WO2019200874A1 - 一种工程菌及其在丹参素生产中的应用 - Google Patents

一种工程菌及其在丹参素生产中的应用 Download PDF

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WO2019200874A1
WO2019200874A1 PCT/CN2018/111895 CN2018111895W WO2019200874A1 WO 2019200874 A1 WO2019200874 A1 WO 2019200874A1 CN 2018111895 W CN2018111895 W CN 2018111895W WO 2019200874 A1 WO2019200874 A1 WO 2019200874A1
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dehydrogenase
escherichia coli
gene
acid
recombinant
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French (fr)
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蔡宇杰
熊天真
刘金彬
丁彦蕊
白亚军
郑晓晖
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江南大学
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Priority claimed from CN201810352649.8A external-priority patent/CN108949646B/zh
Priority claimed from CN201810352693.9A external-priority patent/CN108949653B/zh
Priority claimed from CN201810352695.8A external-priority patent/CN108865960B/zh
Application filed by 江南大学 filed Critical 江南大学
Priority to ES202090050A priority Critical patent/ES2825205B2/es
Priority to KR1020207033050A priority patent/KR102616750B1/ko
Priority to US16/542,787 priority patent/US10870870B2/en
Publication of WO2019200874A1 publication Critical patent/WO2019200874A1/zh

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Definitions

  • the invention relates to an engineering bacteria and the application thereof in the production of Danshensu, and belongs to the technical field of bioengineering.
  • Danshensu extracted from Salvia miltiorrhiza scientific name R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid, D-(+)- ⁇ -(3,4-dihydroxyphenyl Lactic acid, English name: Danshensu, D-DSS, R-DSS, (R)-(+)-3-(3,4-Dihydroxyphenyl)-lactic acid, (R)-(+)-3-(3 4-Dihydroxyphenyl)-2-hydroxypropanoic acid is a dextrophenolic acid compound. There is currently no natural L-tansin.
  • Danshensu is an important active ingredient in the water extract of Salvia miltiorrhiza Bunge. It was obtained from the aqueous extract of Salvia miltiorrhiza in 1980 and identified the structure (Study on the water-soluble active constituents of Salvia miltiorrhiza, II.D(+) ⁇ (3,4-II) The structure of hydroxyphenyl) lactic acid, Journal of Shanghai First Medical College, 1980, 05 (7), 384-385), various studies have shown that Danshensu has important pharmacological effects, in the treatment of cardiovascular and cerebrovascular diseases, etc. Has a unique therapeutic effect.
  • Patent CN201310559498.0 proposes a method for constructing Escherichia coli genetically engineered bacteria to produce Danshensu by glucose fermentation. Since the anabolic pathway involves the use of hydroxylase, the enzyme easily oxidizes the metabolic process product and affects the yield of Danshensu. At the same time, because E. coli fermentation is a high oxygen consumption process, it also oxidizes Danshensu.
  • Patent CN201210190171.6 proposes a method for producing Danshensu by hydrolyzing salvianolic acid B.
  • Salvianolic acid B needs to be extracted from Salvia miltiorrhiza, and there are a large number of side reactions in the chemical hydrolysis process, which is also not suitable for large-scale production.
  • the catalyst for chiral synthesis of Danshensu (patent CN201210420488.4) is extremely expensive and currently only stays at the laboratory level.
  • the present invention Based on the defects of various current methods, the present invention provides an optically pure Danshensu production method based on transaminase, and constructs a multi-enzyme co-expressed engineering bacteria to achieve efficient production of Danshensu.
  • the technical problem to be solved by the present invention is to provide a recombinant strain capable of producing Danshensu at a low cost.
  • the present invention addresses the technical problems of the construction and application of the strain.
  • a first object of the present invention is to provide a recombinant strain capable of producing optical pure Danshensu at a low cost; the recombinant strain simultaneously expresses an ⁇ -hydroxycarboxylic acid dehydrogenase and an L- ⁇ -amino acid aminotransferase, and any one of the following: Glucose dehydrogenase, L-lactate dehydrogenase, L-glutamate dehydrogenase, and knockout genes related to phenolic compound decomposition on the basis of host E. coli.
  • the alpha-hydroxycarboxylic acid dehydrogenase is a D-type alpha-hydroxycarboxylic acid dehydrogenase from Lactobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038 or Lactobacillus fermentum ATCC 14931.
  • the alpha-hydroxycarboxylic acid dehydrogenase is an L-form alpha-hydroxycarboxylic acid dehydrogenase from Bacillus coagulans DSM 1, Weissella confusa strain DSM 20196 or Lactobacillus fermentum ATCC 14931.
  • the ⁇ -hydroxycarboxylic acid dehydrogenase is D- ⁇ -hydroxycarboxylic acid dehydrogenase, and the amino acid sequence thereof is NCBI accession NO. is WP_003643296.1, WP_002335374.1, or EEI22188.
  • the nucleotide sequence of the D- ⁇ -hydroxycarboxylic acid dehydrogenase is NCBI accession NO. is NZ_GL379761 REGION: COMPLEMENT (533562..534560), NZ_KB944641 REGION:161892..162830, ACGI01000078REGION: The sequence of 20793..21791; the nucleotide sequence of L- ⁇ -hydroxycarboxylic acid dehydrogenase is NCBI accession NO. is NZ_ATUM01000014 REGION: 39316..40254, NZ_JQAY01000006 REGION: 69708..70640, NZ_GG669901 REGION:45517. The sequence of .46470.
  • the L-alpha-amino acid aminotransferase is from Escherichia coli BL21, Lactobacillus plantarum ATCC 14917, Lactobacillus paracasei ATCC 334.
  • the amino acid sequence of the L-alpha-amino acid aminotransferase is the sequence of accession NO on NCBI of WP_000462687.71, WP_000486988.1, WP_003643296.1, YP_806114.1.
  • nucleotide sequence of the L-alpha-amino acid aminotransferase is NCBI accession NO: NC_012892 REGION: COMPLEMENT (989603..990793), NC_012892 REGION: 4174517.. 4175710, NZ_GL379768 REGION: complement (121900. .123087), NC_008526REGION: sequence of complement(840419..841594).
  • the glucose dehydrogenase is from Bacillus subtilis ATCC 13952.
  • amino acid sequence of the glucose dehydrogenase is the accession NO of NCBI on the WP_013351020.1 sequence.
  • nucleotide sequence of the glucose dehydrogenase is NCBI accession NO: NZ_CP009748REGION: 386154..38693.
  • the L-lactate dehydrogenase is from Lactococcus lactis ATCC 19257.
  • amino acid sequence of the L-lactate dehydrogenase is the accession NO of NCBI on the WP_003131075.1 sequence.
  • nucleotide sequence of the L-lactate dehydrogenase is the sequence of accession NO on NCBI: NZ_JXJZ01000017 REGION: 18532..19509.
  • the L-glutamate dehydrogenase is from Escherichia coli BL21, Rhodobacter sphaeroides ATCC BAA-808, Clostridium symbiosum ATCC 14940, Bacillus subtilis 168.
  • amino acid sequence of L-glutamate dehydrogenase is NCBI accession NO is WP_000373021.1, WP_011338202.1, WP_003497202.1, WP_010886557.1 sequence.
  • nucleotide sequence of L-glutamate dehydrogenase is NCBI accession NO: NC_012892 REGION: 1787641..1788084, NC_007493 REGION: complement (2129131.. 2130558), NZ_KE992901 REGION: complement (17603..18955), NC_000964 REGION: sequence of complement (2402067..2403350).
  • the recombinant strain encodes an L- ⁇ -amino acid aminotransferase, an ⁇ -hydroxycarboxylic acid dehydrogenase, and any one of the following: glucose dehydrogenase, L-lactate dehydrogenase, L-
  • the gene of glutamate dehydrogenase is ligated to the plasmid to construct a recombinant plasmid for co-expression of the three genes, and then the recombinant plasmid is transformed into the corresponding strain to obtain a recombinant engineering strain.
  • the recombinant strain is constructed using Escherichia coli BL21 (DE3) as a host.
  • the gene related to the decomposition of the phenolic compound is any one or a combination of hpaD and mhpB.
  • the nucleotide sequence of the phenolic material decomposing gene is on NCBI, and the accession NO on NCBI is: NC_012892 REGION: complement (4505585..4506436) and NC_012892 REGION: 339806..340750.
  • the recombinant strain further enhances expression of one or more of a pyruvate transport gene, an L-lactic acid transporter gene, a glutamate transporter gene, a NAD synthesis gene, and a pyridoxal phosphate synthesis gene;
  • the pyruvate transport gene, the L-lactic acid transporter gene, and the glutamate transporter gene are not expressed at the same time.
  • the enhanced expression is by adding a constitutive promoter to the gene to be fortified expression on the Escherichia coli BL21 (DE3) genome.
  • the gene for enhanced expression is a pyruvate transport-related gene, lldP (lactic acid transporter), gltS (glutamate transporter), nadA (NAD synthase), pdxJ (pyridyl phosphate) Any one or more of the synthetic genes); wherein the pyruvate transport-related genes include btsT and ybdD (pyruvate to intracellular transporter genes).
  • the btsT and ybdD accession NO on NCBI is:; nadA is NC_012892REGION: 740487..741530; pdxJ is NC_012892REGION: complement (2567591..2568322).
  • accession NO of the lldP on the NCBI is: NC_012892 REGION: 3646638..3648293; nadA is NC_012892 REGION: 740487..741530; pdxJ is NC_012892 REGION: complement (2567591..2568322).
  • accession NO of the gltS on NCBI is: NC_012892 REGION: complement (3694931..3696136); nadA is NC_012892 REGION: 740487..741530; pdxJ is NC_012892REGION: complement (2567591..2568322).
  • a second object of the present invention is to provide a method for producing Danshensu using the recombinant bacteria of the present invention.
  • the production of danshensu is performed by whole cell transformation.
  • the whole cell transformation production system comprises a cell wet weight of 1-200 g/L and a levodopa concentration of 1-200 g/L;
  • the whole cell transformation production system further comprises: pyruvic acid concentration of 1-200 g/L And glucose concentration is 1-200g / L;
  • the whole cell transformation production system further comprises: L-lactic acid 1-200 g/ L;
  • the whole cell transformation production system further includes: L-glutamic acid 1-200g/L;
  • the system for whole cell transformation produces pH 6.0-9.0; reacts at 15-40 ° C for 1-48 hours.
  • the invention constructs a novel multi-enzyme co-expressing genetic engineering bacteria, realizes the low-cost production of Danshensu, and further promotes the transport and reduction of the substrate by knocking out or enhancing the expression of the related genes on the E. coli genome. Decomposition of the product.
  • the (D/L)- ⁇ -hydroxycarboxylic acid dehydrogenase selected by the invention has the characteristics of poor substrate specificity and strong optical specificity, and can produce optically pure D-danshensu and L-danshensu.
  • the production process is simple and the raw materials are easily available, and has a good industrial application prospect.
  • the functional core of the engineered bacteria of the present invention is that three enzymes can be simultaneously expressed, which are L- ⁇ -amino acid aminotransferase, ⁇ -hydroxycarboxylic acid dehydrogenase and any one of the following: glucose dehydrogenase, L-lactate dehydrogenase , L-glutamate dehydrogenase.
  • the principle is: in the whole cell of engineering bacteria, glucose dehydrogenase or L-lactate dehydrogenase or L-glutamate dehydrogenase dehydrogenate or dehydrogenate L-lactic acid with NAD as coenzyme in the bacteria
  • Enzyme or L-glutamate dehydrogenase produces NADH and the corresponding gluconic acid or pyruvate or alpha-ketoglutarate
  • levodopa is deaminated by L-alpha-amino acid aminotransferase to form 3,4-dihydroxyphenylpyruvate
  • Pyruvic acid obtains ammonia to be converted into alanine
  • ⁇ -hydroxycarboxylic acid dehydrogenase reduces the 3,4-dihydroxyphenylpyruvate to danshensu by NADH produced by the process of glucose dehydrogenation, and simultaneously realizes the regeneration of coenzyme NAD. Further, simultaneously knocking out or enhancing the expression of the gene
  • Lactobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038, Lactobacillus fermentum ATCC 14931, Lactobacillus paracasei ATCC 334, Bacillus subtilis ATCC 13952, Escherichia coli BL21 (DE3), Lactococcus lactis ATCC 19257 were purchased from the American Type Culture Collection ATCC.
  • PETDuet-1, pACYCDue-1, pCOLADuet-1, pRSFDuet-1 plasmid and Escherichia coli BL21 (DE3) were purchased from Novagen.
  • the phenolic substances in the present invention are extremely easily decomposed by the enzyme in Escherichia coli, and the related genes are according to the literature (Biodegradation of Aromatic Compounds by Escherichia coli, Microbiol Mol Biol Rev. 2001, 65(4): 523-569.). Knock out to avoid decomposition of products and substrates.
  • the selected genes were hpaD and mhpB, and the accession NO on NCBI was: NC_012892 REGION: complement (4505585..4506436) and NC_012892REGION: 339806..340750.
  • the substrate In the process of whole cell transformation, the substrate needs to be transported into the cell to carry out. Enhancing the pyruvate transporter helps to maintain the high concentration of the intracellular substrate quickly and for a long time, which is beneficial to the reaction.
  • the genes involved in the selection of pyruvate transport are btsT and ybdD, and the accession NO on NCBI is: NC_012892 REGION: complement (4496239..4498389) and NC_012892 REGION: 592652..592849.
  • Dopa is similar to aromatic amino acids. It needs to absorb amino acids during cell culture. Therefore, the cells themselves express a large number of amino acid transporters without further strengthening expression.
  • NADH ⁇ -hydroxycarboxylic acid dehydrogenase reduction
  • the selected gene is nadA.
  • the accession NO on NCBI is: NC_012892 REGION: 740487..741530.
  • Pyridoxal phosphate (amine) is a coenzyme of L- ⁇ -amino acid aminotransferase, overexpressing the core gene pdxJ in the coenzyme pathway, which is beneficial to the synthesis of levodopa.
  • accession NO on NCBI is: NC_012892REGION: complement (2567591..2568322).
  • L- ⁇ -amino acid aminotransferases are widely present in bacterial, fungal, and mammalian cells. The most active is usually a transaminase with alpha-ketoglutaric acid or oxaloacetate as the acceptor, in which alpha-ketoglutaric acid or oxaloacetate is expensive, and the corresponding glutamic acid or aspartic acid is produced. The value of the acid is much lower than the corresponding precursor.
  • the comprehensive examination of the ⁇ -keto acid corresponding to the 20 natural L-amino acids found that the price of pyruvic acid and alanine is equivalent. Therefore, the application selects pyruvate as the acceptor of ammonia.
  • L- ⁇ -amino acid aminotransferase genes lpt and lct were cloned from Lactobacillus plantarum ATCC 14917 and Lactobacillus paracasei ATCC334, respectively, and the amino acid sequence thereof was the sequence of accession NO. of BD_003643296.1 and YP_806114.1 on NCBI.
  • Two L- ⁇ -amino acid aminotransferase genes ect1 and ect2 were cloned from Escherichia coli BL1 (DE3), and the amino acid sequence was NCBI accession NO WP_000462687.1, WP_000486988.1.
  • the ⁇ -hydroxycarboxylic acid dehydrogenase includes lactate dehydrogenase, ⁇ -hydroxy acid isohexanoate dehydrogenase, mandelic acid dehydrogenase, glyoxylate reductase, etc. These enzymes can be widely used.
  • the action of a variety of substrates to form alpha-hydroxycarboxylic acids is usually named according to the substrate for which it is optimal.
  • the present invention selects an enzyme which is highly optically active and has a strong activity against 3,4-dihydroxyphenylpyruvate, and is used for the production of D or L danshensu.
  • the D-type ⁇ -hydroxycarboxylic acid dehydrogenase genes lpldhd, efmdhd, and lfldhd were cloned from Lactobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038, and Lactobacillus fermentum ATCC 14931, respectively.
  • the amino acid sequence of the amino acid sequence was NCBI accession NO. was WP_003643296.1.
  • the sequence of WP_002335374.1 and EEI22188.1 was provided.
  • the L-type ⁇ -hydroxycarboxylic acid dehydrogenase genes bcldhl, wcldhl and lfldhl were cloned from Bacillus coagulans DSM 1, Weissella confusa strain DSM 20196 and Lactobacillus fermentum ATCC 14931, respectively.
  • the amino acid sequence was accession NO of WP_013858488.1 on NCBI.
  • ⁇ -hydroxycarboxylic acid dehydrogenase requires NADH and/or NADPH as coenzymes, and often has formate dehydrogenase, glucose dehydrogenase, phosphite dehydrogenase, etc., glucose dehydrogenase relative
  • the activity is highest in other enzymes, and thus the present invention obtains the glucose dehydrogenase gene bsgdh (amino acid sequence is WP_013351020.1) from Bacillus subtilis ATCC 13952.
  • L-lactic acid is the most inexpensive organic acid, and the alanine produced by the transamination of pyruvic acid after dehydrogenation has a high added value.
  • L-lactate dehydrogenase is widely present in a variety of microorganisms, and L-lactic acid is used as a substrate to transfer hydrogen generated on L-lactic acid to coenzyme NAD or NADP to generate NADH or NADPH.
  • NADH or NADPH can be used as the hydrogen donor of the aforementioned ⁇ -hydroxy hydroxy acid dehydrogenase.
  • lactate dehydrogenase with NAD (NADP) as a coenzyme tends to synthesize lactic acid using pyruvic acid as a substrate, but in the case of excess lactic acid or the like, lactate dehydrogenase will remove hydrogen from lactic acid to form pyruvic acid.
  • the present invention obtains the L-lactate dehydrogenase gene llldh (amino acid sequence WP_003131075.1) from Lactococcus lactis ATCC 19257.
  • L-glutamic acid is the most inexpensive amino acid, and ⁇ -ketoglutaric acid produced after dehydrogenation can be used as an acceptor for levodopa transamination.
  • L-glutamate dehydrogenase is widely present in almost all organisms, and L-glutamic acid is used as a substrate to transfer hydrogen generated on L-glutamic acid to coenzyme NAD or NADP to generate NADH or NADPH.
  • NADH or NADPH can be used as the hydrogen donor of the aforementioned hydroxy hydroxy acid dehydrogenase.
  • the present invention obtains the L-glutamic acid gene ecgdh (amino acid sequence WP_000373021.1) and rsgdh (amino acid sequence WP_011338202.1) from Escherichia coli BL21, Rhodobacter sphaeroides ATCC BAA-808, Clostridium symbiosum ATCC 14940, and Bacillus subtilis 168, respectively. , csgdh (amino acid sequence is WP_003497202.1), bsgdh (amino acid sequence is WP_010886557.1).
  • E. coli multi-gene co-expression strategy Chinese Journal of Bioengineering, 2012, 32(4): 117-122.
  • the invention adopts Liu Xianglei (synthetic biotechnology to transform E. coli production).
  • the method described in the method of shikimic acid and resveratrol, 2016, Shanghai Pharmaceutical Industry Research Institute, PhD thesis each gene contains a T7 promoter and an RBS binding site, and each gene has a T7 terminator. Theoretically, because each gene has T7 and RBS in front of it, the expression intensity of the gene is not affected by the order.
  • Each plasmid contains three genes (L- ⁇ -amino acid aminotransferase, (D/L)- ⁇ -hydroxycarboxylic acid dehydrogenase, glucose dehydrogenase or L-lactate dehydrogenase or L-glutamate dehydrogenation
  • the gene corresponding to the enzyme is transferred into the E. coli competent cells by heat transfer, and coated on an antibiotic solid plate to obtain a positive transformant, thereby obtaining recombinant Escherichia coli.
  • Culture of cells According to the classical recombinant E. coli culture and induced expression protocol, recombinant E.
  • coli was transferred to LB fermentation medium at a volume ratio of 2% (peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/ In L), when the cell OD 600 reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression culture was induced at 20 ° C for 8 h. After the induction of expression was completed, the cells were collected by centrifugation at 20 ° C, 8000 rpm, and 20 minutes.
  • peptone 10 g/L yeast powder 5 g/L, NaCl 10 g/ In L
  • IPTG was added to a final concentration of 0.4 mM
  • expression culture was induced at 20 ° C for 8 h. After the induction of expression was completed, the cells were collected by centrifugation at 20 ° C, 8000 rpm, and 20 minutes.
  • the cell transformation production system is: cell wet weight is 1-200g / L, levodopa concentration is 1-200g / L;
  • the whole cell transformation production system further comprises: pyruvic acid concentration of 1-200 g/L And glucose concentration is 1-200g / L;
  • the whole cell transformation production system further comprises: L-lactic acid 1-200 g/ L;
  • the whole cell transformation production system further includes: L-glutamic acid 1-200g/L;
  • the system for whole cell transformation produces pH 6.0-9.0, which is reacted at 15-40 ° C for 1-48 hours.
  • Levodopa has a low solubility and is a suspension containing insolubles at high concentrations.
  • the transformant was analyzed by PerkinElmer Series 200 high performance liquid chromatography with a refractive index detector.
  • the chromatographic conditions were as follows: the mobile phase was methanol-0.1% formic acid water (40:60), using a Hanbang Megres C18 column (4.6 ⁇ 250 mm, 5 ⁇ m), a flow rate of 1 ml/min, a column temperature of 30 ° C, and an injection volume of 20 ⁇ l.
  • the solubility of Danshensu is low, and if the transformation process is crystallized, it is measured after dilution.
  • S S is the peak area of S-danshensu in the conversion solution
  • S R is the liquid chromatographic peak area of R-danshen in the conversion solution.
  • HpaD and mhpB on Escherichia coli BL21 were performed according to the method described in the literature Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli. Microbial Cell Factories, 2017, 16(1):68 Single or double knockout.
  • the gene knockout plasmid used in the present invention is pCasRed and pCRISPR-gDNA (hpaD sgRNA) together with a homologous arm (hpaD donor) introduced into Escherichia coli BL21 (DE3), and Cas9/sgRNA induces a host to occur at the hpaD gene locus.
  • hpaD sgRNA, hpaD donor, mhpB sgRNA, mhpB donor are shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, respectively.
  • mhpB is knocked out in the same way.
  • a solution having a pH of 7 was prepared, levodopa or D-danshensu 4 g/L, and the wet cell volume was 200 g/L, and the concentration was measured after standing at 35 ° C for 10 hours.
  • Table 1 shows the reaction system, levodopa and D. - The remaining amount of Danshensu.
  • Escherichia coli BL21 ( ⁇ hpaD ⁇ mhpB, DE3) works best and is named Escherichia coli HM.
  • L- ⁇ -amino acid aminotransferase a variety of L- ⁇ -amino acid aminotransferase genes were cloned from Escherichia coli BL21 (DE3), and the activity of crude enzyme solution was determined by acetone.
  • the acid is a receptor
  • the activity of various enzymes is compared, and L- ⁇ is determined according to the method described in the literature (transaminase-catalyzed asymmetric synthesis of aromatic L-amino acids. Chinese Journal of Bioengineering, 2012, 28(11): 1346-1358.).
  • - Amino acid transaminase activity the results are shown in Table 2. Therefore, the selection of L- ⁇ -amino acid aminotransferase ect1 derived from Escherichia coli for transamination of levodopa is optimal.
  • Induction expression method Transfer recombinant E. coli to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) in a volume ratio of 2%, when the cell OD600 reaches 0.6-0.8 IPTG was added to a final concentration of 0.4 mM, and expression culture was induced at 20 ° C for 8 h. After the induction of expression was completed, the cells were collected by centrifugation at 20 ° C, 8000 rpm, and 20 minutes.
  • Recombinant E. coli construction expressing both ⁇ -hydroxycarboxylic acid dehydrogenase, L- ⁇ -amino acid aminotransferase and glucose dehydrogenase firstly encoding L- ⁇ -amino acid aminotransferase, ⁇ -hydroxycarboxylic acid dehydrogenase and glucose dehydrogenation
  • the gene of the enzyme is ligated to the plasmid.
  • a recombinant plasmid was obtained by co-expression of the three genes, and the plasmid was transformed into Escherichia coli HM, and the positive transformant was obtained by screening with an antibiotic plate to obtain recombinant Escherichia coli.
  • the gene for the carboxylate dehydrogenase is attached to the plasmid.
  • a recombinant plasmid was obtained by co-expression of the three genes, and the plasmid was transformed into Escherichia coli HM, and the positive transformant was obtained by screening with an antibiotic plate to obtain recombinant Escherichia coli.
  • the cells were collected, and the wet weight of the cells was 40 g/L in a reaction volume of 100 ml, the concentration of levodopa was 40 g/L, and the concentration of L-glutamic acid was 30 g/L, pH 8.0.
  • the reaction was carried out at 35 ° C for 12 hours.
  • the yield and configuration of Danshensu were determined by liquid chromatography after the end of the transformation, and the results are shown in Table 7.
  • Escherichia coli was added to the corresponding gene on the Escherichia coli HM genome using the method described in the literature Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli. Microbial Cell Factories, 2017, 16(1):68
  • the medium expression intensity constitutive promoter (PG) before the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) has the sequence shown as SEQ ID NO: 8.
  • the Escherichia coli HM genome was used as a template to amplify the upstream and start with the primers btsT-FF/btsT-FR, btsT-gpdA-F/btsT-gpdA-R, btsT-RF/btsT-RR.
  • the sub- and downstream sequences were fused with btsT-FF and btsT-RR as primers to express the expression cassette containing the gpdA promoter.
  • the Cas9/sgRNA induces a double-strand break in the btsT gene locus, and the recombinase Red integrates the gpdA promoter into the btsT gene. Sequencing verification.
  • the Escherichia coli HM genome was used as a template to amplify the upstream and start with the primers ybdD-FF/ybdD-FR, ybdD-gpdA-F/ybdD-gpdA-R, ybdD-RF/ybdD-RR.
  • the sub- and downstream sequences were fused with ybdD-FF and ybdD-RR as primers to express the expression cassette containing the gpdA promoter.
  • the Cas9/sgRNA induces a double-strand break in the ybdD gene locus, and the recombinase Red integrates the gpdA promoter into the ybdD gene. Sequencing verification.
  • Table 8 below is the corresponding index of the primer name and the sequence number.
  • Example 9 According to the induced expression method described in Example 2, various types of cells were collected for transformation analysis, and the results are shown in Table 9.
  • the whole cell transformation system in the transformation system is: cell wet weight 5g / L, pyruvic acid 50g / L, levodopa 20g / L, glucose 50g / L, pH 8.0, temperature 40 ° C, shaker speed 250 rev / min; Conversion time is 12 hours.
  • the best-performing Escherichia coli HM (PG-btsT, PG-ybdD) was named Escherichia coli HMBY.
  • the upstream, promoter and downstream sequences were amplified using the Escherichia coli HM genome as a template, and the expression cassette containing the gpdA promoter was obtained. Then, after transfecting into Escherichia coli HM together with plasmid pCasRed and pCRISPR-gDNA (including lldPsgRNA), Cas9/sgRNA induces double-strand breaks at the lldP locus. Recombinase Red integrates gpdA promoter into lldP gene and sequence verification.
  • Example 10 According to the induced expression method described in Example 2, various types of cells were collected for transformation analysis, and the results are shown in Table 10.
  • the whole cell transformation system in the transformation system is: cell wet weight 5g / L, L-lactic acid 50g / L, levodopa 20g / L, pH 8.0, temperature 40 ° C, shaker speed 250 rev / min; conversion time 12 hours .
  • the best-performing Escherichia coli HM was named Escherichia coli HML.
  • the upstream, promoter and downstream sequences were amplified using the Escherichia coli HM genome as a template, and the expression cassette containing the gpdA promoter was obtained. Then, after transfecting into Escherichia coli HM together with the plasmid pCasRed and pC CRISPR-gDNA (containing gltS sgRNA), the Cas9/sgRNA induces a double-strand break in the gltS gene locus, and the recombinase Red integrates the gpdA promoter into the gltS gene. Sequencing verification.
  • Example 11 According to the induced expression method described in Example 2, various types of cells were collected for transformation analysis, and the results are shown in Table 11.
  • the whole cell transformation system in the transformation system is: cell wet weight 5g / L, L-glutamic acid 1g / L, levodopa 20g / L, pH 8.0, temperature 40 ° C, shaker speed 250 rev / min; conversion time 12 hours.
  • the best-performing Escherichia coli HM was named Escherichia coli HMG.
  • the nadA and pdxJ genes in Escherichia coli HMBY were pre-expressed with the medium expression intensity constitutive promoter (PG) of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), and the sequence is SEQ ID NO: 8 is shown.
  • the plasmid is then introduced.
  • nadA gene When the expression of nadA gene was enhanced, the Escherichia coli HMBY genome was used as a template to amplify the upstream and start with the primers nadA-FF/nadA-FR, nadA-gpdA-F/nadA-gpdA-R, nadA-RF/nadA-RR.
  • the sub- and downstream sequences were fused with nadA-FF and nadA-RR as primers to express the expression cassette containing the gpdA promoter.
  • the Cas9/sgRNA induces a double-strand break in the nadA gene locus, and the recombinase Red integrates the gpdA promoter into the nadA gene. Sequencing verification.
  • the Escherichia coli HMBY genome was used as a template, and the primers pdxJ-FF/pdxJ-FR, pdxJ-gpdA-F/pdxJ-gpdA-R, pdxJ-RF/pdxJ-RR were amplified and amplified.
  • the sub- and downstream sequences were fused with pdxJ-FF and pdxJ-RR as primers to express the expression cassette containing the gpdA promoter.
  • the Cas9/sgRNA induces a double-strand break at the pdxJ locus, and the recombinant enzyme Red integrates the gpdA promoter into the pdxJ gene. Sequencing verification.
  • Table 12 shows the corresponding indexes of the primer name and the sequence number.
  • nadA-gpdA-F SEQ ID NO: 4
  • nadA-gpdA-R SEQ ID NO: 5
  • nadA-RF SEQ ID NO: 6
  • nadA-RR SEQ ID NO:7
  • the co-expression plasmid is introduced.
  • various types of cells were collected for transformation analysis, and the results are shown in Table 13.
  • the whole cell transformation system in the transformation system is: cell wet weight of 20g / L, pyruvic acid 100g / L, levodopa 120g / L, glucose 200g / L, pH 9.0, temperature of 30 ° C, shaker speed of 250 rev / min
  • the conversion time is 24 hours.
  • Escherichia coli NP The best Escherichia coli HMBY (PG-nadA, PG-pdxJ) was named Escherichia coli NP.
  • the co-expression plasmid is introduced.
  • various types of cells were collected for transformation analysis, and the results are shown in Table 14.
  • the whole cell transformation system in the transformation system is: cell wet weight 20g / L, L-lactic acid 100g / L, levodopa 120g / L, pH 9.0, temperature 30 ° C, shaker speed 250 rev / min; conversion time 24 hour.
  • Escherichia coli HML The best Escherichia coli HML (PG-nadA, PG-pdxJ) was named Escherichia coli NL.
  • the co-expression plasmid is introduced.
  • various types of cells were collected for transformation analysis, and the results are shown in Table 15.
  • the whole cell transformation system in the transformation system is: cell wet weight of 20g / L, L-glutamic acid 200g / L, levodopa 120g / L, pH 9.0, temperature of 30 ° C, shaker speed of 250 rev / min; Time is 24 hours.
  • Escherichia coli HNP The best Escherichia coli HMG (PG-nadA, PG-pdxJ) was named Escherichia coli HNP.
  • the expression method was induced according to Example 2, and the cells were collected after induction of expression by Escherichia coli NP/pCOLADuet-1-lfldhd-ect1-bsgdh.
  • the cell wet weight was 1 g/L
  • pyruvic acid was 1 g/L.
  • levodopa 1g / L glucose 1g / L, pH 6.0, temperature 15 ° C, shaking speed 250 rev / min; conversion time 1 hour.
  • the concentration of R-danshensu was 77 mg/L, and e.e%>99.9.
  • the cells were collected after induction of expression by Escherichia coli NL/pCOLADuet-1-lfldhd-ect1-llldh.
  • the cell wet weight was 1 g/L
  • L-lactic acid was 1 g/ L
  • levodopa 1 g / L pH 6.0
  • temperature 15 ° C shaking speed 250 rpm / conversion time 1 hour.
  • the concentration of R-danshensu was 77 mg/L, and e.e%>99.9.
  • the expression method was induced according to Example 2, and the cells were collected after induction of expression by Escherichia coli HNP/pCOLADuet-1-efmdhd-bsgdh-lct.
  • the cell wet weight was 1 g/L, L-glutamic acid. 1 g / L, levodopa 1 g / L, pH 6.0, temperature 15 ° C, shaker speed 250 rpm / conversion time 1 hour.
  • the concentration of S-danshensu was 93 mg/L, and e.e%>99.9.
  • the cells were collected after the expression of the strain in Table 16 was completed, and the cell wet weight was 200 g/L, pyruvic acid 200 g/L, levodopa 200 g/L, glucose in a 100 ml reaction system. 200 g / L, pH 8.5, temperature 40 ° C, shaking speed 250 rpm / conversion time 48 hours. The precipitate was completely diluted and dissolved, and the result was measured.
  • the cells were collected after the induction of the expression in Table 17, and the cell wet weight was 200 g/L, L-lactic acid 200 g/L, and levodopa 200 g/L in a 100 ml reaction system. pH 8.5, temperature 40 ° C, shaker speed 250 rpm / conversion time 48 hours. The precipitate was completely diluted and dissolved, and the result was measured.
  • the cells were collected after the induction of expression in the strain in Table 18.
  • the cell wet weight was 200 g/L, L-glutamic acid 20 g/L, and levodopa 200 g/ L, pH 8.5, temperature 40 ° C, shaker speed 250 rpm / conversion time 48 hours.
  • the precipitate was completely diluted and dissolved, and the result was measured.

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Abstract

提供了一种重组大肠杆菌及其在丹参素生产中的应用，所述重组大肠杆菌同时表达α-­­­羟基羧酸脱氢酶和L-­­α-­­氨基酸转氨酶，以及葡萄糖脱氢酶、L-­­乳酸脱氢酶和L­­-谷氨酸脱氢酶中的任意一种，并在宿主大肠杆菌的基础上敲除了酚类化合物分解相关的基因。

Description

一种工程菌及其在丹参素生产中的应用 技术领域
本发明涉及一种工程菌及其在丹参素生产中的应用，属于生物工程技术领域。
背景技术
提取自丹参的丹参素，学名R-(+)-3-(3，4-二羟基苯基)-2-羟基丙酸、D-(+)-β-(3，4-二羟基苯基)乳酸，英文名为：Danshensu、D-DSS、R-DSS、(R)-(+)-3-(3，4-Dihydroxyphenyl)-lactic acid、(R)-(+)-3-(3，4-Dihydroxyphenyl)-2-hydroxypropanoic acid，是一种右旋酚酸类化合物。目前不存在天然的左旋丹参素。
丹参素是丹参水提液中的重要有效成份，国内于1980年从丹参水提液中得到并鉴定了结构(丹参水溶性有效成分的研究，Ⅱ.D(+)β(3，4-二羟基苯基)乳酸的结构，上海第一医学院学报，1980，05(7)，384-385)，各种研究表明丹参素具有重要的药理药效作用，在心脑血管疾病的治疗等方面具有独特治疗作用。
当前丹参素主要从丹参中提取得到(专利CN200810038853.9)。丹参素在丹参中的含量较低，并且丹参种植成本高且产量有限，因此当前丹参素不仅价格高而且远远无法满足市场的需求。专利CN201310559498.0提出了一种构建大肠杆菌基因工程菌利用葡萄糖发酵产丹参素的方法，由于合成代谢途径涉及利用羟基化酶，该酶很容易使代谢过程产物氧化而影响丹参素的产率，同时由于大肠杆菌发酵是高耗氧过程，也会氧化丹参素，因此当前该方法产率较低，成本将会高于植物提取过程。专利CN201210190171.6提出了水解丹酚酸B生产丹参素的方法，丹酚酸B需从丹参提取，并且化学水解过程有大量副反应，同样不适用于规模化生产。手性合成丹参素(专利CN201210420488.4)的催化剂极为昂贵，当前也仅停留在实验室水平。
早在1988年Roth等人提出了先以化学法处理左旋多巴得到对应的3，4-二羟基苯丙酮酸，再酶法合成S-(+)-3-(3，4-二羟基苯基)-2-羟基丙酸(S-DSS，L-DSS)的方法(Enzymatic Synthesis of(S)-(-)-3-(3，4-Dihydroxyphenyl)lactic Acid，Arch.Pharm.(Weinheim)321，179-180(1988))。Z.Findrik，等人采用蛇毒氨基酸氧化酶将左旋多巴转化成3，4-二羟基苯丙酮酸，然后再用D-乳酸脱氢酶还原生成D-(3，4-二羟基苯基)乳酸(Modelling and Optimization of the(R)-(+)-3,4-dihydroxyphenyllactic Acid Production Catalyzed with D-lactateDehydrogenase from Lactobacillus leishmannii Using Genetic Algorithm，Chem.Biochem.Eng.Q.19(4)351–358(2005))。这两种方法制备3，4-二羟基苯丙酮酸中间体的成本较高，且操作复杂。
发明内容
基于目前各种方法的缺陷，本发明提了了一种基于转氨酶的光学纯的丹参素生产方法，并构建了多酶共表达的工程菌，实现了丹参素的高效生产。本发明所要解决的技术问题是提供一种能低成本生产丹参素的重组菌。同时本发明要解决该菌株的构建和应用的技术问题。
本发明的第一个目的是提供能低成本生产光学纯丹参素的重组菌；所述重组菌同时表达了α-羟基羧酸脱氢酶和L-α-氨基酸转氨酶，以及以下任意一种：葡萄糖脱氢酶、L-乳酸脱氢酶、L-谷氨酸脱氢酶，并在宿主大肠杆菌的基础上敲除了酚类化合物分解相关的基因。
在一种实施方式中，所述α-羟基羧酸脱氢酶为D型α-羟基羧酸脱氢酶，来自Lactobacillus plantarum ATCC 14917、Enterococcus faecalis ATCC 35038或者Lactobacillus fermentum ATCC 14931。
在一种实施方式中，所述α-羟基羧酸脱氢酶为L型α-羟基羧酸脱氢酶，来自Bacillus coagulans DSM 1、Weissella confusa strain DSM 20196或者Lactobacillus fermentum ATCC 14931。
在一种实施方式中，所述α-羟基羧酸脱氢酶为D-α-羟基羧酸脱氢酶，其氨基酸序列是NCBI上accession NO.为WP_003643296.1、WP_002335374.1、或EEI22188.1的序列；α-羟基羧酸脱氢酶为L-α-羟基羧酸脱氢酶，其氨基酸序列是NCBI上accession NO为WP_013858488.1、WP_003607654.1或WP_035430779.1的序列。
在一种实施方式中，D-α-羟基羧酸脱氢酶的核苷酸序列是NCBI上accession NO.为NZ_GL379761 REGION:COMPLEMENT(533562..534560)、NZ_KB944641 REGION:161892..162830、ACGI01000078REGION:20793..21791的序列；L-α-羟基羧酸脱氢酶的核苷酸序列是NCBI上accession NO.为NZ_ATUM01000014 REGION:39316..40254、NZ_JQAY01000006 REGION:69708..70640、NZ_GG669901 REGION:45517..46470的序列。
在一种实施方式中，所述L-α-氨基酸转氨酶来自Escherichia coli BL21、Lactobacillus plantarum ATCC 14917、Lactobacillus paracasei ATCC 334。
在一种实施方式中，L-α-氨基酸转氨酶的氨基酸序列是NCBI上accession NO为WP_000462687.1、WP_000486988.1、WP_003643296.1、YP_806114.1的序列。
在一种实施方式中，L-α-氨基酸转氨酶的核苷酸序列是NCBI上accession NO为：NC_012892 REGION:COMPLEMENT(989603..990793)、NC_012892 REGION:4174517..4175710、NZ_GL379768REGION:complement(121900..123087)、NC_008526REGION:complement(840419..841594)的序列。
在一种实施方式中，所述葡萄糖脱氢酶来自Bacillus subtilis ATCC 13952。
在一种实施方式中，所述葡萄糖脱氢酶的氨基酸序列是NCBI上accession NO为WP_013351020.1序列。
在一种实施方式中，所述葡萄糖脱氢酶的核苷酸序列是NCBI上accession NO为：NZ_CP009748REGION:386154..38693。
在一种实施方式中，所述L-乳酸脱氢酶来自Lactococcus lactis ATCC 19257。
在一种实施方式中，所述L-乳酸脱氢酶的氨基酸序列是NCBI上accession NO为WP_003131075.1序列。
在一种实施方式中，所述L-乳酸脱氢酶的核苷酸序列是NCBI上accession NO为：NZ_JXJZ01000017 REGION:18532..19509的序列。
在一种实施方式中，所述L-谷氨酸脱氢酶来自Escherichia coli BL21、Rhodobacter sphaeroides ATCC BAA-808、Clostridium symbiosum ATCC 14940、Bacillus subtilis 168。
在一种实施方式中，L-谷氨酸脱氢酶的氨基酸序列是NCBI上accession NO为WP_000373021.1、WP_011338202.1、WP_003497202.1、WP_010886557.1序列。
在一种实施方式中，L-谷氨酸脱氢酶的核苷酸序列是NCBI上accession NO为：NC_012892 REGION:1786741..1788084、NC_007493 REGION:complement(2129131..2130558)、NZ_KE992901 REGION:complement(17603..18955)、NC_000964 REGION:complement(2402067..2403350)的序列。
在一种实施方式中，所述重组菌，是将编码L-α-氨基酸转氨酶、α-羟基羧酸脱氢酶和以下任意一种：葡萄糖脱氢酶、L-乳酸脱氢酶、L-谷氨酸脱氢酶的基因，都连接到质粒上，构建得到三基因共表达重组质粒，然后将重组质粒转化相应菌株，得到重组工程菌。
在一种实施方式中，所述重组菌是以Escherichia coli BL21(DE3)为宿主构建得到的。
在一种实施方式中，所述酚类化合物分解相关的基因为hpaD、mhpB中的任意一种或者两种组合。
在一种实施方式中，所述酚类物质分解基因的核苷酸序列是NCBI上,NCBI上accession NO为：NC_012892 REGION:complement(4505585..4506436)和NC_012892 REGION:339806..340750。
在一种实施方式中，所述重组菌还强化表达了丙酮酸转运基因、L-乳酸转运基因、谷氨酸转运基因、NAD合成基因、磷酸吡哆醛合成基因一种或者多种；其中，丙酮酸转运基因、L-乳酸转运基因、谷氨酸转运基因不同时表达。
在一种实施方式中，所述强化表达是通过将Escherichia coli BL21(DE3)基因组上所要强化表达的基因前增加组成型启动子。
在一种实施方式中，所述强化表达的基因为丙酮酸转运相关的基因、lldP(乳酸转运基因)、gltS(谷氨酸转运基因)、nadA(NAD合成基因)、pdxJ(磷酸吡多醛合成基因)中的任意一种或多种；其中，所述丙酮酸转运相关的基因包括btsT和ybdD(丙酮酸向胞内转运基因)。
在一种实施方式中，所述btsT和ybdD在NCBI上accession NO为：；nadA为NC_012892REGION:740487..741530；pdxJ为NC_012892REGION:complement(2567591..2568322)。
在一种实施方式中，所述lldP在NCBI上accession NO为：NC_012892 REGION:3646638..3648293；nadA为NC_012892 REGION:740487..741530；pdxJ为NC_012892 REGION:complement(2567591..2568322)。
在一种实施方式中，所述gltS在NCBI上accession NO为：NC_012892 REGION:complement(3694931..3696136)；nadA为NC_012892 REGION:740487..741530；pdxJ为NC_012892REGION:complement(2567591..2568322)。
本发明的第二个目的是提供一种生产丹参素的方法，所述方法是利用本发明的重组菌。
在一种实施方式中，所述生产丹参素，是进行全细胞转化生产。
在一种实施方式中，所述全细胞转化生产的体系中，包括细胞湿重为1-200g/L和左旋多巴浓度为1-200g/L；
当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和葡萄糖脱氢酶时，所述全细胞转化生产的体系中还包括：丙酮酸浓度为1-200g/L和葡萄糖浓度为1-200g/L；
当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-乳酸脱氢酶时，所述全细胞转化生产的体系中还包括：L-乳酸1-200g/L；
当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-谷氨酸脱氢酶时，所述全细胞转化生产的体系中还包括：L-谷氨酸1-200g/L；
所述全细胞转化生产的体系pH 6.0-9.0；于15-40℃反应，时间1-48小时。
本发明的有益效果：
本发明构建了一种新型的多酶共表达基因工程菌，实现了丹参素的低成本生产，进一步地，通过敲除或强化表达大肠杆菌基因组上的相将关基因促进底物的转运及减少产物的分解。本发明选择的(D/L)-α-羟基羧酸脱氢酶均具有底物专一性差，光学专一性强的特点，可生产光学纯的D-丹参素和L-丹参素。该生产过程简单且原料易得，具有良好的工业化应用前景。
具体实施方案
本发明的工程菌的功能核心在于可以同时表达3种酶，分别为L-α-氨基酸转氨酶、α-羟基羧酸脱氢酶和以下任意一种：葡萄糖脱氢酶、L-乳酸脱氢酶、L-谷氨酸脱氢酶。其原理为：在工程菌全细胞内，葡萄糖脱氢酶或L-乳酸脱氢酶或L-谷氨酸脱氢酶以菌体内的NAD为辅酶将对应的葡萄糖脱氢或L-乳酸脱氢酶或L-谷氨酸脱氢酶生成NADH和对应的葡萄糖酸或丙酮酸或α-酮戊二酸；左旋多巴被L-α-氨基酸转氨酶脱氨生成3,4-二羟基苯丙酮酸，丙酮酸获得氨转化成丙氨酸；α-羟基羧酸脱氢酶利用葡萄糖脱氢过程生成的NADH将3,4-二羟基苯丙酮酸还原成丹参素、同时实现了辅酶NAD的再生。进一步地，同时敲除或强化表达大肠杆菌基因组上的相将关基因促进底物的转运及减少产物的分解。
为解决上述技术问题，本发明采用的技术方案如下：
1.本发明所涉及的菌株及质粒
购自美国菌种保藏中心ATCC的Lactobacillus plantarum ATCC 14917、Enterococcus faecalis ATCC 35038、Lactobacillus fermentum ATCC 14931、Lactobacillus paracasei ATCC 334、 Bacillus subtilis ATCC 13952、Escherichia coli BL21(DE3)、Lactococcus lactis ATCC 19257。购自德国微生物菌种保藏中心DSMZ的Bacillus coagulans DSM 1、Weissella confusa strain DSM 20196。购自Novagen公司的pETDuet-1、pACYCDue-1、pCOLADuet-1、pRSFDuet-1质粒和Escherichia coli BL21(DE3)。
2.大肠杆菌中相关基因的敲除及组成型强化表达
(1)大肠杆菌酚类化合物分解相关的基因的敲除
本发明中的酚类物质都极易被大肠杆菌中的酶分解，根据文献(Biodegradation of Aromatic Compounds by Escherichia coli，Microbiol Mol Biol Rev.2001,65(4):523–569.)，将相关基因敲除，避免产物和底物的分解。选择的基因是hpaD和mhpB,NCBI上accession NO为：NC_012892REGION:complement(4505585..4506436)和NC_012892REGION:339806..340750。
(2)大肠杆菌丙酮酸转运基因的组成型强化表达
在全细胞转化过程中，需把底物转运至细胞内才能进行，增强丙酮酸转运蛋白有助于快速并长时间维持胞内底物的高浓度，有利于反应的进行。选择丙酮酸转运相关的基因是btsT和ybdD，NCBI上accession NO为：NC_012892 REGION:complement(4496239..4498389)和NC_012892 REGION:592652..592849。多巴与芳香族氨基酸类似，细胞培养过程中需要吸收氨基酸等，因此菌体本身会表达大量的氨基酸转运蛋白，无须再强化表达。
(3)大肠杆菌辅酶合成相关重要基因的组成型强化表达
在α-羟基羧酸脱氢酶还原过程中需要以NADH为辅酶，强化表达大肠杆菌NAD合成途径的关键酶，可以提高菌体内的NAD水平，从而有利于丹参素的生成。选择的基因有nadA。NCBI上accession NO为：NC_012892 REGION:740487..741530。
磷酸吡多醛(胺)是L-α-氨基酸转氨酶的辅酶，过表达该辅酶途径中的核心基因pdxJ，有利于左旋多巴的合成。NCBI上accession NO为：NC_012892REGION:complement(2567591..2568322)。
3.酶的选择
(1)L-α-氨基酸转氨酶的选择
L-α-氨基酸转氨酶广泛存在于细菌、真菌、哺乳动物细胞中。通常活力最高的是以α-酮戊二酸或草酰乙酸为受氨体的转氨酶，在这过程中α-酮戊二酸或草酰乙酸价格昂贵，而对应生成的谷氨酸或天冬氨酸价值均远低于对应的前体，综合考查20种天然L-氨基酸对应的α-酮酸发现，丙酮酸与丙氨酸的价格相当，因此本申请选择以丙酮酸为氨的受体，从而实现丙氨酸与丹参素的联产。从Lactobacillus plantarum ATCC 14917、Lactobacillus paracasei ATCC334中分别克隆得到L-α-氨基酸转氨酶基因lpt、lct，其氨基酸序列是NCBI上accession NO.为WP_003643296.1、YP_806114.1的序列。从Escherichia coli BL1(DE3)中克隆得到两个L-α-氨基酸转氨酶基因ect1、ect2，其氨基酸序列是NCBI上accession NO为WP_000462687.1、WP_000486988.1。
(2)α-羟基羧酸脱氢酶的选择
根据最适底物的情况，α-羟基羧酸脱氢酶包含有乳酸脱氢酶、α-羟酸异己酸脱氢酶、扁桃酸脱氢酶、乙醛酸还原酶等，这些酶能广泛作用于多种底物生成α-羟基羧酸，通常根据其最适作用的底物来命名。本发明从中选择光学性强且对3，4-二羟基苯丙酮酸具有较强活性的酶，用于D或L丹参素的生产。从Lactobacillus plantarum ATCC 14917、Enterococcus faecalis ATCC 35038、Lactobacillus fermentum ATCC 14931中分别克隆得到D型α-羟基羧酸脱氢酶基因lpldhd、efmdhd、lfldhd，其氨基酸序列是NCBI上accession NO.为WP_003643296.1、WP_002335374.1、EEI22188.1的序列。从Bacillus coagulans DSM 1、Weissella confusa strain DSM 20196、Lactobacillus fermentum ATCC 14931中分别克隆得到L型α-羟基羧酸脱氢酶基因bcldhl、wcldhl、lfldhl，其氨基酸序列是NCBI上accession NO为WP_013858488.1、WP_003607654.1、WP_035430779.1的序列。
(3)葡萄糖脱氢酶的选择
在生物转化反应中，α-羟基羧酸脱氢酶需要以NADH和/或NADPH为辅酶，采常有甲酸脱氢酶、葡萄糖脱氢酶、亚磷酸酸脱氢酶等，葡萄糖脱氢酶相对于其它来酶来说活力最高，因此本发明从Bacillus subtilis ATCC 13952得到葡萄糖脱氢酶基因bsgdh(氨基酸序列是WP_013351020.1)。
(4)L-乳酸脱氢酶的选择
L-乳酸是最为廉价的有机酸，脱氢后成的丙酮酸再转氨生成的丙氨酸具有较高的附加值。L-乳酸脱氢酶广泛存在多种微生物中，以L-乳酸为底物将L-乳酸上生成的氢传递给辅酶NAD或NADP，从而生成NADH或NADPH。NADH或者NADPH可以作为前述的α-羟基羟酸脱氢酶的供氢体。通常来讲以NAD(NADP)为辅酶的乳酸脱氢酶更趋向于以丙酮酸为底物合成乳酸，但当乳酸过量等情况下乳酸脱氢酶会脱掉乳酸的氢生成丙酮酸。本发明从Lactococcus lactis ATCC 19257中得到L-乳酸脱氢酶基因llldh(氨基酸序列是WP_003131075.1)。
(5)L-谷氨酸脱氢酶的选择
L-谷氨酸是最为廉价的一种氨基酸，脱氢后生成的α-酮戊二酸可作为左旋多巴转氨脱氨的受氨体。L谷氨酸脱氢酶广泛存在于几乎所有生物中，以L-谷氨酸为底物将L-谷氨酸上生成的氢传递给辅酶NAD或NADP，从而生成NADH或NADPH。NADH或者NADPH可以作为前述的羟基羟酸脱氢酶的供氢体。本发明从Escherichia coli BL21、Rhodobacter sphaeroides ATCC BAA-808、Clostridium symbiosum ATCC 14940、Bacillus subtilis 168中分别得到L-谷氨酸基因ecgdh(氨基酸序列是WP_000373021.1)、rsgdh(氨基酸序列是WP_011338202.1)、csgdh(氨基酸序列是WP_003497202.1)、bsgdh(氨基酸序列是WP_010886557.1)。
4.三酶共表达体系的构建及细胞的培养
目前大肠杆菌多基因共表达有多种方法(大肠杆菌多基因共表达策略，中国生物工程杂志，2012，32(4):117-122)，本发明采用刘向磊(合成生物学技术改造大肠杆菌生产莽草酸及白藜芦醇，2016，上海医药工业研究院，博士论文)所述方法构建，每个基因前均包含T7启动子和RBS结合点，每个基因后有一个T7终止子。理论上来讲因为每个基因前都有T7和RBS，因此基因的表达强度受排列次序的影响不大。每个质粒上包含三个基因(L-α-氨基酸转氨酶、(D/L)-α-羟基羧酸脱氢酶、葡萄糖脱氢酶或L-乳酸脱氢酶或L-谷氨酸脱氢酶对应的基因)，将构建好的质粒热转导入大肠杆菌感受态细胞中，并涂布于抗生素固体平板上，筛选得到阳性转化子，即得到重组大肠杆菌。细胞的培养：根据经典的重组大肠杆菌培养及诱导表达方案，将重组大肠杆菌按体积比为2％的量转接到LB发酵培养基(蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L)中，当细胞OD 600达到0.6-0.8后，加入终浓度为0.4mM的IPTG，在20℃诱导表达培养8h。诱导表达结束后，20℃、8000rpm、20分钟离心收集细胞。
5.全细胞转化生产纯丹参素
细胞转化生产的体系为：细胞湿重为1-200g/L，左旋多巴浓度为1-200g/L；
当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和葡萄糖脱氢酶时，所述全细胞转化生产的体系中还包括：丙酮酸浓度为1-200g/L和葡萄糖浓度为1-200g/L；
当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-乳酸脱氢酶时，所述全细胞转化生产的体系中还包括：L-乳酸1-200g/L；
当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-谷氨酸脱氢酶时，所述全细胞转化生产的体系中还包括：L-谷氨酸1-200g/L；
所述全细胞转化生产的体系pH 6.0-9.0，于15-40℃反应，时间1-48小时。
转化结束后液相色谱测定丹参素产量及构型。左旋多巴溶解度较低，高浓度情况下是含有不溶物的混悬液。
6.样品的检测分析
丹参素定量分析：转化液采用PerkinElmer Series 200高效液相色谱仪检测分析，配示差折光检测器。色谱条件为：流动相是甲醇-0.1％甲酸水(40:60)、采用汉邦Megres C18色谱柱(4.6×250mm，5μm)，流速1ml/min、柱温30℃、进样量20μl。
手性分析：PerkinElmer Series 200高效液相色谱仪检测分析，配示紫外检测器，Chiralcel OD-H手性柱(4.6×250mm)，流动相体积比为正己烷:异丙醇:三氟乙酸＝80:20:0.1，流速为0.5mL/min，柱温25℃，进样量20μL，检测波长280nm。
丹参素溶解度较低，转化过程如有结晶析出，则稀释后测定。
丹参素的光学纯度通过对映体过量值(％e.e)来评价。
当生产R-丹参素时，
对映体过量值％e.e＝[(S R-S S)/(S R+S S)×100％]
当生产S-丹参素时，
对映体过量值％e.e＝[(S S-S R)/(S R+S S)×100％]
式中S S为转化液中S-丹参素的峰面积，S R为转化液中R-丹参素的液相色谱峰面积。
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚明白，以下结合实施例，对本发明进行详细的说明。应当说明的是，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1
根据文献Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli.Microbial Cell Factories,2017,16(1):68所述的方法将Escherichia coli BL21(DE3)上的hpaD和mhpB进行单或双敲除。其中，本发明所用基因敲除的质粒为pCasRed与pCRISPR-gDNA(hpaD sgRNA)与同源臂(hpaD donor)一起导入Escherichia coli BL21(DE3)上，Cas9/sgRNA诱发宿主在hpaD基因位点发生双链断裂，重组酶Red将hpaD donor整合到hpaD基因上，实现基因的敲除，并测序验证。hpaD sgRNA、hpaD donor、mhpB sgRNA、mhpB donor分别如序列表SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12所示。mhpB以同样的方式敲除。
配置pH为7的溶液，左旋多巴或D-丹参素4g/L，湿菌体量200g/L，35℃放置10小时后测定浓度，表1中显示了反应体系中，左旋多巴和D-丹参素的剩余量。
表1 不同菌株对底物和产物分解后的剩余浓度
  左旋多巴g/L D-丹参素g/L
Escherichia coli BL21(DE3) 1.6 1.4
Escherichia coli BL21(ΔhpaDΔmhpB,DE3) 3.7 3.5
Escherichia coli BL21(ΔhpaD,DE3) 2.0 2.8
Escherichia coli BL21(ΔmhpB,DE3) 1.7 1.5
很显然Escherichia coli BL21(ΔhpaDΔmhpB,DE3)效果最好，将之命名为Escherichia coli HM。
实施例2
L-α-氨基酸转氨酶的筛选，分别从大肠杆菌和乳杆菌中克隆多种L-α-氨基酸转氨酶基因，并在Escherichia coli BL21(DE3)中得到表达，破胞测定粗酶液活性，以丙酮酸为受体时比较各种酶的活性，根据文献(转氨酶催化不对称合成芳香族L-氨基酸.生物工程学报，2012，28(11):1346-1358.)所述的方法测定L-α-氨基酸转氨酶的活性，结果如表2所示。因此选择来源于大肠杆菌的L-α-氨基酸转氨酶ect1用于左旋多巴的转氨脱氨是最佳的。
以α-酮戊二酸为受体时比较各种酶的活性，根据文献(转氨酶催化不对称合成芳香族L-氨基酸.生物工程学报，2012，28(11):1346-1358.)所述的方法测定L-α-氨基酸转氨酶的活性，结果如表3所示。因此选择来源于Lactobacillus paracasei ATCC 334的L-α-氨基酸转氨酶lct用于左旋多巴的转氨脱氨是最佳的。
诱导表达方法：将重组大肠杆菌按体积比为2％的量转接到LB发酵培养基(蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L)中，当细胞OD600达到0.6-0.8后，加入终浓度为0.4mM的IPTG，在20℃诱导表达培养8h。诱导表达结束后，20℃、8000rpm、20分钟离心收集细胞。
表2 不同L-α-氨基酸转氨酶的活性比较
重组菌 活性U/ml
Escherichia coli BL21(DE3)/pETDuet-1-ect1 2
Escherichia coli BL21(DE3)/pETDuet-1-ect2 0.01
Escherichia coli BL21(DE3)/pETDuet-1-lpt 1.2
Escherichia coli BL21(DE3)/pETDuet-1-lct 0.6
表3 不同L-α-氨基酸转氨酶的活性比较
重组菌 活性U/ml
Escherichia coli BL21/pETDuet-1-ect1 2.1
Escherichia coli BL21/pETDuet-1-ect2 0.9
Escherichia coli BL21/pETDuet-1-lpt 2.4
Escherichia coli BL21/pETDuet-1-lct 3.1
实施例3
α-羟基羧酸脱氢酶酶学性质的比较。通常这种酶类也可能具有还原丙酮酸生成乳酸的能力，因此不能还原或极微弱还原丙酮酸的酶是比较好的。以丙酮酸为底物，比较了不同酶的还原能力，根据文献(粘质沙雷氏菌H3010发酵型D-乳酸脱氢酶基因的克隆表达、纯化及酶学性质研究.工业微生物,2012,42(04):30-37.)所述的方法测定以NAD为辅酶还原丙酮酸的活性，实验结果如表4所示。
表4 各种α-羟基羧酸脱氢酶还原丙酮酸活性比较
重组菌 活性U/ml
Escherichia coli HM/pETDuet-1-lpldhd 6.6
Escherichia coli HM/pETDuet-1-efmdhd 0
Escherichia coli HM/pETDuet-1-lfldhd 0.7
Escherichia coli HM/pETDuet-1-bcldhl 5.2
Escherichia coli HM/pETDuet-1-wcldhl 0.2
Escherichia coli HM/pETDuet-1-lfldhl 6.3
实施例4
同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和葡萄糖脱氢酶的重组大肠杆菌构建：首先将编码L-α-氨基酸转氨酶、α-羟基羧酸脱氢酶和葡萄糖脱氢酶的基因，连接到质粒上。得到三基因共表达重组质粒，将质粒转化大肠杆菌Escherichia coli HM，利用抗生素平板筛选得到阳性转化子，即得到重组大肠杆菌。
将重组大肠杆菌诱导表达完成后收集菌体，于100ml反应体积中，细胞湿重40g/L，左旋多巴40g/L，丙酮酸30g/L，葡萄糖30g/L，pH 8.0，于35℃反应，时间12小时。转化结束后液相色谱测定丹参素产量及构型，结果如表5所示。
表5 各种重组菌的比较
Figure PCTCN2018111895-appb-000001
Figure PCTCN2018111895-appb-000002
实施例5
同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-乳酸脱氢酶的重组大肠杆菌构建：首先将编码L-乳酸脱氢酶、L-α-氨基酸转氨酶、α-羟基羧酸脱氢酶的基因，连接到质粒上。得到三基因共表达重组质粒，将质粒转化大肠杆菌Escherichia coli HM，利用抗生素平板筛选得到阳性转化子，即得到重组大肠杆菌。
将重组大肠杆菌诱导表达完成后收集菌体，于100ml反应体积中，细胞湿重为40g/L，左旋多巴浓度为40g/L，丙酮酸浓度为30g/L，pH 8.0，于35℃反应，时间12小时。转化结束后液相色谱测定丹参素产量及构型，结果如表6所示。
表6 各种重组菌的比较
Figure PCTCN2018111895-appb-000003
实施例6
同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-谷氨酸脱氢酶重组大肠杆菌构建：首先将编码L-α-氨基酸转氨酶、α-羟基羧酸脱氢酶和L-谷氨酸脱氢酶的基因，连接到质粒上。得到三基因共表达重组质粒，将质粒转化大肠杆菌Escherichia coli HM，利用抗生素平板筛选得到阳性转化子，即得到重组大肠杆菌。
将重组大肠杆菌诱导表达完成后收集菌体，于100ml反应体积中，细胞湿重为40g/L，左旋多巴浓度为40g/L，L-谷氨酸浓度为30g/L，pH 8.0，于35℃反应，时间12小时。转化结束后液相色谱测定丹参素产量及构型，结果如表7所示。
表7 各种重组菌的比较
Figure PCTCN2018111895-appb-000004
Figure PCTCN2018111895-appb-000005
实施例7
采用文献Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli.Microbial Cell Factories,2017,16(1):68所述的方法，将Escherichia coli HM基因组上对应基因前增加大肠杆菌的3-磷酸甘油醛脱氢酶基因(gpdA)前的中等表达强度组成型启动子(PG)，序列如SEQ ID NO:8所示。
强化基因btsT表达时，以Escherichia coli HM基因组为模板，以引物btsT-FF/btsT-FR、btsT-gpdA-F/btsT-gpdA-R、btsT-RF/btsT-RR,扩增出上游、启动子、下游序列，并以btsT-FF和btsT-RR为引物融合为含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含btsT sgRNA)一起转入Escherichia coli HM后，Cas9/sgRNA诱发宿主在btsT基因位点发生双链断裂，重组酶Red将gpdA启动子整合到btsT基因之前，并测序验证。
强化基因ybdD表达时，以Escherichia coli HM基因组为模板，以引物ybdD-FF/ybdD-FR、ybdD-gpdA-F/ybdD-gpdA-R、ybdD-RF/ybdD-RR,扩增出上游、启动子、下游序列，并以ybdD-FF和ybdD-RR为引物融合为含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含ybdD sgRNA)一起转入Escherichia coli HM后，Cas9/sgRNA诱发宿主在ybdD基因位点发生双链断裂，重组酶Red将gpdA启动子整合到ybdD基因之前，并测序验证。
下表8为引物名称与序列表序号的对应索引。
表8 引物名称与序列表序号对照
名称 序列表中编号
btsT sgRNA SEQ ID NO:20
btsT-FF SEQ ID NO:22
btsT-FR SEQ ID NO:23
btsT-gpdA-F SEQ ID NO:24
btsT-gpdA-R SEQ ID NO:25
btsT-RF SEQ ID NO:26
btsT-RR SEQ ID NO:27
ybdD sgRNA SEQ ID NO:21
ybdD-FF SEQ ID NO:28
ybdD-FR SEQ ID NO:29
ybdD-gpdA-F SEQ ID NO:30
ybdD-gpdA-R SEQ ID NO:31
ybdD-RF SEQ ID NO:32
ybdD-RR SEQ ID NO:33
根据实施例2所述的诱导表达方法，收集各类细胞进行转化分析，结果如表9所示。转化体系中全细胞转化体系为：细胞湿重5g/L，丙酮酸50g/L，左旋多巴20g/L，葡萄糖50g/L，pH 8.0，温度为40℃，摇床转速250转/分钟；转化时间12小时。
表9 转化结果比较
Figure PCTCN2018111895-appb-000006
将效果最好的Escherichia coli HM(PG-btsT,PG-ybdD)命名为Escherichia coli HMBY。
强化基因lldP表达时，以Escherichia coli HM基因组为模板，扩增出上游、启动子、下游序列，再得到含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含lldPsgRNA)一起转入Escherichia coli HM后，Cas9/sgRNA诱发宿主在lldP基因位点发生双链断裂，重组酶Red将gpdA启动子整合到lldP基因之前，并测序验证。
根据实施例2所述的诱导表达方法，收集各类细胞进行转化分析，结果如表10所示。转化体系中全细胞转化体系为：细胞湿重5g/L，L-乳酸50g/L，左旋多巴20g/L，pH 8.0，温度为40℃，摇床转速250转/分钟；转化时间12小时。
表10 转化结果比较
Figure PCTCN2018111895-appb-000007
将效果最好的Escherichia coli HM(PG-lldP)命名为Escherichia coli HML。
强化基因gltS表达时，以Escherichia coli HM基因组为模板，扩增出上游、启动子、下游序列，再得到含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含gltS sgRNA)一起转入Escherichia coli HM后，Cas9/sgRNA诱发宿主在gltS基因位点发生双链断裂，重组酶Red将gpdA启动子整合到gltS基因之前，并测序验证。
根据实施例2所述的诱导表达方法，收集各类细胞进行转化分析，结果如表11所示。转化体系中全细胞转化体系为：细胞湿重5g/L，L-谷氨酸1g/L，左旋多巴20g/L，pH 8.0，温度为40℃，摇床转速250转/分钟；转化时间12小时。
表11 转化结果比较
Figure PCTCN2018111895-appb-000008
将效果最好的Escherichia coli HM(PG-gltS)命名为Escherichia coli HMG。
实施例8
根据实例7的方法将Escherichia coli HMBY中nadA、pdxJ基因前增加大肠杆菌的3-磷酸甘油醛脱氢酶基因(gpdA)前的中等表达强度组成型启动子(PG)，序列如SEQ ID NO:8所示。然后再将质粒导入。
强化基因nadA表达时，以Escherichia coli HMBY基因组为模板，以引物nadA-FF/nadA-FR、nadA-gpdA-F/nadA-gpdA-R、nadA-RF/nadA-RR,扩增出上游、启动子、下游序列，并以nadA-FF和nadA-RR为引物融合为含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含nadA sgRNA)一起转入Escherichia coli HMBY后，Cas9/sgRNA诱发宿主在nadA基因位点发生双链断裂，重组酶Red将gpdA启动子整合到nadA基因之前，并测序验证。
强化基因pdxJ表达时，以Escherichia coli HMBY基因组为模板，以引物pdxJ-FF/pdxJ-FR、pdxJ-gpdA-F/pdxJ-gpdA-R、pdxJ-RF/pdxJ-RR,扩增出上游、启动子、下游序列，并以pdxJ-FF和pdxJ-RR为引物融合为含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含pdxJ sgRNA)一起转入Escherichia coli HMBY后，Cas9/sgRNA诱发宿主在pdxJ基因位点发生双链断裂，重组酶Red将gpdA启动子整合到pdxJ基因之前，并测序验证。
下表12为引物名称与序列表序号的对应索引。
表12 引物名称与序列表序号对照
名称 序列表中编号
pdxJ sgRNA SEQ ID NO:13
nadA sgRNA SEQ ID NO:1
pdxJ-FF SEQ ID NO:14
pdxJ-FR SEQ ID NO:15
pdxJ-gpdA-F SEQ ID NO:16
pdxJ-gpdA-R SEQ ID NO:17
pdxJ-RF SEQ ID NO:18
pdxJ-RR SEQ ID NO:19
nadA-FF SEQ ID NO:2
nadA-FR SEQ ID NO:3
nadA-gpdA-F SEQ ID NO:4
nadA-gpdA-R SEQ ID NO:5
nadA-RF SEQ ID NO:6
nadA-RR SEQ ID NO:7
基因改造完成后，将共表达质粒导入。根据实施例2所述的诱导表达方法，收集各类细胞进行转化分析，结果如表13所示。转化体系中全细胞转化体系为：细胞湿重为20g/L，丙酮酸100g/L，左旋多巴120g/L，葡萄糖200g/L，pH 9.0，温度为30℃，摇床转速250转/分钟；转化时间24小时。
表13 转化结果比较
Figure PCTCN2018111895-appb-000009
将最好的Escherichia coli HMBY(PG-nadA,PG-pdxJ)命名为Escherichia coli NP。
基因改造完成后，将共表达质粒导入。根据实施例2所述的诱导表达方法，收集各类细胞进行转化分析，结果如表14所示。转化体系中全细胞转化体系为：细胞湿重为20g/L，L-乳酸100g/L，左旋多巴120g/L，pH 9.0，温度为30℃，摇床转速250转/分钟；转化时间24小时。
表14 转化结果比较
Figure PCTCN2018111895-appb-000010
将最好的Escherichia coli HML(PG-nadA,PG-pdxJ)命名为Escherichia coli NL。
基因改造完成后，将共表达质粒导入。根据实施例2所述的诱导表达方法，收集各类细胞进行转化分析，结果如表15所示。转化体系中全细胞转化体系为：细胞湿重为20g/L，L-谷氨酸200g/L，左旋多巴120g/L，pH 9.0，温度为30℃，摇床转速250转/分钟；转化时间24小时。
表15 转化结果比较
Figure PCTCN2018111895-appb-000011
将最好的Escherichia coli HMG(PG-nadA,PG-pdxJ)命名为Escherichia coli HNP。
实施例9
根据实施例2所述诱导表达方法，将Escherichia coli NP/pCOLADuet-1-lfldhd-ect1-bsgdh诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重1g/L，丙酮酸1g/L，左旋多巴1g/L，葡萄糖1g/L，pH 6.0，温度为15℃，摇床转速250转/分钟；转化时间1小时。测定结果，R-丹参素浓度为77mg/L，e.e％>99.9。
根据实施例2所述诱导表达方法，将Escherichia coli NL/pCOLADuet-1-lfldhd-ect1-llldh诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重1g/L，L-乳酸1g/L，左旋多巴1g/L，pH 6.0，温度为15℃，摇床转速250转/分钟；转化时间1小时。测定结果，R-丹参素浓度为77mg/L，e.e％>99.9。
根据实施例2所述诱导表达方法，将Escherichia coli HNP/pCOLADuet-1-efmdhd-bsgdh-lct诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重1g/L，L-谷氨酸1g/L，左旋多巴1g/L，pH 6.0，温度为15℃，摇床转速250转/分钟；转化时间1小时。测定结果，S-丹参素浓度为93mg/L，e.e％>99.9。
实施例10
根据实施例2所述诱导表达方法，将表16中菌株诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重200g/L，丙酮酸200g/L，左旋多巴200g/L，葡萄糖200g/L，pH 8.5，温度为40℃，摇床转速250转/分钟；转化时间48小时。将沉淀全部稀释溶解后测定结果。
表16 转化结果比较
Figure PCTCN2018111895-appb-000012
根据实施例2所述诱导表达方法，将表17中菌株诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重200g/L，L-乳酸200g/L，左旋多巴200g/L，pH 8.5，温度为40℃，摇床转速250转/分钟；转化时间48小时。将沉淀全部稀释溶解后测定结果。
表17 转化结果比较
Figure PCTCN2018111895-appb-000013
Figure PCTCN2018111895-appb-000014
根据实施例1所述诱导表达方法，将表18中菌株诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重200g/L，L-谷氨酸20g/L，左旋多巴200g/L，pH 8.5，温度为40℃，摇床转速250转/分钟；转化时间48小时。将沉淀全部稀释溶解后测定结果。
表18 转化结果比较
Figure PCTCN2018111895-appb-000015
以上所述的酶及其共表达基因工程菌的改造和构建、菌体的培养基组成及培养方法和全细胞生物转化仅为本发明的较佳实施例而已，并不用于限制本发明，理论上来讲其它的细菌、丝状真菌、放线菌、动物细胞均可进行基因组的改造，并用于多基因共表达的全细胞催化。凡在本发明的原则和精神之内所作的任何修改、等同替换。
Figure PCTCN2018111895-appb-000016
Figure PCTCN2018111895-appb-000017
Figure PCTCN2018111895-appb-000018
Figure PCTCN2018111895-appb-000019
Figure PCTCN2018111895-appb-000020
Figure PCTCN2018111895-appb-000021
Figure PCTCN2018111895-appb-000022
Figure PCTCN2018111895-appb-000023

Claims (10)

  1. 一种重组大肠杆菌，其特征在于，所述重组大肠杆菌同时表达了α-羟基羧酸脱氢酶和L-α-氨基酸转氨酶，以及以下任意一种：葡萄糖脱氢酶、L-乳酸脱氢酶、L-谷氨酸脱氢酶，并在宿主大肠杆菌的基础上敲除了酚类化合物分解相关的基因。
  2. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述酚类分解基因为hpaD、mhpB中的任意一种或者两种组合。
  3. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述重组大肠杆菌还强化表达了丙酮酸转运基因、L-乳酸转运基因、谷氨酸转运基因、NAD合成基因、磷酸吡哆醛合成基因一种或者多种；其中，丙酮酸转运基因、L-乳酸转运基因、谷氨酸转运基因不同时表达。
  4. 根据权利要求3所述的重组大肠杆菌，其特征在于，所述强化表达的基因为丙酮酸转运相关的基因、lldP、gltS、nadA、pdxJ中的任意一种或多种，其中，所述丙酮酸转运相关的基因包括btsT和ybdD。
  5. 根据权利要求4所述的重组大肠杆菌，其特征在于，所述强化表达是通过将宿主大肠杆菌基因组上所要强化表达的基因前增加组成型启动子。
  6. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述葡萄糖脱氢酶、L-乳酸脱氢酶、L-谷氨酸脱氢酶中的任意一种和α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶是通过pCOLADuet共表达的。
  7. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述宿主菌为Escherichia coli BL21。
  8. 一种生产丹参素的方法，其特征在于，所述方法是利用权利要求1-7任一所述的重组菌。
  9. 根据权利要求8所述的方法，其特征在于，所述方法是进行全细胞转化生产。
  10. 根据权利要求9所述的方法，其特征在于，所述全细胞转化生产的体系中，包括细胞湿重为1-200g/L和左旋多巴浓度为1-200g/L；
    当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和葡萄糖脱氢酶时，所述全细胞转化生产的体系中还包括：丙酮酸浓度为1-200g/L和葡萄糖浓度为1-200g/L；
    当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-乳酸脱氢酶时，所述全细胞转化生产的体系中还包括：L-乳酸1-200g/L；
    当所述重组菌同时表达α-羟基羧酸脱氢酶、L-α-氨基酸转氨酶和L-谷氨酸脱氢酶时，所述全细胞转化生产的体系中还包括：L-谷氨酸1-200g/L；
    所述全细胞转化生产的体系pH6.0-9.0；于15-40℃反应，时间1-48小时。
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