003497202.1) and bsgdh (whose amino acid sequence is WP_010886557.1) respectively from E. coli BL21, Rhodobacter sphaeroides ATCC BAA-808, Clostridium symbiosum ATCC 14940 and Bacillus subtilis 168.
    [0134] 4. Construction of a three enzyme co-expression system and cell culture
    [0136] At present, there are many methods for the co-expression of multiple E. coli genes ( E. coli Multi-gene Co-expression Strategy, China Biotechnology, 2012, 32 (4): 117-122). The present invention adopts a method described by Liu Xianglei (Production of Shikimic Acid and Resveratrol by Transformation of E. coli by Synthetic Biotechnology, 2016, Shanghai Pharmaceutical Industry Research Institute, PhD thesis). Each gene contains a T7 promoter and an RBS binding site on the front, and each gene has a T7 terminator on the back. Theoretically, since each gene has T7 and RBS in the front, the intensity of expression of the gene is less affected by the order. Each plasmid contains three genes (genes corresponding to L- a -amino acid transaminase, (D / L) - a -hydroxycarboxylate dehydrogenase, and glucose dehydrogenase or L-lactate dehydrogenase or L-glutamate dehydrogenase). The constructed plasmid is hot transferred to competent E. coli cells, and competent E. coli cells are coated on a solid plate with antibiotics. Positive transformants are obtained by selection, that is, recombinant E. coli is obtained. Cells are grown according to a standard recombinant E. coli culture and induction expression program. Recombinant E. coli is transferred to LB fermentation medium (containing 10 g / l peptone, 5 g / l yeast powder, and 10 g / l NaCl) at a 2% volumetric ratio. After the cell OD 600 reaches 0.6-0.8, IPTG is added with a final concentration of 0.4 mM, and expression is carried out by induction and culture at 20 ° C for 8 h. After completion of induction expression, cells are harvested by centrifugation at 20 ° C and 8000 rpm for 20 minutes.
    [0137] 5. Production by whole cell transformation of pure danshensu
    [0138] In a cell transformation production system, the wet weight of cells is 1-200 g / l, and the levodopa concentration is 1-200 g / l.
    [0140] When the recombinant strain expresses simultaneously a -hydroxycarboxylate dehydrogenase, L- a -amino acid transaminase and glucose dehydrogenase, in the whole cell transformation production system, the pyruvic acid concentration is 1-200 g / l, and the concentration of glucose is 1-200 g / l.
    [0142] When the recombinant strain simultaneously expressed -hidroxicarboxilato dehydrogenase, L- -amino acid transaminase to L-lactate dehydrogenase and the production system for processing whole cell further it includes L-lactic acid of 1 200 g / l.
    [0144] When the recombinant strain simultaneously expresses a -hydroxycarboxylate dehydrogenase, L- a- amino acid transaminase and L-glutamate dehydrogenase, the whole cell transformation production system further includes 1-200 g / L L-glutamic acid.
    [0146] The system that produces whole cell transformation has a pH of 6.0-9.0, and reacts at 15-40 ° C for 1-48 hours.
    [0148] After the end of transformation, the yield and danshensu configuration are determined by liquid chromatography. Levodopa has a low solubility and is a suspension that contains insoluble matter at high concentrations.
    [0150] 6. Detection and analysis of samples
    [0152] Quantitative danshensu analysis: The transformed broth is detected and analyzed by a PerkinElmer 200 series high performance liquid chromatograph with a differential refractive index detector. The chromatographic conditions are as follows: the mobile phase is methanol-0.1% formic acid in water (40:60), a C18 Hanbon Megres chromatographic column is used (4.6 ^ 250 mm, 5. ^ M), the flow rate is 1 ml / min, the column temperature is 30 ° C, and the injection volume is 20 ^.
    [0153] Chiral analysis: PerkinElmer 200 series high performance liquid chromatograph with UV detector is used for detection and analysis, Chiralcel OD-H chiral column (4.6x250mm) is used, the volumetric phase ratio n-hexane with respect to isopropanol with respect to trifluoroacetic acid is 80: 20: 0.1, the flow rate is 0.5 ml / min, the column temperature is 25 ° C, the injection volume is 20.1, and the detection wavelength is 280 nm.
    [0155] Danshensu has a relatively low solubility, and if crystallization occurs in the transformation process, the measurement is carried out after dilution.
    [0157] The optical purity of danshensu is evaluated by an enantiomeric excess value (% e.e.).
    [0159] When R-danshen is produced,
    [0161] the enantiomeric excess value,% ee = [(S r -S s ) / (S r + S s ) x 100%]
    [0163] When S-danshen occurs,
    [0165] the enantiomeric excess value,% ee = [(S s -S r ) / (S r + S s ) x 100%]
    [0167] where Ss is the S-danshensu peak area in the transformed broth, and S r is the R-danshen liquid chromatography peak area in the transformed broth.
    [0169] In order to clarify the technical problems to be solved, the technical solutions and the beneficial effects of the present invention, the present invention will be described in detail below, with reference to examples. It should be noted that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention.
    [0171] Example 1
    [0173] Using a method according to the literature, Large scale validation of an efficient CRISPR / Cas-based multi gene editing protocol in E. coli. Microbial Cell Factories, 2017, 16 (1): 68, HpaD and mhpB are individually or double inactivated in E. coli BL21 (DE3). The gene knockout plasmids used in the present invention are pCasRed and pCRISPR-gDNA (hpaD sgRNA) which are introduced into E. coli BL21 (DE3) together with a homologous arm (hpaD donor). Cas9 / sRNA induces a double-stranded break at the hpaD gene locus in the host, the Red recombinase integrates the hpaD donor on the hpaD gene to effect gene knockout, and the gene is verified by sequencing. The hpaD sgRNA, hpaD donor, mhpB sgRNA, and mhpB donor, respectively, as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. MhpB is disabled in the same way.
    [0175] A solution having a pH of 7 is prepared with the 4 g / l levodopa or D-danshensu, the amount of wet cells is 200 g / l, and the concentration is measured after the solution is put at 35 ° C for 10 hours. The residual amounts of levodopa and D-danshenin in the reaction system are shown in Table 1.
    [0177] Table 1 Residual concentrations of substrates and decomposed products by different strains
    [0181] Obviously, E. coli BL21 (AhpaDAmhpB, DE3) has the best effect and is called E. coli HM.
    [0183] Example 2
    [0185] While selecting L- -amino acid transaminase to a variety of genes cloned L - -amino acid transaminase to from E. coli and lactobacilli, respectively, and expressed in E. coli BL21 (DE3). The activity of the crude enzyme is determined by cellular disruption, and the activities of various enzymes are compared with pyruvic acid as a receptor. The activity of L- a- amino acid transaminase is determined according to the literature (Transaminase-catalyzed asymmetric synthesis of aromatic L-amino acids. Chinese Journal of Biotechnology, 2012, 28 (11): 1346-1358.). The results are shown in Table 2. Therefore, it is preferred to select E. coli- derived L- a -amino acid transaminase ect1 for transamination and deamination of levodopa.
    [0187] The activities of various enzymes compared to -cetoglutárico as acid receptor. The activity of L- a- amino acid transaminase is determined according to the method described in the literature (Transaminase-catalyzed asymmetric synthesis of aromatic L-amino acids. Chinese Journal of Biotechnology, 2012, 28 (11): 1346-1358.). The results are shown in Table 3. Therefore, it is preferred to select L- a -amino acid transaminase lct derived from Lactobacillus paracasei ATCC 334 for the transamination and deamination of levodopa.
    [0188] The induction expression method is: Recombinant E. coli is transferred to LB fermentation medium (containing 10 g / l peptone, 5 g / l yeast powder, and 10 g / l NaCl) at a volumetric ratio of two%. After the cell OD 600 reaches 0.6-0.8, IPTG is added to a final concentration of 0.4 mM, and expression is carried out by induction and culture at 20 ° C for 8 h. After completion of induction expression, cells are harvested by centrifugation at 20 ° C and 8000 rpm for 20 minutes.
    [0189] Table 2 Comparison of the activity of different L- a -aminoacid transaminases
    [0193] Table 3 Comparison of the activity of different L- a -aminoacid transaminases
    [0197] Example 3
    [0199] The enzymatic properties of a dehydrogenase -hidroxicarboxilato compared. Usually this enzyme may also have the ability to reduce pyruvic acid to produce lactic acid, so an enzyme that cannot or very weakly reduces pyruvic acid is preferred. Using pyruvic acid as a substrate, the reducing capacity of different enzymes is compared. Using a method according to the bibliography (Study on cloning expression, purification and enzymatic property of Serratia marcescens H3010 fermented D-lactate dehydrogenase gene. Industrial Microbiology, 2012, 42 (04): 30-37.), The activity of NAD is determined as coenzyme to reduce pyruvic acid, and the experimental results are shown in Table 4.
    [0201] Table 4 Comparison of activity to various -hidroxicarboxilato dehydrogenases in reducing pyruvic acid
    [0205] Example 4
    [0207] It is constructed the recombinant E. coli expressing simultaneously -hidroxicarboxilato dehydrogenase, transaminase and L- glucose dehydrogenase to -amino acid: first, the genes encoding L- -amino acid transaminase to, a dehydrogenase and glucose dehydrogenase bind -hidroxicarboxilato to a plasmid to obtain a recombinant three gene co-expression plasmid. The plasmid is transformed into E. coli HM, and positive transformants are obtained by selection with an antibiotic plate to obtain recombinant E. coli.
    [0209] After the induction expression of the recombinant E. coli is complete, cells are harvested, and in a reaction volume of 100 ml with the wet weight of cells of 40 g / l, levodopa of 40 g / l, pyruvic acid of 30 g / l, glucose of 30 g / l and pH of 8.0, the reaction is carried out at 35 ° C for 12 hours. After the end of transformation, the yield and configuration of the danshensu are determined by liquid chromatography, and the results are shown in Table 5.
    [0211] Table 5 Comparison of various recombinant strains
    [0213]
    [0215] Example 5
    [0216] It is constructed the recombinant E. coli expressing simultaneously -hidroxicarboxilato dehydrogenase, L- -amino acid transaminase to L-lactate dehydrogenase and: first, the genes coding for L-lactate bind dehydrogenase, L- -amino acid transaminase to -hidroxicarboxilato dehydrogenase and a plasmid to obtain a recombinant plasmid coexpression of three genes. The plasmid is transformed to give E. coli HM, and positive transformants are obtained by selection with an antibiotic plate to obtain recombinant E. coli.
    [0218] After the induction expression of the recombinant E. coli is completed, cells are harvested, and in a reaction volume of 100 ml with the wet weight of cells of 40 g / l, the levodopa concentration of 40 g / l, the pyruvic acid concentration of 30 g / l, and pH of 8.0, the reaction is carried out at 35 ° C for 12 hours. After the end of the transformation, the yield and configuration of the danshensu are determined by liquid chromatography, and the results are shown in Table 6.
    [0220] Table 6 Comparison of various recombinant strains
    [0224] Example 6
    [0226] It is constructed the recombinant E. coli expressing simultaneously -hidroxicarboxilato dehydrogenase, L- -amino acid transaminase to glutamate dehydrogenase and L-: first, the genes encoding for the L- -amino acid transaminase to, to bind dehydrogenase -hidroxicarboxilato and L-glutamate dehydrogenase to a plasmid to obtain a recombinant plasmid for the co-expression of three genes. The plasmid is transformed into E. coli HM, and positive transformants are obtained by selection with an antibiotic plate to obtain recombinant E. coli.
    [0228] After the induction expression of the recombinant E. coli is completed, cells are harvested, and in a reaction volume of 100 ml with the wet weight of cells of 40 g / l, the levodopa concentration of 40 g / l, the L-glutamic acid concentration of 30 g / l, and pH of 8.0, the reaction is carried out at 35 ° C for 12 hours. After the end of transformation, the yield and danshensu configuration are determined by liquid chromatography, and the results are shown in Table 7.
    [0230] Table 7 Comparison of various recombinant strains
    [0234] Example 7
    [0236] Using the method described in the literature Large scale validation of an efficient CRISPR / Cas-based multi gene editing protocol in E. coli. Microbial Cell Factories, 2017, 16 (1): 68, a constitutive promoter of medium expression intensity (PG) in front of the E. coli glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) is added in front of the corresponding gene in the genome of E. coli HM , and the sequence is as shown in SEQ ID NO: 8.
    [0238] When the expression of the btsT gene is enhanced, the E. coli HM genome is used as a template, btsT-FF / btsT-FR, btsT-gpdA-F / btsT-gpdA-R and btsT-RF / btsT-RR are used as primers, 5 'downstream sequences, promoters and downstream sequences are amplified, and btsT-FF and btsT-RR are used as primers to fuse an expression cassette containing the gpdA promoter. Then, after transferring the expression cassette into E. coli HM along with plasmids pCasRed and pCRISPR-gDNA (containing btsT sgRNAs), Cas9 / sRNA induces double-stranded break at the host's btsT gene locus, Red recombinase. integrates the gpdA promoter in front of the btsT gene, and the gene is verified by sequencing.
    [0240] When the expression of the ybdD gene is enhanced, the E. coli HM genome is used as the template, ybdD-FF / ybdD-FR, ybdD-gpdA-F / ybdD-gpdA-R and ybdD-RF / ybdD-RR are used. as primers, 5 'sequences, promoters and 3' sequences are amplified, and ybdD-FF and ybdD-RR are used to fuse an expression cassette containing the gpdA promoter. Then, after transferring the expression cassette into E. coli HM along with plasmids pCasRed and pCRISPR-gDNA (containing ybdD sRNAs), Cas9 / sRNA induces double-stranded break at the host's ybdD gene locus, Red recombinase integrates the gpdA promoter in front of the ybdD gene, and the gene is verified by sequencing.
    [0242] Table 8 below shows the corresponding indexes of the primer name and the sequence identity number.
    [0244] Table 8 Primer name and sequence identity number
    [0248] According to the induction expression method described in Example 2, various types of cells are harvested for transformation analysis, and the results are shown in Table 9. In the whole cell transformation system with the wet weight of cells of 5 g / l, pyruvic acid of 50 g / l, levodopa of 20 g / l, glucose of 50 g / l, pH of 8.0, temperature of 40 ° C, and stirrer speed of 250 rpm, the time transformation is 12 hours.
    [0250] Table 9 Comparison of transformation results
    [0252]
    [0254] The E. coli HM (PG-btsT, PG-ybdD) with the best effect is called E. coli HMBY.
    [0255] When the expression of the lldP gene is enhanced, the E. coli HM genome is used as a template, 5 'sequences, promoters and 3' sequences are amplified, and an expression cassette containing the gpdA promoter. Then, after transferring the expression cassette into E. coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing lldP sRNAs), Cas9 / sgRNA induces double-stranded breakage at the host lldP gene locus, Red recombinase. integrates the gpdA promoter in front of the lldP gene, and the gene is verified by sequencing.
    [0257] According to the induction expression method described in Example 2, various types of cells are harvested for transformation analysis, and the results are shown in Table 10. In the whole cell transformation system with the wet weight of cells of 5 g / l, L-lactic acid of 50 g / l, levodopa of 20 g / l, pH of 8.0, temperature of 40 ° C, and stirrer speed of 250 rpm, the transformation time is 12 hours.
    [0258] Table 10 Comparison of transformation results
    [0260]
    [0262] The E. coli HM (PG-lldP) with the best effect is called E. coli HML.
    [0264] When the expression of the gltS gene is enhanced, the E. coli HM genome is used as a template, 5 'sequences, promoters and 3' sequences are amplified, and an expression cassette containing the gpdA promoter. Then, after transferring the expression cassette into E. coli HM along with plasmids pCasRed and pCRISPR-gDNA (containing gltS sRNAs), Cas9 / sRNA induces double-stranded breakage at the host's gltS gene locus, Red recombinase. integrates the gpdA promoter in front of the gltS gene, and the gene is verified by sequencing.
    [0266] According to the induction expression method described in Example 2, various cell types are harvested for transformation analysis, and the results are shown in Table 11. In the whole cell transformation system with the wet weight of 5 g / l cells, 1 g / l L-glutamic acid, 20 g / l levodopa, pH 8.0, temperature 40 ° C, and shaker speed of 250 rpm, the transformation time is 12 hours.
    [0267] Table 11 Comparison of transformation results
    [0269]
    [0271] The E. coli HM (PG-gltS) with the best effect is called E. coli HMG.
    [0273] Example 8
    [0275] According to the method as in example 7, a constitutive promoter of the medium expression intensity (PG) is added in front of the E. coli glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) in front of the nadA and pdxJ genes in E coli HMBY, and the sequence is shown in SEQ ID NO: 8. The plasmid is then introduced.
    [0277] When the expression of the nadA gene is enhanced, the E. coli HMBY genome is used as a template, nadA-FF / nadA-FR, nadA-gpdA-F / nadA-gpdA-R and nadA-RF / nadA-RR are used as primers, sequences downstream, promoters and sequences downstream are amplified, and nadA-FF and nadA-RR are used as primers to fuse an expression cassette containing the gpdA promoter. Then, after transferring the expression cassette into E. coli HMBY together with plasmids pCasRed and pCRISPR-gDNA (containing nadA sRNAs), Cas9 / sRNA induces double-stranded break at the host's nadA gene locus, Red recombinase. integrates the gpdA promoter in front of the nadA gene, and the gene is verified by sequencing.
    [0279] When the expression of the pdxJ gene is enhanced, the E. coli HMBY genome is used as a template, pdxJ-FF / pdxJ-FR, pdxJ-gpdA-F / pdxJ-gpdA-R and pdxJ-RF / pdxJ-RR are used as primers, 5 'upstream sequences, promoters and downstream sequences are amplified, and pdxJ-FF and pdxJ-RR are used as primers to fuse an expression cassette containing the gpdA promoter. Then, after the expression cassette was transferred into E. coli HMBY together with plasmids pCasRed and pCRISPR-gDNA (which contain pdxJ sgRNA), Cas9 / sgRNA induces double-stranded break at the host's pdxJ gene locus, Red recombinase integrates the gpdA promoter in front of the pdxJ gene, and the gene is verified by sequencing.
    [0281] Table 12 below shows the corresponding indexes of primer name and sequence identity number.
    [0283] Table 12 Primer name and sequence identity number
    [0287] After completion of the genetic modification, the co-expression plasmid is introduced. According to the induction expression method described in Example 2, various types of cells are harvested for transformation and analysis, and the results are shown in Table 13. In the whole cell transformation system with the wet weight of cells of 20 g / l, pyruvic acid of 100 g / l, levodopa of 120 g / l, glucose of 200 g / l, pH of 9.0, temperature of 30 ° C, and stirrer speed of 250 rpm, the time transformation is 24 hours.
    [0289] Table 13 Comparison of transformation results
    [0291]
    [0293] The E. coli HMBY (PG-nadA, PG-pdxJ) with the best effect is called E. coli NP.
    [0294] After completion of the genetic modification, the co-expression plasmid is introduced. According to the induction expression method as described in Example 2, various types of cells are collected for transformation and analysis, and the results are shown in Table 14. In the whole cell transformation system with weight 20 g / l wet cell, 100 g / l L-lactic acid, 120 g / l levodopa, pH 9.0, temperature 30 ° C, and shaker speed of 250 rpm, transformation time it is 24 hours.
    [0296] Table 14 Comparison of transformation results
    [0298]
    [0299]
    [0301] The best performing E. coli HML (PG-nadA, PG-pdxJ) is called E. coli NL.
    [0303] After completion of the genetic modification, the co-expression plasmid is introduced. According to the induction expression method described in Example 2, various types of cells are harvested for transformation and analysis, and the results are shown in Table 15. In the whole cell transformation system with the wet weight of cells 20 g / l, 200 g / l L-glutamic acid, 20 g / l levodopa, pH 9.0, temperature 30 ° C, and stirrer speed of 250 rpm, transformation time is 24 hours .
    [0305] Table 15 Comparison of transformation results
    [0309] The best performing E. coli HMG (PG-nadA, PG-pdxJ) is called E. coli HNP.
    [0310] Example 9
    [0312] According to the induction expression method as described in Example 2, cells are harvested after completion of induction expression of E. coli NP / pCOLADuet-1-lfldhd-ect1-bsgdh, and in a reaction system of 100 ml with 1 g / l wet cell weight, 1 g / l pyruvic acid, 1 g / l levodopa, 1 g / l glucose, pH 6.0, temperature 15 ° C, and stirrer speed 250 rpm, the transformation time is 1 hour. As a result of the measurement, the concentration of R-danshensu is 77 mg / l, and the% ee> 99.9.
    [0314] According to the method of expression by induction as described in Example 2, collect cells after completion of the expression by induction of E. coli NL / pCOLADuet-1-lfldhd-ect1-llldh, and in a 100 ml reaction system with the wet weight of cells of 1 g / l, L-lactic acid of 1 g / l, levodopa of 1 g / l, pH of 6.0, temperature of 15 ° C and stirrer speed of 250 rpm, the transformation time is 1 hour. As a result of the measurement, the concentration of R-danshensu is 77 mg / l, and the% ee> 99.9.
    [0316] According to the induction expression method as described in Example 2, cells are harvested after completion of the induction expression of E. coli HNP / pCOLADuet-1-efmdhd-bsgdh-lct, and in a reaction system of 100 ml with the wet cell weight of 1 g / l, L-glutamic acid of 1 g / l, levodopa of 1 g / l, pH of 6.0, temperature of 15 ° C and stirrer speed of 250 rpm, the transformation time is 1 hour. As a result of the measurement, the concentration of S-danshensu is 93 mg / l, and the% ee> 99.9.
    [0318] Example 10
    [0320] According to the induction expression method as described in Example 2, cells are harvested after completion of the induction expression of the strains in Table 16, and in a 100 ml reaction system with the wet weight of cells from 200 g / l, pyruvic acid 200 g / l, levodopa 200 g / l, glucose 200 g / l, pH 8.5, temperature 40 ° C and stirrer speed of 250 rpm, transformation time is 48 hours. Results are measured after the precipitate is diluted and completely dissolved.
    [0322] Table 16 Comparison of transformation results
    [0324]
    [0325]
    [0327] According to the induction expression method as described in Example 2, cells are harvested after completion of the induction expression of the strains in Table 17, and in a 100 ml reaction system with the wet weight of cells from 200 g / l, 200 g / l L-lactic acid, 200 g / l levodopa, pH of 8.5, temperature of 40 ° C and stirrer speed of 250 rpm, the transformation time is 48 hours. Results are measured after the precipitate is diluted and completely dissolved.
    [0329] Table 17 Comparison of transformation results
    [0333] According to the induction expression method as described in Example 1, cells are harvested after completion of the induction expression of the strains in Table 18, and in a 100 ml reaction system with the wet weight of cells from 200 g / l, 20 g / l L-glutamic acid, 200 g / l levodopa, pH 8.5, temperature 40 ° C and shaker speed of 250 rpm, the transformation time is 48 hours. Results are measured after the precipitate is diluted and completely dissolved.
    [0334] Table 18 Comparison of transformation results
    权利要求:
    Claims (10)
    [1]
    1. Recombinant Eschenchia coli , which simultaneously expresses α-hydroxycarboxylate dehydrogenase, La-amino acid transaminase, and any one of glucose dehydrogenase, L-lactate dehydrogenase and L-glutamate dehydrogenase, in which a gene related to the breakdown of phenolic compounds is deactivated based on a host E. coli.
    [2]
    2. Recombinant Eschenchia coli according to claim 1, wherein the phenol breakdown gene is any one or a combination of hpaD and mhpB.
    [3]
    Recombinant Eschenchia coli according to claim 1, wherein the recombinant E. coli further enhances the expression of one or more pyruvic acid transporter genes, an L-lactic acid transporter gene, a glutamic acid transporter gene , a NAD synthesis gene and a pyridoxal phosphate synthesis gene; and the pyruvic acid transporter gene, the L-lactic acid transporter gene and the glutamic acid transporter gene are not expressed at the same time.
    [4]
    4. Recombinant Eschenchia coli according to claim 3, wherein the gene whose expression is to be enhanced is any one or more genes related to pyruvic acid transport, lldP, gltS, nadA and pdxJ, and genes related to transport of pyruvic acid include btsT and ybdD.
    [5]
    Recombinant Eschenchia coli according to claim 4, wherein the enhanced expression adds a constitutive promoter in front of the gene whose expression is to be enhanced in the host E. coli genome.
    [6]
    6. Recombinant Eschenchia coli according to claim 1, wherein any one of glucose dehydrogenase, L-lactate dehydrogenase and L-glutamate dehydrogenase is coexpressed with α-hydroxycarboxylate dehydrogenase and L-amino acid transaminase by pCOLADuet.
    [7]
    7. The recombinant Eschenchia coli according to claim 1, wherein the host strain is E. coli BL21.
    [8]
    8. Method for producing danshensu, using the recombinant strain according to any one of claims 1-7.
    [9]
    A method according to claim 8, wherein the method carries out production by whole cell transformation.
    [10]
    A method according to claim 9, wherein in a whole cell transformation production system, the wet weight of cells is 1-200 g / l, and the levodopa concentration is 1-200 g / l;
    When the recombinant strain simultaneously expresses α-hydroxycarboxylate dehydrogenase, La-amino acid transaminase and glucose dehydrogenase, in the whole cell transformation production system, the pyruvic acid concentration is 1-200 g / L, and the glucose concentration is from 1-200 g / l;
    when the recombinant strain simultaneously expresses α-hydroxycarboxylate dehydrogenase, L-α-amino acid transaminase and L-lactate dehydrogenase, the whole cell transformation production system further comprises 1-200 g / L L-lactic acid;
    when the recombinant strain simultaneously expresses α-hydroxycarboxylate dehydrogenase, L-α-amino acid transaminase and L-glutamate dehydrogenase, the whole cell transformation production system further comprises 1-200 g / l L-glutamic acid; Y
    The whole cell transformation production system has a pH of 6.0 9.0, and reacts at 15-40 ° C for 1-48 hours.
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    同族专利:
    公开号 | 公开日
    WO2019200874A1|2019-10-24|
    KR20210003816A|2021-01-12|
    ES2825205B2|2022-01-17|
    US20190376100A1|2019-12-12|
    US10870870B2|2020-12-22|
    ES2825205R1|2021-06-11|
    引用文献:
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    CN103667371B|2013-11-11|2016-03-16|天津大学|A kind of biological production of Salvianic acidA|
    CN105624217B|2016-02-15|2019-03-15|江南大学|A kind of method of microorganism conversion|
    CN105543290B|2016-02-15|2019-07-23|江南大学|A kind of method of microorganism conversion|
    CN105543292B|2016-02-15|2019-03-08|江南大学|A kind of method of microorganism conversion|
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