WO2019200873A1 - 一种重组大肠杆菌及使用其生产丹参素的方法 - Google Patents

一种重组大肠杆菌及使用其生产丹参素的方法 Download PDF

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WO2019200873A1
WO2019200873A1 PCT/CN2018/111884 CN2018111884W WO2019200873A1 WO 2019200873 A1 WO2019200873 A1 WO 2019200873A1 CN 2018111884 W CN2018111884 W CN 2018111884W WO 2019200873 A1 WO2019200873 A1 WO 2019200873A1
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escherichia coli
gene
dehydrogenase
expression
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 CN201810352668.0A external-priority patent/CN108949648B/zh
Priority claimed from CN201810352680.1A external-priority patent/CN108949650B/zh
Priority claimed from CN201810352697.7A external-priority patent/CN108949655B/zh
Priority claimed from CN201810352742.9A external-priority patent/CN108949657B/zh
Application filed by 江南大学 filed Critical 江南大学
Priority to DE112018007299.1T priority Critical patent/DE112018007299T5/de
Priority to JP2020558016A priority patent/JP7075505B2/ja
Priority to US16/536,406 priority patent/US10829790B2/en
Publication of WO2019200873A1 publication Critical patent/WO2019200873A1/zh

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Definitions

  • the invention relates to a method for producing Danshensu, belonging 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 provides a method for producing optically pure Danshensu, and constructs an engineering bacteria co-expressed by multiple enzymes, thereby realizing 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 ⁇ -hydroxycarboxylic acid dehydrogenase and L-amino acid oxidase, and any one of the following: a source of L-glutamate dehydrogenase, exogenous L-lactate dehydrogenase, glucose dehydrogenase, tyrosine phenol lyase, wherein the expression of tyrosine phenol lyase simultaneously expresses L-lactate dehydrogenase; The gene related to the decomposition of phenolic compounds is knocked out on the basis of the 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_GL379761REGION: COMPLEMENT (533562..534560), NZ_KB944641REGION: 161892..162830, ACGI01000078REGION: 20793.
  • the sequence of .21791; the nucleotide sequence of L- ⁇ -hydroxycarboxylic acid dehydrogenase is the sequence of accession NO. for NCBI on NZ_ATUM01000014REGION: 39316..40254, NZ_JQAY01000006REGION: 69708..70640, NZ_GG669901REGION:45517..46470.
  • 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_012892REGION: 1787641..1788084, NC_007493 REGION: complement (2129131.. 2130558), NZ_KE992901 REGION: complement (17603. .18955), NC_000964REGION: sequence of complement (2402067..2403350).
  • the L-amino acid oxidase is from hydrogen peroxide-free L-oxygen in Proteus mirabilis ATCC 29906, Cosenzaea myxofaciens ATCC 19692, Morganella morganii ATCC 49993, Providencia rettgeri DSM 1131 or Ignatzschineria larvae DSM 13226 Acid oxidase.
  • the amino acid sequence of the L-amino acid oxidase is the sequence of accession NO on the NCBI of WP_004244224.1, OAT30292.1, EFE55026.1, WP_036414800.1, or WP_026879504.1.
  • the nucleotide sequence of the L-amino acid oxidase is as in the sequence listing: NZ_GG668576REGION: 1350390.. 1351805, LXEN01000066 REGION: 20563.. 21963, ACCI02000030 REGION: 21025.. 22443, NZ_LAGC01000006 REGION: 309569.. 310993 , NZ_KI783332REGION: 35799..37217.
  • 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_JXJZ01000017REGION: 18532..19509.
  • the tyrosine phenol lyase is from Erwinia herbicola ATCC 214344.
  • amino acid sequence of the tyrosine phenol lyase is NC3 and the accession NO is P31011.2.
  • 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 recombinant strain is a gene encoding an enzyme encoding L-amino acid oxidase, ⁇ -hydroxycarboxylic acid dehydrogenase, and L-glutamic acid dehydrogenation, and is ligated to a plasmid to construct The three genes co-express the recombinant plasmid, and then the recombinant plasmid is transformed into the corresponding strain to obtain a recombinant engineering strain.
  • the recombinant strain is a gene encoding an enzyme encoding L-amino acid oxidase, ⁇ -hydroxycarboxylic acid dehydrogenase, and L-lactic acid dehydrogenase, and is ligated to a plasmid to construct a three gene.
  • the recombinant plasmid is co-expressed, and then the recombinant plasmid is transformed into the corresponding strain to obtain a recombinant engineered strain.
  • the recombinant bacterium comprising a gene encoding an enzyme encoding tyrosine phenol lyase, L-amino acid oxidase, alpha-hydroxycarboxylic acid dehydrogenase, and L-lactic acid, is linked to The plasmid is then transformed into a host E. coli to obtain a recombinant engineered strain.
  • the ⁇ -hydroxycarboxylic acid dehydrogenase gene and the L-lactate dehydrogenase gene are expressed after ligation to the plasmid pETDuet-1
  • the L-amino acid oxidase and the tyrosine phenol lyase gene are Expression after ligation to plasmid pACYCDue-1.
  • the recombinant strain comprises ligating a gene encoding L-amino acid oxidase, ⁇ -hydroxycarboxylic acid dehydrogenase, and glucose dehydrogenase to a plasmid to construct a three-gene co-expression recombination.
  • the plasmid is then transformed into the corresponding strain to obtain a recombinant engineered 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.
  • nucleotide sequence of the phenol compound-decomposing gene is NCBI accession NO: NC_012892 REGION: complement (4505585..4506436) and NC_012892REGION: 339806..340750.
  • the recombinant Escherichia coli further enhances expression of one or more of a glutamate transport gene, a lactate transporter gene, a catechol transporter gene, a NAD synthesis gene, and a FAD synthesis gene;
  • the benzenediol transport gene simultaneously expresses the lactic acid transporter gene, and the glutamate transporter gene is expressed at the same time as the lactic acid transporter gene.
  • 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 any one or more of gltS (glutamate transport gene), nadA (NAD synthetic gene), and ribF (FAD synthetic gene).
  • accession NO of the gltS on NCBI is: NC_012892 REGION: complement (3694931..3696136); nadA is NC_012892REGION: 740487..741530; ribF is NC_012892REGION: 25479..26420.
  • the gene for enhanced expression is any one or more of lldP (lactic acid transporter gene), nadA (NAD synthetic gene), and ribF (FAD synthetic gene).
  • accession NO of the lldP on NCBI is: NC_012892REGION: 3646638..3648293; nadA is NC_012892REGION: 740487..741530; ribF is NC_012892REGION: 25479..26420.
  • the enhanced expression gene is lldP (lactic acid transporter gene), hpaX (catechol transporter gene), mhpT (catechol transporter gene), nadA (NAD synthetic gene), pdxJ ( Any one or more of a pyridoxal phosphate synthesis gene) and a ribF (FAD synthetic gene).
  • accession NO of the lldP on the NCBI is: NC_012892REGION: 3646638..3648293; hpaX is; NC_012892REGION: complement (4502025..4503401); mhpT is NC_012892REGION: 344788..345999; nadA is NC_012892REGION: 740487..741530; pdxJ is NC_012892REGION:complement(2567591..2568322); ribF is NC_012892REGION:25479..26420.
  • the recombinant strain is based on an E. coli host that knocks out hpaD and mhpB, and enhances expression of lldP, hpaX, mhpT, nadA, pdxJ, and ribF, and simultaneously expresses tyrosine phenol cleavage.
  • 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 is produced.
  • the system includes cell wet weight 1-200g / L, levodopa 1-200g / L, L-glutamic acid 1-200g / L, pH 6.0-9.0; reaction at 15-40 ° C, time 1-48 hours .
  • the whole cell transformation production system including cell wet weight of 1-200g / L, levodopa concentration of 1-200g / L, L-lactic acid concentration of 1-200g / L, pH 4.0-9.0; reaction at 15-40 ° C, time 1-48 hour.
  • the whole cell In the system for transformation production the wet weight of the cells is 1-200 g/L, the concentration of catechol is 1-200 g/L, the concentration of L-lactic acid is 1-200 g/L, pH 6.0-9.0, and the concentration of amino ions is 1- 30 g / L; reaction at 15-40 ° C, time 1-48 hours. After the end of the transformation, the yield and configuration of Danshensu were determined by liquid chromatography.
  • the whole cell transformation produces a system including cell wet
  • the weight is 1-200 g/L
  • the concentration of levodopa is 1-200 g/L
  • the glucose concentration is 1-200 g/L
  • pH 6.0-9.0 the reaction is carried out at 15-40 ° C for 1-48 hours.
  • a third object of the present invention is to provide a recombinant strain of the present invention or an application of the method of the present invention in the fields of chemical industry, food, medicine, and the like.
  • the invention constructs a novel three-enzyme co-expressing genetic engineering bacteria, which can be applied to the production of optically pure 3-(3,4-dihydroxyphenyl)-2-hydroxypropionic acid.
  • 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-danshen. Further, the production efficiency of the recombinant bacteria is improved by knocking out or enhancing the expression of the phase-inducing gene on the E. coli genome to promote substrate transport and reduce product decomposition.
  • the method for producing Danshensu and ⁇ -ketoglutaric acid by using the recombinant bacteria of the invention is simple and the raw materials are easily available, and has good industrial application prospects.
  • the functional core of the engineered bacteria of the present invention is that a plurality of enzymes can be simultaneously expressed, which are L-amino acid oxidase and ⁇ -hydroxycarboxylic acid dehydrogenase, respectively, and any one of the following: exogenous L-glutamate dehydrogenase, Exogenous L-lactate dehydrogenase, glucose dehydrogenase, tyrosine phenol lyase, and L-lactate dehydrogenase, wherein tyrosine phenol lyase and L-lactate dehydrogenase are simultaneously expressed.
  • any one of L-glutamate dehydrogenase, L-lactate dehydrogenase, and glucose dehydrogenase will use the NAD in the bacteria as a coenzyme to the corresponding L-glutamine.
  • the phenolic substances in the present invention are highly susceptible to decomposition by enzymes in Escherichia coli, and related genes are knocked according to the literature (Biodegradation of Aromatic Compounds by Escherichia coli, Microbiol Mol Biol Rev. 2001, 65(4): 523-569.). In addition, 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 enhanced glutamate transporter helps to maintain the high concentration of the intracellular substrate quickly and for a long time, which is beneficial to the reaction.
  • the gene associated with the selection of glutamate transport is gltS, and the accession NO on NCBI is: NC_012892 REGION: complement (3694931.. 3696136).
  • 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 accessionNO on NCBI is: NC_012892REGION: 740487..741530.
  • FAD is a coenzyme of L-amino acid oxidase, overexpressing the important gene ribF in the coenzyme pathway, which is beneficial to strengthen L-amino acid oxidase activity.
  • accessionNO on NCBI is: NC_012892REGION: 25479..26420.
  • L-amino acid oxidase is widely found in bacteria, fungi, mammalian cells, snake venom, insect toxins and algae (L-amino acid oxidase as biocatalyst: a dream too far. Appl. Microbiol. Biotechnol. 2013, 97:9323-41 ).
  • the L-amino acid oxidase transfers the hydrogen on the ⁇ -amino group and C ⁇ to the FAD, and most of them utilize the molecular oxygen direct redox type FAD to regenerate the oxidized FAD and simultaneously generate hydrogen peroxide.
  • Electrons are transported to the cytochrome oxidase via the respiratory chain, reducing molecular oxygen to water, thereby not generating hydrogen peroxide.
  • This enzyme is mainly present in deformation.
  • Proteus sp. Providencia sp., Morganella sp., etc. (Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris. J.Struct .Biol. 2016, 195: 306-15).
  • the present invention selects five L-oxyacid oxidases which do not produce hydrogen peroxide, and clones L from Proteus mirabilis ATCC 29906, Cosenzaea myxofaciens ATCC 19692, Providencia rettgeri DSM 1131, Morganella morganii ATCC 49993, Ignatzschineria larvae DSM 13226, respectively.
  • - amino acid oxidase genes pmaao, cmaao, praao, mmaao, ilaao, the amino acid sequence of which is the sequence of accession NO. on NCBI WP_004244224.1, OAT30925.1, EFE55026.1, WP_036414800.1 or WP_026879504.1, these enzymes are It has the characteristics of wide substrate and strong activity.
  • 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.
  • L-glutamic acid is the most inexpensive amino acid.
  • the dehydrogenated ⁇ -ketoglutaric acid has a high added value.
  • L-glutamic acid oxidase is used to oxidize L-glutamic acid to produce ⁇ - Ketoglutaric acid, in which hydrogen removed from L-glutamic acid is wasted.
  • 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).
  • L-lactic acid is the most inexpensive organic acid, and pyruvic acid formed after dehydrogenation has a high added value. At present, L-lactic acid is mainly used to oxidize L-lactic acid to produce pyruvic acid, and in the process, hydrogen desorbed from L-lactic acid is wasted. There are also methods for producing keto acid by yeast fermentation. 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 acid dehydrogenase.
  • lactate dehydrogenase with NAD (NADP) as a coenzyme tends to synthesize lactic acid with pyruvic acid as a substrate, but when lactate is excessively introduced, some lactate dehydrogenase will remove hydrogen from lactic acid to form pyruvic acid.
  • NADP NAD
  • the present invention obtains the L-lactate dehydrogenase gene llldh (amino acid sequence WP_003131075.1) from Lactococcus lactis ATCC 19257.
  • Tyrosine phenol lyase (TPL, EC 4.1.99.2), also known as ⁇ -tyrosinase, tyrosine phenol lyase can catalyze the ⁇ -elimination of L-tyrosine to form phenol, acetone Acid and ammonia can also catalyze the ⁇ -elimination reaction of dopa to produce catechol, pyruvic acid and ammonia. The reaction is reversible, and catechol, pyruvic acid and ammonia can be produced by tyrosine phenol lyase to form L-dopa.
  • the tyrosine phenol lyase gene ehtpl was cloned from Erwiniaherbicola ATCC 214344, and its amino acid sequence was P31011.2.
  • ⁇ -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. Therefore, the present invention obtains the glucose dehydrogenase gene bsgdh from Bacillus subtilis ATCC 13952 (amino acid sequence is WP_013351020.1).
  • 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).
  • each gene contains T7 promoter and an RBS binding point.
  • the intensity of gene expression is not affected by the order of alignment.
  • Three genes were included in each plasmid, and the constructed plasmid was heat-transferred into E.
  • 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.
  • 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.
  • L-glutamate dehydrogenase genes were cloned from each strain and expressed in Escherichia coli BL21 (DE3).
  • 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 OD 600 reaches 0.6-0.8 Thereafter, IPTG was added to a final concentration of 0.4 mM, and expression culture was induced at 20 ° C for 8 hours. After the induction of expression was completed, the cells were collected by centrifugation at 20 ° C, 8000 rpm, and 20 minutes.
  • HpaD and mhpB on Escherichia coli BL21 were singled 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 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: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, 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, the wet cell volume was 200 g/L, and the concentration was measured after standing at 35 ° C for 10 hours.
  • Table 2 shows the levodopa and D- in the reaction system. The remaining amount of Danshensu.
  • Escherichiacoli BL21 (DE3) 1.5 1.5 Escherichiacoli BL21 ( ⁇ hpaD ⁇ mhpB, DE3) 3.6 3.6 Escherichiacoli BL21 ( ⁇ hpaD, DE3) 2.1 2.7 Escherichiacoli BL21 ( ⁇ mhpB, DE3) 1.6 1.6
  • Escherichia coli BL21 ( ⁇ hpaD ⁇ mhpB, DE3) works best and is named Escherichia coli HM.
  • 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 recombinant plasmid was co-expressed with the three genes, and the plasmid was transformed into Escherichia coli HM, and the positive transformants were obtained by using chloramphenicol and ampicillin plates to obtain recombinant Escherichia coli.
  • the genes for oxidase, ⁇ -hydroxycarboxylic acid dehydrogenase and L-lactate dehydrogenase were ligated to the pETDuet-1 or pACYCDuet-1 plasmid, respectively.
  • Two kinds of double gene co-expression recombinant plasmids were obtained, and the two plasmids were transformed into Escherichia coli HM, and positive transformants were obtained by using chloramphenicol and ampicillin plates to obtain recombinant Escherichia coli.
  • the cells were collected, and the cell wet weight was 20 g/L in a reaction volume of 100 ml, the concentration of catechol was 10 g/L, the concentration of L-lactic acid was 10 g/L, and the pH was 8.0. The ion concentration was 30 g/L; 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 5.
  • Recombinant E. coli construction expressing both ⁇ -hydroxycarboxylic acid dehydrogenase, L-amino acid oxidase and glucose dehydrogenase first encoding L-amino acid oxidase, ⁇ -hydroxycarboxylic acid dehydrogenase and glucose dehydrogenase The gene is ligated into the pETDuet-1 or pACYCDuet-1 plasmid.
  • the recombinant plasmid was co-expressed with the three genes, and the plasmid was transformed into Escherichia coli HM, and the positive transformants were obtained by using chloramphenicol and ampicillin plates to obtain recombinant Escherichia coli.
  • 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 OD 600 reaches 0.6-0.8 Thereafter, IPTG was added to a final concentration of 0.4 mM, and expression culture was induced at 20 ° C for 8 hours. After the induction of expression was completed, the cells were collected by centrifugation at 20 ° C, 8000 rpm, and 20 minutes.
  • the cells were collected, and the cell wet weight was 40 g/L in a reaction volume of 100 ml, the concentration of levodopa was 40 g/L, the glucose concentration was 30 g/L, and the pH was 8.0, and the reaction was carried out at 35 ° C. Time is 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 6.
  • 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 A medium expression intensity constitutive promoter (PG) before the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), the sequence of which is shown in SEQ ID NO: 15.
  • PG medium expression intensity constitutive promoter
  • gpdA glyceraldehyde-3-phosphate dehydrogenase gene
  • the Escherichia coli HM genome was used as a template, and the primers gltS-FF/gltS-FR, gltS-gpdA-F/gltS-gpdA-R, gltS-RF/gltS-RR were amplified and amplified.
  • the sub- and downstream sequences were fused with gltS-FF and gltS-RR as primers to express the expression cassette containing the gpdA promoter.
  • 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.
  • Table 7 shows the corresponding indexes of the primer name and the sequence number.
  • Example 8 Expression was induced according to the method described in Example 1, and various types of cells were collected for transformation analysis, and the results are shown in Table 8.
  • the whole cell transformation system in the transformation system is: cell wet weight 5g / L, L-glutamic 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-1.
  • 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 lldP sgRNA), Cas9/sgRNA induces a double-strand break at the lldP locus, and recombinase Red integrates the gpdA promoter into the lldP gene. Sequencing verification.
  • 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-2.
  • hpaX When the expression of hpaX was enhanced, a similar method of enhancing the expression of lldP was used to first amplify the upstream, promoter and downstream sequences, and primers were designed to be fused to the expression cassette containing the gpdA promoter. Then, after transfer into Escherichia coli HM together with plasmid pCasRed and pC CRISPR-gDNA (containing hpaX sgRNA), Cas9/sgRNA induces double-strand break at the hpaX gene locus, and recombinase Red integrates gpdA promoter into hpaX gene. Sequencing verification
  • mhpT When the expression of mhpT was enhanced, a similar method of enhancing the expression of lldP was used to first amplify the upstream, promoter and downstream sequences, and primers were designed to be fused to the expression cassette containing the gpdA promoter. Then, after transfecting into Escherichia coli HM with plasmid pCasRed and pCRISPR-gDNA (including mhpT sgRNA), Cas9/sgRNA induces double-strand breaks in the mhpT gene locus. Recombinase Red integrates gpdA promoter into mhpT and sequence verification
  • Example 10 Expression was induced according to the method described in Example 2, and 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 10g / L, L-lactic acid 200g / L, catechol 10g / L, pH 8.0, temperature 40 ° C, shaker speed 250 rev / min; conversion time 12 hour.
  • the best-performing Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT) was named Escherichia coli HMLHM.
  • the nadA and ribF genes in Escherichia coli HML 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: 15 is shown.
  • the plasmid is then introduced.
  • nadA gene the Escherichia coli HML genome was used as a template, and the primers nadA-FF/nadA-FR, nadA-gpdA-F/nadA-gpdA-R, nadA-RF/nadA-RR were amplified upstream and started.
  • 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 at the nadA gene locus, and the recombinase Red integrates the gpdA promoter into the nadA gene. Sequencing verification.
  • the Escherichia coli HML genome was used as a template, and the primers ribF-FF/ribF-FR, ribF-gpdA-F/ribF-gpdA-R, ribF-RF/ribF-RR were amplified and amplified.
  • the sub- and downstream sequences were fused with ribF-FF and ribF-RR as primers to express the expression cassette containing the gpdA promoter.
  • the Cas9/sgRNA induces a double-strand break in the ribF gene locus, and the recombinase Red integrates the gpdA promoter into the ribF gene. Sequencing verification.
  • Table 11 shows the corresponding indexes of the primer name and the sequence number.
  • the co-expression plasmid is introduced. Expression was induced according to the method described in Example 1, and various types of cells were collected for transformation analysis, and the results are shown in Table 12.
  • the whole cell transformation system in the transformation system is: cell wet weight of 20g / L, L-glutamic acid 120g / L, levodopa 120g / L, pH 9.0, temperature of 30 ° C, shaking speed of 250 rev / min; The time of 24 hours, the conversion results are compared as shown in Table 12.
  • the best Escherichia coli HML (PG-nadA, PG-ribF) was named Escherichia coli HNR-1.
  • the co-expression plasmid is introduced. Expression was induced according to the method described in Example 1, and various types of cells were collected for transformation analysis, and the results are shown in Table 7.
  • 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 Hours, the conversion results are compared as shown in Table 13.
  • the best Escherichia coli HML (PG-nadA, PG-ribF) was named Escherichia coli HNR-2.
  • the co-expression plasmid is introduced. Expression was induced according to the method described in Example 1, and 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 of 20g / L, L-lactic acid 200g / L, catechol 200g / L, pH 9.0, temperature of 30 ° C, shaking speed of 250 rev / min; conversion time 24 hours.
  • Escherichia coli HMLHM The best Escherichia coli HMLHM (PG-nadA, PG-ribF, PG-pdxJ) was named Escherichia coli NPR.
  • the co-expression plasmid is introduced. Expression was induced according to the method described in Example 1, and 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 was: cell wet weight of 20 g/L, glucose 100 g/L, levodopa 120 g/L, pH 9.0, temperature of 30 ° C, shaking speed of 250 rpm, and conversion time of 24 hours.
  • Escherichia coli NR The best Escherichia coli HM (PG-nadA, PG-ribF) was named Escherichia coli NR.
  • the expression method was induced according to Example 1, and the cells were collected after induction of expression by Escherichia coli HNR/pCOLADuet-1-efmdhd-bsgdh-cmaao.
  • 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 R-danshensu was 93 mg/L, and e.e%>99.9.
  • the expression method was induced according to Example 1, and the cells were collected after induction of expression by Escherichia coli HNR/pCOLADuet-1-efmdhd-llldh-cmaao.
  • the cell wet weight was 1 g/L, L-lactic acid 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 93 mg/L, and e.e%>99.9.
  • the cells were collected after induction of Escherichia coli NPR/pETDuet-1-wcldhl-llldh+pACYCDuet-1-cmaao-ehtpl, and the cell wet weight was 1 g/L in a 100 ml reaction system.
  • L-lactic acid 1 g / L
  • catechol 1 g / L
  • pH 6.0 pH 6.0
  • temperature 15 ° C shaker speed 250 rpm / conversion time 1 hour.
  • the concentration of S-danshensu was 78 mg/L.
  • the cells were collected after induction of expression by Escherichia coli NR/pCOLADuet-1-efmdhd-bsgdh-cmaao, and the wet weight of the cells was 1 g/L and glucose 1 g/L in a 100 ml reaction system.
  • the concentration of S-danshensu was 93 mg/L, and e.e%>99.9.
  • the cells were collected after the induction of expression in the strain in Table 16, and the cell wet weight was 200 g/L, L-glutamic acid 200 g/L, levodopa 200 g/ in a 100 ml reaction system. L, pH 8.5, temperature 40 ° C, shaker speed 250 rpm / conversion time 48 hours. The results were measured after all the precipitates were diluted and dissolved, and the results are shown in Table 16.
  • the cells were collected after the induction of expression in the strain in Table 17, and the wet weight of the cells 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 induction of expression in Table 18, and the cell wet weight was 200 g/L, L-lactic acid 200 g/L, and catechol 200 g/L in a 100 ml reaction system. , 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 induction of expression in the strains in Table 19.
  • the cell wet weight was 200 g/L, glucose 200 g/L, levodopa 200 g/L, pH 8.5.
  • the temperature was 40 ° C, the shaking speed was 250 rpm, and the conversion time was 48 hours.
  • the precipitate was completely diluted and dissolved, and the result was measured.

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Abstract

提供了一种基因工程菌，该菌共表达α-羟基羧酸脱氢酶、L-氨基酸氧化酶和选自由外源L-谷氨酸脱氢酶、外源L-乳酸脱氢酶，或葡萄糖脱氢酶与酪氨酸酚裂解酶的共表达组合组成的群组中的任一种，同时敲除了酚类化合物分解相关的基因。该工程菌可用于生产光学纯的3-(3,4-二羟基苯基)-2-羟基丙酸。还提供了使用所述工程菌生产丹参素的方法。

Description

[根据细则37.2由ISA制定的发明名称]　一种重组大肠杆菌及使用其生产丹参素的方法 技术领域
本发明涉及一种丹参素的生产方法，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，本发明提供了一种光学纯的丹参素的生产方法，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-氨基酸氧化酶，以及以下任意一种：外源L-谷氨酸脱氢酶、外源L-乳酸脱氢酶、葡萄糖脱氢酶、酪氨酸酚裂解酶，其中表达酪氨酸酚裂解酶时同时表达L-乳酸脱氢酶；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 ATCC14931。
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 ATCC14931。
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_GL379761REGION:COMPLEMENT(533562..534560)、NZ_KB944641REGION: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_ATUM01000014REGION:39316..40254、NZ_JQAY01000006REGION:69708..70640、NZ_GG669901REGION:45517..46470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_012892REGION:1786741..1788084、NC_007493REGION:complement(2129131..2130558)、NZ_KE992901REGION:complement(17603..18955)、NC_000964REGION:complement(2402067..2403350)的序列。
在一种实施方式中，所述L-氨基酸氧化酶来自Proteus mirabilis ATCC 29906、Cosenzaea myxofaciens ATCC 19692、Morganella morganii ATCC 49993、Providencia rettgeri DSM 1131或Ignatzschineria larvae DSM 13226中的不产过氧化氢的L-氧基酸氧化酶。
在一种实施方式中，L-氨基酸氧化酶的氨基酸序列是NCBI上accession NO为WP_004244224.1、OAT30925.1、EFE55026.1、WP_036414800.1或WP_026879504.1的序列。
在一种实施方式中，L-氨基酸氧化酶的核苷酸序列如序列表中的：NZ_GG668576REGION:1350390..1351805、LXEN01000066REGION:20563..21963、ACCI02000030REGION:21025..22443、NZ_LAGC01000006REGION:309569..310993、NZ_KI783332REGION:35799..37217。
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_JXJZ01000017REGION:18532..19509的序列。
在一种实施方式中，所述酪氨酸酚裂解酶来自于Erwinia herbicola ATCC 214344。
在一种实施方式中，所述酪氨酸酚裂解酶的氨基酸序列是NCBI上accession NO为P31011.2。
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，所述重组菌，是将编码L-氨基酸氧化酶、α-羟基羧酸脱氢酶和L-谷氨酸脱氢的酶的基因，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，是将编码L-氨基酸氧化酶、α-羟基羧酸脱氢酶和L-乳酸脱氢的酶的基因，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，包括将编码酪氨酸酚裂解酶、L-氨基酸氧化酶、α-羟基羧酸脱氢酶和L-乳酸脱氢的酶的基因，连接到2个质粒上，然后将重组质粒转化宿主大肠杆菌，得到重组工程菌。
在一种实施方式中，所述α-羟基羧酸脱氢酶基因和L-乳酸脱氢酶基因是连接到质粒pETDuet-1后表达、L-氨基酸氧化酶和酪氨酸酚裂解酶基因是连接到质粒pACYCDue-1后表达。
在一种实施方式中，所述重组菌，包括将编码L-氨基酸氧化酶、α-羟基羧酸脱氢酶和葡萄糖脱氢酶的基因，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，所述酚类化合物分解相关的基因的核苷酸序列是NCBI上accession NO为：NC_012892REGION:complement(4505585..4506436)和NC_012892REGION:339806..340750。
在一种实施方式中，所述重组大肠杆菌还强化表达了谷氨酸转运基因、乳酸转运基因、邻苯二酚转运基因、NAD合成基因、FAD合成基因一种或者多种；其中，表达邻苯二酚转运基因时同时表达乳酸转运基因，谷氨酸转运基因与乳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gltS(谷氨酸转运基因)、nadA(NAD合成基因)、ribF(FAD合成基因)中的任意一种或多种。
在一种实施方式中，所述gltS在NCBI上accession NO为：NC_012892REGION:complement(3694931..3696136)；nadA为NC_012892REGION:740487..741530；ribF为NC_012892REGION:25479..26420。
在一种实施方式中，所述强化表达的基因为lldP(乳酸转运基因)、nadA(NAD合成基因)、ribF(FAD合成基因)中的任意一种或多种。
在一种实施方式中，所述lldP在NCBI上accession NO为：NC_012892REGION:3646638..3648293；nadA为NC_012892REGION:740487..741530；ribF为NC_012892REGION:25479..26420。
在一种实施方式中，所述强化表达的基因为lldP(乳酸转运基因)、hpaX(邻苯二酚转运基因)、mhpT(邻苯二酚转运基因)、nadA(NAD合成基因)、pdxJ(磷酸吡多醛合成基因)、ribF(FAD合成基因)中的任意一种或多种。
在一种实施方式中，所述lldP在NCBI上accession NO为：NC_012892REGION:3646638..3648293；hpaX为；NC_012892REGION:complement(4502025..4503401)；mhpT为NC_012892REGION:344788..345999；nadA为NC_012892REGION:740487..741530；pdxJ为NC_012892REGION:complement(2567591..2568322)；ribF为NC_012892REGION:25479..26420。
在一种实施方式中，所述重组菌是在敲除了hpaD和mhpB的大肠杆菌宿主的基础上，强化表达了lldP、hpaX、mhpT、nadA、pdxJ和ribF，并且同时表达了酪氨酸酚裂解酶、L-氨基酸氧化酶、L-乳酸脱氢酶和α-羟基羧酸脱氢酶。
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，当所述重组大肠杆菌同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶和外源L-谷氨酸脱氢酶时，所述全细胞转化生产的体系中，包括细胞湿重1-200g/L，左旋多巴1-200g/L，L-谷氨酸1-200g/L，pH 6.0-9.0；于15-40℃反应，时间1-48小时。
在一种实施方式中，当所述重组大肠杆菌同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶和外源L-乳酸脱氢酶时，所述全细胞转化生产的体系中，包括细胞湿重为1-200g/L，左旋多巴浓度为1-200g/L，L-乳酸浓度为1-200g/L，pH 4.0-9.0；于15-40℃反应，时间1-48小时。
在一种实施方式中，当所述重组大肠杆菌同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶、酪氨酸酚裂解酶和L-乳酸脱氢酶时，所述全细胞转化生产的体系中，细胞湿重为1-200g/L，邻苯二酚浓度为1-200g/L，L-乳酸浓度为1-200g/L，pH 6.0-9.0，氨根离子浓度1-30g/L；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。
在一种实施方式中，当所述重组大肠杆菌同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶和葡萄糖脱氢酶时，所述全细胞转化生产的体系中，包括细胞湿重为1-200g/L，左旋多巴浓度为1-200g/L，葡萄糖浓度为1-200g/L，pH 6.0-9.0；于15-40℃反应，时间1-48小时。
本发明的第三个目的是提供本发明重组菌或者本发明方法在化工、食品、医药等领域的应用。
本发明的有益效果：
本发明构建了一种新型的三酶共表达基因工程菌，该菌可应用于光学纯的3-(3,4-二羟基苯基)-2-羟基丙酸的生产。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。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，提高了重组菌的生产效率。利用本发明的重组菌转化生产丹参素和α-酮戊二酸的方法简单且原料易得，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多种酶，分别为L-氨基酸氧化酶和α-羟基羧酸脱氢酶，以及以下任意一种：外源L-谷氨酸脱氢酶、外源L-乳酸脱氢酶、葡萄糖脱氢酶、酪氨酸酚裂解酶和L-乳酸脱氢酶，其中酪氨酸酚裂解酶和L-乳酸脱氢酶同时表达。其原理为：在工程菌全细胞内，L-谷氨酸脱氢酶、L-乳酸脱氢酶、葡萄糖脱氢酶中的任意一种以菌体内的NAD为辅酶将对应的L-谷氨酸脱氢、L-乳酸脱氢、葡萄糖脱氢生成对应的α-酮戊二酸、丙酮酸、葡萄糖酸和NADH；酪氨酸酚裂解酶催化丙酮酸、氨根离子、邻苯二酚生成左旋多巴；左旋多巴被L-氨基酸氧化酶脱氨生成3,4-二羟基苯丙酮酸；α-羟基羧酸脱氢酶利用谷氨酸脱氢过程生成的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。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，从而提高了目标物的产量。
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、Bacillus subtilis ATCC 13952、Escherichia coli BL21(DE3)、Proteus mirabilis ATCC 29906、Cosenzaea myxofaciens ATCC19692、Morganella morganii ATCC 49993、Lactococcus lactis ATCC 19257、Erwinia herbicola ATCC 214344、Aeromonas phenologenes ATCC 7966。购自德国微生物菌种保藏中心DSMZ的Bacillus coagulans DSM 1、Weissella confusa strain DSM 20196、Providencia rettgeri DSM1131、Ignatzschineria larvae DSM 13226。购自Novagen公司的pETDuet-1、pACYCDue-1、pCOLADuet-1、pRSFDuet-1质粒和Escherichia coli BL21(DE3)。pCasRed、pCRISPR-gDNA购自镇江爱必梦生物科技有限公司。
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 ofAromatic 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)大肠杆菌谷氨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，增强谷氨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。选择谷氨酸转运相关的基因是gltS，NCBI上accession NO为：NC_012892REGION:complement(3694931..3696136)。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accessionNO为：NC_012892REGION:740487..741530。
FAD是L-氨基酸氧化酶的辅酶，过表达该辅酶途径中的重要基因ribF，有利于强化L-氨基酸氧化酶活性。NCBI上accessionNO为：NC_012892REGION:25479..26420。
3.酶的选择
(1)L-氨基酸氧化酶的选择
L-氨基酸氧化酶广泛存在于细菌、真菌、哺乳动物细胞、蛇毒、昆虫毒素及藻类中(L-amino acid oxidase as biocatalyst:a dream too far.Appl.Microbiol.Biotechnol.2013,97:9323-41)。L-氨基酸氧化酶将α氨基和C α上的氢转移到FAD上，绝大部份利用分子氧直接氧化还原型FAD，再生氧化型FAD，同时生成过氧化氢。例如Poljanac等采用东部菱背响尾蛇毒L-氨基酸氧化酶氧化多巴生成3,4-二羟基苯丙酮酸，然后再加乳酸脱氢酶和甲酸脱氢酶生成3,4-二羟基苯乳酸，在此过程中另外必须添加过氧化氢酶以消除过氧化氢的毒性(Modelling and Optimization of the(R)-(+)-3,4-Dihydroxyphenyllactic Acid Production Catalyzed,Chem.Biochem.Eng.Q.2005,19(4)351–358)。另外还有一类L-氨基酸氧化酶与细胞膜上电子传递链相关，电子经过呼吸链传递给细胞色素氧化酶，使分子氧还原为水，从而不生成过氧化氢，这种酶类主要存在于变形杆菌属(Proteus sp.)、普罗威登菌属(Providencia sp.)、摩根菌属(Morganella sp.)等细菌中(Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.J.Struct.Biol.2016,195:306-15)。本发明选择了5种不产过氧化氢的L-氧基酸氧化酶，从Proteus mirabilis ATCC 29906、Cosenzaea myxofaciens ATCC 19692、Providencia rettgeri DSM 1131、Morganella morganii ATCC 49993、Ignatzschineria larvae DSM 13226中分别克隆得到L-氨基酸氧化酶基因pmaao、cmaao、praao、mmaao、ilaao，其氨基酸序列是NCBI上accession NO.为WP_004244224.1、OAT30925.1、EFE55026.1、WP_036414800.1或WP_026879504.1的序列，这些酶都具有底物广泛和活性强的特点。
(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)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，脱氢后成的α-酮戊二酸具有较高的附加值，目前主要以L-谷氨酸氧化酶氧化L-谷氨酸生产α-酮戊二酸，在此过程中L-谷氨酸上脱下的氢被浪费了。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)L-乳酸脱氢酶的选择
L-乳酸是最为廉价的有机酸，脱氢后成的丙酮酸具有较高的附加值。目前主要以L-乳酸氧化酶氧化L-乳酸生产丙酮酸，在此过程中L-乳酸上脱下的氢被浪费了。也有以酵母发酵生产酮酸的方法。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，但当乳酸过量进也有一些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)酪氨酸酚裂解酶的选择
酪氨酸酚裂解酶(Tyrosine phenol lyase,TPL,E.C.4.1.99.2)，又名β-酪氨酸酶，酪氨酸酚裂解酶可以催化L-酪氨酸发生β-消去反应生成苯酚、丙酮酸和氨，也可催化多巴发生β-消去反应生成邻苯二酚、丙酮酸和氨。该反应是可逆的，邻苯二酚、丙酮酸和氨可在酪氨酸酚裂解酶催化下生成L-多巴。本发明从Erwiniaherbicola ATCC 214344中分别克隆得到酪氨酸酚裂解酶基因ehtpl，其氨基酸序列是P31011.2。
(6)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.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，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，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.样品的检测分析
丹参素的定量分析：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
L-谷氨酸脱氢酶的筛选，分别从各菌株中克隆多种L-谷氨酸脱氢酶基因，并在Escherichia coli BL21(DE3)中得到表达。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。
根据文献(纳豆芽孢杆菌谷氨酸脱氢酶基因的克隆、表达及酶活性测定.上海交通大学学报·农业科学版，2010，1:82-86.)破胞测定粗酶液活性，所述的方法测定L-谷氨酸脱氢酶以NAD为辅酶的活性，结果如表1所示。因此选择来源于枯草芽孢杆菌的L-谷氨酸脱氢酶bsgdh用于丹参素的生产为最佳。
表1不同L-氨基酸脱氢酶的活性比较
重组菌 活性U/ml
EscherichiacoliBL21(DE3)/pETDuet-1-ecgdh 0.3
EscherichiacoliBL21(DE3)/pETDuet-1-rsgdh 1.1
EscherichiacoliBL21(DE3)/pETDuet-1-csgdh 1.6
EscherichiacoliBL21(DE3)/pETDuet-1-bsgdh 3.5
实施例2
根据文献Large scale validation ofan 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:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19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2中显示了反应体系中左旋多巴和D-丹参素的剩余量。
表2 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
EscherichiacoliBL21(DE3) 1.5 1.5
EscherichiacoliBL21(ΔhpaDΔmhpB,DE3) 3.6 3.6
EscherichiacoliBL21(ΔhpaD,DE3) 2.1 2.7
EscherichiacoliBL21(ΔmhpB,DE3) 1.6 1.6
Escherichia coli BL21(ΔhpaDΔmhpB,DE3)效果最好，将之命名为Escherichia coli HM。
实施例3
同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶和外源L-谷氨酸脱氢酶的重组大肠杆菌构建：首先将编码酪氨酸酚裂解酶、L-氨基酸氧化酶、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3所示。
表3 各种重组菌的比较
Figure PCTCN2018111884-appb-000001
同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶和外源L-乳酸脱氢酶的重组大肠杆菌构建：首先将编码L-氨基酸氧化酶、α-羟基羧酸脱氢酶和L-乳酸脱氢酶的基因，连接到pETDuet-1或pACYCDuet-1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，利用氯霉素和氨苄青霉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-乳酸浓度为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4所示。
表4 各种重组菌的比较
Figure PCTCN2018111884-appb-000002
Figure PCTCN2018111884-appb-000003
同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶、酪氨酸酚裂解酶和L-乳酸脱氢酶的重组大肠杆菌构建：首先将编码酪氨酸酚裂解酶、L-氨基酸氧化酶、α-羟基羧酸脱氢酶和L-乳酸脱氢酶的基因，分别连接到pETDuet-1或pACYCDuet-1质粒上。得到两种双基因共表达重组质粒，将两种质粒转化大肠杆菌Escherichia coli HM，利用氯霉素和氨苄青霉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20g/L，邻苯二酚浓度为10g/L，L-乳酸浓度为10g/L，pH 8.0，氨根离子浓度30g/L；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 PCTCN2018111884-appb-000004
同时表达了α-羟基羧酸脱氢酶、L-氨基酸氧化酶和葡萄糖脱氢酶的重组大肠杆菌构建：首先将编码L-氨基酸氧化酶、α-羟基羧酸脱氢酶和葡萄糖脱氢酶的基因，连接到pETDuet-1 或pACYCDuet-1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，利用氯霉素和氨苄青霉素平板筛选得到阳性转化子，即得到重组大肠杆菌。
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。
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，葡萄糖浓度为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 PCTCN2018111884-appb-000005
实施例4
采用文献Large scale validation ofan 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:15所示。
强化基因gltS表达时，以Escherichia coli HM基因组为模板，以引物gltS-FF/gltS-FR、gltS-gpdA-F/gltS-gpdA-R、gltS-RF/gltS-RR,扩增出上游、启动子、下游序列，并以gltS-FF和gltS-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。
下表7为引物名称与序列表序号的对应索引。
表7 引物名称与序列表序号对照
名称 序列表中编号
gltS sgRNA SEQ ID NO:1
gltS-FF SEQ ID NO:3
gltS-FR SEQ ID NO:4
gltS-gpdA-F SEQ ID NO:5
gltS-gpdA-R SEQ ID NO:6
gltS-RF SEQ ID NO:7
gltS-RR SEQ ID NO:8
根据实施例1所述的方法诱导表达，收集各类细胞进行转化分析，结果如表8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8 转化结果比较
Figure PCTCN2018111884-appb-000006
将效果最好的Escherichia coli HM(PG-gltS)命名为Escherichia coli HML-1。
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lldP sgRNA)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所述的方法诱导表达，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，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9 转化结果比较
Figure PCTCN2018111884-appb-000007
将效果最好的Escherichia coli HM(PG-lldP)命名为Escherichia coli HML-2。
强化基因hpaX表达时，采用类似强化基因lldP表达的方法，先扩增出上游、启动子、下游序列，并设计引物融合为含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含hpaX sgRNA)一起转入Escherichia coli HM后，Cas9/sgRNA诱发宿主在hpaX基因位点发生双链断裂，重组酶Red将gpdA启动子整合到hpaX基因之前，并测序验证
强化基因mhpT表达时，采用类似强化基因lldP表达的方法，先扩增出上游、启动子、下游序列，并设计引物融合为含有gpdA启动子的表达框。然后与质粒pCasRed、pCRISPR-gDNA(含mhpT sgRNA)一起转入Escherichia coli HM后，Cas9/sgRNA诱发宿主在mhpT基因位点发生双链断裂，重组酶Red将gpdA启动子整合到mhpT之前，并测序验证
根据实施例2所述的方法诱导表达，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10g/L，L-乳酸200g/L，邻苯二酚10g/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 PCTCN2018111884-appb-000008
将效果最好的Escherichia coli HM(PG-lldP,PG-hpaX,PG-mhpT)命名为Escherichia coli HMLHM。
实施例5
根据实例4的方法将Escherichia coli HML中nadA、ribF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:15所示。然后再将质粒导入。
强化基因nadA表达时，以Escherichia coli HML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 HML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ribF表达时，以Escherichia coli HML基因组为模板，以引物ribF-FF/ribF-FR、ribF-gpdA-F/ribF-gpdA-R、ribF-RF/ribF-RR,扩增出上游、启动子、下游序列，并以ribF-FF和ribF-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ribF sgRNA)一起转入Escherichia coli HML后，Cas9/sgRNA诱发宿主在ribF基因位点发生双链断裂，重组酶Red将gpdA启动子整合到ribF基因之前，并测序验证。
下表11为引物名称与序列表序号的对应索引。
表11 引物名称与序列表序号对照
名称 序列表中编号
ribF sgRNA SEQ ID NO:20
nadA sgRNA SEQ ID NO:2
ribF-FF SEQ ID NO:21
ribF-FR SEQ ID NO:22
ribF-gpdA-F SEQ ID NO:23
ribF-gpdA-R SEQ ID NO:24
ribF-RF SEQ ID NO:25
ribF-RR SEQ ID NO:26
nadA-FF SEQ ID NO:9
nadA-FR SEQ ID NO:10
nadA-gpdA-F SEQ ID NO:11
nadA-gpdA-R SEQ ID NO:12
nadA-RF SEQ ID NO:13
nadA-RR SEQ ID NO:14
基因改造完成后，将共表达质粒导入。根据实施例1所述的方法诱导表达，收集各类细胞进行转化分析，结果如表12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120g/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，转化结果比较如表12所示。
表12 转化结果比较
Figure PCTCN2018111884-appb-000009
将最好的Escherichia coli HML(PG-nadA,PG-ribF)命名为Escherichia coli HNR-1。
基因改造完成后，将共表达质粒导入。根据实施例1所述的方法诱导表达，收集各类细胞进行转化分析，结果如表7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，转化结果比较如表13所示。
表13 转化结果比较
Figure PCTCN2018111884-appb-000010
Figure PCTCN2018111884-appb-000011
将最好的Escherichia coli HML(PG-nadA,PG-ribF)命名为Escherichia coli HNR-2。
基因改造完成后，将共表达质粒导入。根据实施例1所述的方法诱导表达，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200g/L，邻苯二酚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14 转化结果比较
Figure PCTCN2018111884-appb-000012
将最好的Escherichia coli HMLHM(PG-nadA,PG-ribF,PG-pdxJ)命名为Escherichia coli NPR。
基因改造完成后，将共表达质粒导入。根据实施例1所述的方法诱导表达，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，葡萄糖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15 转化结果比较
Figure PCTCN2018111884-appb-000013
Figure PCTCN2018111884-appb-000014
将最好的Escherichia coli HM(PG-nadA,PG-ribF)命名为Escherichia coli NR。
实施例6
根据实施例1所述诱导表达方法，将Escherichia coli HNR/pCOLADuet-1-efmdhd-bsgdh-cmaao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，R-丹参素浓度为93mg/L，e.e％>99.9。
根据实施例1所述诱导表达方法，将Escherichia coli HNR/pCOLADuet-1-efmdhd-llldh-cmaao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93mg/L，e.e％>99.9。
根据实施例1所述诱导表达方法，将Escherichia coli NPR/pETDuet-1-wcldhl-llldh+pACYCDuet-1-cmaao-ehtpl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，邻苯二酚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78mg/L。
根据实施例1所述诱导表达方法，将Escherichia coli NR/pCOLADuet-1-efmdhd-bsgdh-cmaao诱导表达完成后收集菌体，于100ml反应体系中，细胞湿重1g/L，葡萄糖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7
根据实施例1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，L-谷氨酸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，结果如表16所示。
表16 转化结果比较
Figure PCTCN2018111884-appb-000015
根据实施例1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 PCTCN2018111884-appb-000016
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-乳酸200g/L，邻苯二酚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 PCTCN2018111884-appb-000017
根据实施例1所述诱导表达方法，将表19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，葡萄糖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19 转化结果比较
Figure PCTCN2018111884-appb-000018
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 PCTCN2018111884-appb-000019
Figure PCTCN2018111884-appb-000020
Figure PCTCN2018111884-appb-000021
Figure PCTCN2018111884-appb-000022
Figure PCTCN2018111884-appb-000023
Figure PCTCN2018111884-appb-000024
Figure PCTCN2018111884-appb-000025

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-氨基酸氧化酶，以及以下任意一种：外源L-谷氨酸脱氢酶、外源L-乳酸脱氢酶、葡萄糖脱氢酶、酪氨酸酚裂解酶，其中表达酪氨酸酚裂解酶时同时表达L-乳酸脱氢酶；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谷氨酸转运基因、乳酸转运基因、邻苯二酚转运基因、NAD合成基因、FAD合成基因一种或者多种；其中，表达邻苯二酚转运基因时同时表达乳酸转运基因，谷氨酸转运基因与乳酸转运基因不同时表达。
  4. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述强化表达的基因为gltS、nadA、ribF中的任意一种或多种；所述强化表达的基因为lldP、nadA、ribF中的任意一种或多种；所述强化表达的基因为lldP、hpaX、mhpT、nadA、pdxJ、ribF中的任意一种或多种。
  5. 根据权利要求3或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L-谷氨酸脱氢酶、L-乳酸脱氢酶、葡萄糖脱氢酶、酪氨酸酚裂解酶、L-乳酸脱氢酶、α-羟基羧酸脱氢酶、L-氨基酸氧化酶是通过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，所述宿主大肠杆菌为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. 权利要求1-7任一所述的重组大肠杆菌或者权利要求8-9任一所述的方法在化工、食品、制备药物领域的应用。
PCT/CN2018/111884 2018-04-19 2018-10-25 一种重组大肠杆菌及使用其生产丹参素的方法 WO2019200873A1 (zh)

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CN101565694A (zh) * 2008-04-25 2009-10-28 天津天士力制药股份有限公司 丹酚酸酶及丹酚酸酶和人参皂苷混合酶及其转化药材的方法
CN103667371A (zh) * 2013-11-11 2014-03-26 天津大学 一种丹参素的生物生产方法
CN107299072A (zh) * 2017-08-02 2017-10-27 江南大学 一种工程菌及其应用
CN107916245A (zh) * 2017-10-31 2018-04-17 天津大学前沿技术研究院有限公司 一种生产l‑酪氨酸重组工程菌的方法及该重组工程菌的应用

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CN103667371A (zh) * 2013-11-11 2014-03-26 天津大学 一种丹参素的生物生产方法
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