CN108949653B - Engineering bacterium and application thereof in production of tanshinol - Google Patents
Engineering bacterium and application thereof in production of tanshinol Download PDFInfo
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Abstract
The invention discloses an engineering bacterium and an application thereof in danshensu production, belonging to the technical field of biological engineering.A novel three-enzyme co-expression genetic engineering bacterium is constructed, and can be applied to the production of optically pure danshensu.A (D/L) - α -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.
Description
Technical Field
The invention relates to an engineering bacterium and application thereof in danshensu production, belonging to the technical field of biological engineering.
Background
Salvianic acid extracted from Salvia miltiorrhiza Bunge with the scientific names of R- (+) -3- (3,4-Dihydroxyphenyl) -2-hydroxypropionic acid and D- (+) - β - (3,4-Dihydroxyphenyl) lactic acid, wherein the English names of the R- (+) -3- (3,4-Dihydroxyphenyl) -lactic acid, D-DSS, R-DSS, (R) - (+) -3- (3,4-Dihydroxyphenyl) -lactic acid and (R) - (+) -3- (3,4-Dihydroxyphenyl) -2-hydroxypropanoic acid are dextro phenolic acid compounds.
The danshensu is an important effective component in the water extract of salvia miltiorrhiza, the structure (research on water-soluble effective components of salvia miltiorrhiza, II, D (+) β (3,4-dihydroxyphenyl) lactic acid structure, reported by Shanghai first college of medicine, 1980, 05(7), 384-plus 385) is obtained and identified from the water extract of salvia miltiorrhiza in 1980 at home, and various researches show that the danshensu has important pharmacological and pharmacodynamic effects and unique treatment effects on the aspects of treating cardiovascular and cerebrovascular diseases and the like.
Danshensu is mainly extracted from Salvia miltiorrhiza Bunge (patent CN 200810038853.9). The content of the tanshinol in the salvia miltiorrhiza is low, the planting cost of the tanshinol is high, and the yield is limited, so that the current tanshinol is high in price and can not meet the market demand far. Patent CN201310559498.0 proposes a method for producing tanshinol by glucose fermentation by constructing escherichia coli genetic engineering bacteria, wherein the anabolic pathway involves the use of hydroxylase, which can easily oxidize the metabolic process product to affect the yield of tanshinol, and meanwhile, since escherichia coli fermentation is a high oxygen consumption process and can also oxidize tanshinol, the yield of the method is lower at present, and the cost is higher than that of the plant extraction process. Patent CN201210190171.6 proposes a method for producing danshensu by hydrolyzing salvianolic acid B, which needs to be extracted from Salvia miltiorrhiza Bunge, has a large amount of side reactions in the chemical hydrolysis process, and is not suitable for large-scale production. The catalyst for chiral synthesis of danshensu (patent CN201210420488.4) is very expensive and currently only stays at laboratory level.
Roth et al, 1988, proposed a method of treating levodopa chemically to give the corresponding 3, 4-dihydroxyphenylpyruvic Acid and enzymatically synthesizing S- (+) -3- (3,4-Dihydroxyphenyl) -2-hydroxypropionic Acid (S-DSS, &lTtTtranslation = L "&gTtL &lTt/T gTt-DSS) (enzymic Synthesis of (S) - (-) -3- (3,4-Dihydroxyphenyl) lactic Acid, Arch. Pharm. (Weinheim)321, 179-180(1988), Z.Findrik et al, converting levodopa to 3, 4-dihydroxyphenylpyruvic Acid Using an amino Acid oxidase, then reducing D- (3,4-Dihydroxyphenyl) lactic Acid Using D-lactate dehydrogenase to produce D- (3,4-Dihydroxyphenyl) lactic Acid (crystallization and crystallization of (R-4-dihydroxy-propionic Acid) (3, 4-dihydroxy-propionic Acid, 3, 19, 3-dihydroxy-propionic Acid, 3, 4-propionic Acid, 3, 4-dihydroxy-propionic Acid, 3.
Disclosure of Invention
Based on the defects of various methods at present, the invention provides a method for producing optically pure tanshinol, constructs multienzyme coexpression engineering bacteria, and realizes the high-efficiency production of tanshinol. The technical problem to be solved by the invention is to provide a recombinant bacterium capable of producing danshensu at low cost. Meanwhile, the invention aims to solve the technical problems of construction and application of the strain.
The first purpose of the invention is to provide a recombinant bacterium capable of producing optical pure danshensu at low cost, wherein the recombinant bacterium simultaneously expresses 3 enzymes, namely L- α -amino acid transaminase, L-glutamate dehydrogenase and α -hydroxycarboxylic acid dehydrogenase, and genes related to phenolic compound decomposition are knocked out on the basis of host escherichia coli.
In one embodiment, the α -hydroxycarboxylic acid dehydrogenase is a D-form α -hydroxycarboxylic acid dehydrogenase from L actinobacillus planterum ATCC 14917, Enterococcus faecalis ATCC 35038, or L actinobacillus fermentum ATCC 14931.
In one embodiment, the α -hydroxycarboxylic acid dehydrogenase is a L-type α -hydroxycarboxylic acid dehydrogenase from Bacillus coagulousns DSM1, Weissella convusas strain DSM 20196 or L actinobacillus ATCC 14931.
In one embodiment, the α -hydroxycarboxylic acid dehydrogenase is D- α -hydroxycarboxylic acid dehydrogenase whose amino acid sequence is the sequence of access No. WP _003643296.1, WP _002335374.1, or EEI22188.1 at NCBI and α -hydroxycarboxylic acid dehydrogenase is L- α -hydroxycarboxylic acid dehydrogenase whose amino acid sequence is the sequence of access No. WP _013858488.1, WP _003607654.1, or WP _035430779.1 at NCBI.
In one embodiment, the nucleotide sequence of the D- α -hydroxycarboxylic acid dehydrogenase is the sequence of the accession No. NZ _ G L379761 REGION at NCBI COMP L EMENT (533562..534560), NZ _ KB944641REGION:161892..162830, ACGI01000078REGION:20793..21791, and the nucleotide sequence of the L- α -hydroxycarboxylic acid dehydrogenase is the sequence of the accession No. NZ _ ATUM01000014REGION at NCBI 39316..40254, NZ _ JQAY01000006REGION:69708..70640, NZ _ GG669901REGION:45517.. 46470.
In one embodiment, the L-glutamate dehydrogenase is derived from Escherichia coli B L21, Rhodobacter sphaeroides ATCC BAA-808, Clostridium symbiosum ATCC 14940, Bacillus subtilis 168.
In one embodiment, the amino acid sequence of L-glutamate dehydrogenase is the sequence of accession NO WP _000373021.1, WP _011338202.1, WP _003497202.1, WP _010886557.1 at NCBI.
In one embodiment, the nucleotide sequence of L-glutamate dehydrogenase is the sequence of access NO: NC-012892 REGION:1786741. 1788084, NC-007493 REGION: compensation (2129131. 2130558), NZ-KE 992901REGION: compensation (17603. 18955), NC-000964 REGION: compensation (2402067. 2403350) at NCBI.
In one embodiment, the L- α -amino acid transaminase is from Escherichia coli B L21, L Acobacterium plantarum ATCC 14917, L Acobacterium paracasei ATCC 334.
In one embodiment, the amino acid sequence of the L- α -amino acid transaminase is the sequence with access NO WP _000462687.1, WP _000486988.1, WP _003643296.1, YP _806114.1 at NCBI.
In one embodiment, the nucleotide sequence of the L- α -amino acid transaminase is the sequence of accession NO. NC-012892 REGION: COMP L EMENT (989603..990793), NC-012892 REGION:4174517..4175710, NZ-G L379768 REGION: compensation (121900..123087), NC-008526 REGION: compensation (840419..841594) at NCBI.
In one embodiment, the recombinant bacterium is a recombinant engineering bacterium obtained by connecting genes encoding L- α -amino acid transaminase, α -hydroxycarboxylic acid dehydrogenase and L-glutamic acid dehydrogenase to a plasmid to construct a three-gene co-expression recombinant plasmid, and then transforming the recombinant plasmid into a corresponding strain.
In one embodiment, the recombinant bacterium is constructed by using Escherichia coli B L21 (DE3) as a host.
In one embodiment, the gene involved in the degradation of the phenolic compound is any one of hpaD, mhpB, or a combination of both.
In one embodiment, the nucleotide sequence of the gene associated with phenolic compound breakdown is at NCBI, where access NO is: NC _012892REGION: completion (4505585..4506436) and NC _012892REGION:339806.. 340750.
In one embodiment, the recombinant bacterium further enhances expression of one or more of a glutamate transporter gene, an NAD synthetic gene, and a pyridoxal phosphate synthetic gene.
In one embodiment, the enhanced expression is achieved by adding a constitutive promoter in front of the gene to be enhanced on the genome of Escherichia coli B L21 (DE 3).
In one embodiment, the gene whose expression is enhanced is any one or more of gltS (glutamate transporter gene), nadA (NAD synthesis gene), pdxJ (pyridoxal phosphate synthesis).
In one embodiment, the gltS access NO at NCBI is: NC _012892REGION: compensation (3694931.. 3696136); nadA is NC _012892REGION 740487.. 741530; pdxJ is NC _012892REGION: completion (2567591.. 2568322).
The second purpose of the invention is to provide a method for producing danshensu, which utilizes the recombinant bacterium of the invention.
In one embodiment, the production of tanshinol is by whole cell transformation.
In one embodiment, the whole cell transformation production system comprises 1-200 g/L wet weight of cells, 1-200 g/L g/L of levodopa and 1-200 g/L of glutamic acid, and has pH of 6.0-9.0, and the reaction is carried out at 15-40 ℃ for 1-48 hours.
The invention has the beneficial effects that:
the (D/L) - α -hydroxycarboxylic acid dehydrogenase selected by the invention has the characteristics of poor substrate specificity and strong optical specificity, and can be used for producing optically pure D-tanshinol and L-tanshinol.
Detailed description of the preferred embodiments
The functional core of the engineering bacteria is that 3 enzymes, namely L- α -amino acid transaminase, α 1-hydroxycarboxylic acid dehydrogenase and α 0-glutamic acid dehydrogenase, can be expressed simultaneously, the principle is that in the whole cell of the engineering bacteria, L-glutamic acid dehydrogenase takes NAD in the bacteria as coenzyme to dehydrogenate L-glutamic acid to generate α -ketoglutaric acid and NADH, L- α -amino acid transaminase converts levodopa to 3,4-dihydroxy phenylpyruvic acid and realizes the regeneration of L-glutamic acid, so that the concentration of L-glutamic acid in a conversion system is kept constant, α -hydroxycarboxylic acid dehydrogenase reduces 3,4-dihydroxy phenylpyruvic acid to danshensu by utilizing the NADH generated in the process of dehydrogenating the glutamic acid, and the regeneration of the coenzyme NAD is realized at the same time, and the related genes on the genome of the escherichia coli are knocked out or enhanced to promote the transportation of substrates and reduce the decomposition of products.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
1. the invention relates to a strain and a plasmid
L Acobacterium plantarum ATCC 14917, L Acobacterium paracasei ATCC334, Enterococcus faecalis ATCC 35038, L Acobacterium fermentum ATCC 14931, Bacillus subtilis ATCC 13952, Escherichia coli B L21 (DE3), Bacillus coagulum DSM1, Weissella confluesa strain DSM 20196, which are available from the German collection of microorganisms DSMZ, pETDuet-1, pACYCDue-1, pCO L ADuet-1, SFpRDuet-1 plasmids and Escherichia coli B L21 (DE3), which are available from Novagen.
2. Knockout and constitutive enhanced expression of related genes in escherichia coli
(1) Knock-out of genes involved in the decomposition of phenolic compounds in E.coli
The phenolic substances in the invention are all easily decomposed by enzymes in Escherichia coli, and according to the literature (Biogradatino Aromatic Compounds by Escherichia coli, Microbiol Mol Biol Rev.2001,65(4): 523-. The genes selected were hpaD and mhpB, with access NO at NCBI: NC _012892REGION: completion (4505585..4506436) and NC _012892REGION:339806.. 340750.
(2) Constitutive enhanced expression of glutamate transporter in Escherichia coli
In the whole cell transformation process, the substrate is required to be transported into the cell, and the enhancement of the glutamate transporter is beneficial to maintaining the high concentration of the intracellular substrate rapidly and for a long time and is beneficial to the reaction. The glutamate transport related gene was chosen to be gltS, and access NO at NCBI was: NC _012892REGION: completion (3694931.. 3696136). Dopa is similar to aromatic amino acid, and amino acid and the like need to be absorbed in the cell culture process, so that the thalli can express a large amount of amino acid transporters without enhancing the expression.
(3) Constitutive enhanced expression of important genes related to Escherichia coli coenzyme synthesis
NADH is used as coenzyme in the reduction process of α -hydroxycarboxylic acid dehydrogenase, the key enzyme of the NAD synthetic pathway of escherichia coli is enhanced and expressed, the level of NAD in the bacteria can be improved, and the generation of tanshinol is facilitated.
Pyropyraldehyde (amine) phosphate is a coenzyme of L- α -amino acid transaminase, and a core gene pdxJ in the pathway of the coenzyme is overexpressed, so that levodopa is synthesized, and the accession NO on NCBI is NC-012892 REGION: compensation (2567591.. 2568322).
3. Selection of enzymes
(1) Selection of L- α -amino acid transaminase
L- α -amino acid transaminase is widely present in bacteria, fungi and mammalian cells, and the transaminase with α -ketoglutarate or oxaloacetate as an ammonia acceptor is usually the most active transaminase cloned from L actinobacillus plantarum ATCC 14917 and L actinobacillus paracasei ATCC334 to obtain L- α -amino acid transaminase genes lpt and lct respectively, wherein the amino acid sequences of the genes are sequences WP _003643296.1 and YP _806114.1 on NCBI, and two L- α -amino acid transaminase genes ect1 and ect2 are cloned from Escherichia coli B L1 (DE3) and have amino acid sequences WP _000462687.1 and WP _000486988.1 on NCBI.
(2) α selection of hydroxycarboxylic acid dehydrogenases
The α -hydroxycarboxylic acid dehydrogenase includes lactate dehydrogenase, α -hydroxyacid isocaproate dehydrogenase, mandelate dehydrogenase, glyoxylate reductase, etc., which can act widely on various substrates to produce α -hydroxycarboxylic acid, and are usually named according to the most effective substrates thereof.the present invention selects an enzyme which is optically strong and has a strong activity on 3, 4-dihydroxyphenylpyruvic acid for the production of D or L tanshins.D-type α -hydroxycarboxylic acid dehydrogenase genes lpnddhdhdhdhd and lddhfld are cloned from L actinobacillus plantarum ATCC 14917, Enterococcus faecalis ATCC 35038 and L actinobacillus ferum ATCC 14931 respectively, and the amino acid sequences thereof are those of accession No. 003643296.1, dhdhdhwp and oldwp 6368 on NCBI, and EEI 48 are those of accession No. 003643296.1, dhldwp6326, DSM 6396, accession No. 68, DSM 6396, 1498-1498, and 1498-1498 on NCBI.
(3) L selection of glutamate dehydrogenase
L-glutamic acid is the cheapest amino acid, α -ketoglutaric acid produced after dehydrogenation can be used as an acceptor for transamination of levodopa L glutamate dehydrogenase is widely present in almost all organisms, L-glutamic acid is used as a substrate to transfer hydrogen produced on L-glutamic acid to coenzyme NAD or NADP, thereby producing NADH or NADPH.NADH or NADPH can be used as a hydrogen donor for the aforementioned hydroxy acid dehydrogenase the present invention obtains L-glutamic acid gene ecgdh (amino acid sequence is WP _000373021.1), rsgdh (amino acid sequence is WP _011338202.1), csgdh (amino acid sequence is WP _003497202.1), bsgdh (amino acid sequence is WP _010886557.1) from Escherichia coli B L, Rhodobacterium sphingoides ATCC BAA-808, Clostridium symbolosum ATCC 14940, Bacillus subtilis168, respectively.
4. Construction of Co-expression System and culture of cells
At present, multiple Escherichia coli polygene coexpression methods (Escherichia coli polygene coexpression strategy, China journal of bioengineering, 2012, 32(4):117-122) are provided), the invention adopts Liu-Nei (Liu-Nei-LeiThe method comprises the steps of) constructing by using a T7 promoter and an RBS binding site in front of each gene, theoretically, because each gene is provided with a T7 and an RBS in front of each gene, the expression intensity of the genes is not greatly influenced by the arrangement sequence, each plasmid comprises three genes, the constructed plasmids are thermally transduced into escherichia coli competent cells and coated on an antibiotic solid plate, and positive transformants are obtained by screening, namely recombinant escherichia coli are obtained, the cells are cultured, according to a classical recombinant escherichia coli culture and induced expression scheme, the recombinant escherichia coli is transferred into a L B fermentation culture medium (peptone 10 g/L, yeast powder 5 g/L and NaCl 10 g/L) according to the volume ratio of 2 percent, and when the OD of the cells is up to OD600After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20 minutes at 20 ℃.
5. Production of optical pure danshensu by whole cell transformation
The cell transformation production system comprises the steps of enabling the wet weight of cells to be 1-200 g/L, enabling the concentration of levodopa to be 1-200 g/L-glutamic acid to be 1-200 g/L, enabling the concentration of pH to be 6.0-9.0, enabling the reaction to be carried out at 15-40 ℃ for 1-48 hours, and determining the yield and configuration of the tanshinol by liquid chromatography after transformation is finished.
6. Detection analysis of samples
The quantitative analysis of danshensu is carried out by detecting and analyzing the transformation solution by PerkinElmer Series 200 high performance liquid chromatograph equipped with ultraviolet detector, wherein the chromatographic conditions comprise that the mobile phase is methanol-0.1% formic acid water (40:60), and a Hanbang Megres C18 chromatographic column (4.6 × 250mm, 5 μm), the flow rate is 1ml/min, the column temperature is 30 ℃, the sample injection amount is 20 μ l, and the sample injection amount is 280 nm.
Chiral analysis, Perkinelmer Series 200 high performance liquid chromatograph detection analysis, ultraviolet detector, Chiralcel OD-H chiral column (4.6 × 250mm), mobile phase volume ratio of n-hexane, isopropanol, trifluoroacetic acid 80:20:0.1, flow rate of 0.5m L/min, column temperature of 25 ℃, sample injection amount of 20 mu L, and detection wavelength of 280 nm.
The solubility of danshensu is low, and if crystal is separated out in the conversion process, the danshensu is measured after dilution.
The optical purity of tanshinol was assessed by the enantiomeric excess value (% e.e).
When the R-danshensu is produced,
enantiomeric excess% e.e ═ S [ [ (S)R-SS)/(SR+SS)×100％]
When the S-danshensu is produced,
enantiomeric excess% e.e ═ S [ [ (S)S-SR)/(SR+SS)×100％]
In the formula SSIs the peak area of S-tanshinol in the transformation solution, SRIs the peak area of the liquid chromatogram of the R-danshensu in the transformation liquid.
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more apparent, the present invention is described in detail below with reference to the embodiments. It should be noted that the specific embodiments described herein are only for explaining the present invention and are not used to limit the present invention.
Example 1
L-screening glutamate dehydrogenase, cloning multiple L-glutamate dehydrogenase genes from each strain, and expressing in Escherichia coli B L21 (DE 3). Induced expression method, transferring recombinant Escherichia coli 2% by volume into L B fermentation medium (peptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L), when cell OD is reached600After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20 minutes at 20 ℃.
Crude enzyme solution activity was determined by cell disruption according to the literature (cloning, expression and enzyme activity determination of the Bacillus natto glutamate dehydrogenase gene, proceedings of Shanghai university of transportation, agricultural science edition, 2010, 1:82-86.) the activity of L-glutamate dehydrogenase with NAD as a coenzyme was determined, and the results are shown in Table 1. therefore L-glutamate dehydrogenase bsgdh derived from Bacillus subtilis was selected to be optimal for the production of tanshinol.
TABLE 1 comparison of the activities of various L-glutamate dehydrogenases
| Recombinant bacterium | Activity U/ml |
| Escherichia coli BL21(DE3)/pETDuet-1-ecgdh | 0.4 |
| Escherichia coli BL21(DE3)/pETDuet-1-rsgdh | 1.5 |
| Escherichia coli BL21(DE3)/pETDuet-1-csgdh | 2.1 |
| Escherichia coli BL21(DE3)/pETDuet-1-bsgdh | 2.8 |
Example 2
L- α -amino acid transaminase screening, cloning several α 0- α 1-amino acid transaminase genes from E.coli and Lactobacillus, respectively, according to the method described in example 1, expression was obtained in Escherichia coli B L21 (DE3), cells were collected, crude enzyme liquid activity was determined by cell disruption, activities of various enzymes were compared with α -ketoglutarate as an acceptor, and L- α -amino acid transaminase activity was determined according to the method described in the literature (transaminase catalyzing asymmetric synthesis of aromatic L-amino acid. Bioengineering Proc., 2012, 28(11): 1346-1358), and the results are shown in Table 2. L- α -amino acid transaminase lct from L actinobacillus paracasei ATCC334 was therefore selected to be optimal for transamination and deamination of levodopa.
TABLE 2 comparison of the activities of the different L- α -amino acid transaminases
| Recombinant bacterium | Activity 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 |
Example 3
The hpaD and mhpB on Escherichia coli B L21 (DE3) were single-or double-knocked out according to the method described in document L image scale validation of an effective CRISPR/Cas-based multigene editing protocol in Escherichia coli, Microbiological Cell Factores, 2017,16(1):68, wherein the plasmids used in the present invention were pCasRed and pCRISPR-gDNA (hpaD sgRNA) introduced together with the homology arm (hpaD doror) onto Escherichia coli B L21 (DE3), and 9/sgRNA induced double-strand breaks in the hpaD gene locus of the host, and Red integrated the hpaD doror into the hppBD gene, realizing the knock-out of the gene, and sequencing the hpaD sgRNA, hPD multigene, mpBR, mpBID, SEQ ID NO:12, mNO: SEQ ID NO:10, respectively.
Preparing a solution with the pH value of 7, 4 g/L of levodopa or D-danshensu, and 200 g/L of wet bacterium, standing at 35 ℃ for 10 hours, and then measuring the concentration, wherein the table 3 shows the residual amount of levodopa and D-danshensu in the reaction system.
TABLE 3 residual concentrations of different strains after substrate and product decomposition
| Levodopa g/L | D-danshensu g/L | |
| Escherichia coli BL21(DE3) | 1.6 | 1.8 |
| Escherichia coli BL21(ΔhpaDΔmhpB,DE3) | 3.7 | 3.5 |
| Escherichia coli BL21(ΔhpaD,DE3) | 2.2 | 2.6 |
| Escherichia coli BL21(ΔmhpB,DE3) | 1.9 | 2.1 |
Escherichia coli B L21 (Δ hpaD Δ mhpB, DE3) was the most effective and was named Escherichia coli HM.
Example 4
The recombinant colibacillus is constructed by connecting genes coding L- α -amino acid transaminase, α -hydroxycarboxylic acid dehydrogenase and L-glutamic acid dehydrogenase to a plasmid to obtain a three-gene coexpression recombinant plasmid, transforming the plasmid into Escherichia coli HM, and screening by using an antibiotic plate to obtain a positive transformant, thus obtaining the recombinant colibacillus.
After the induction expression of the recombinant escherichia coli is finished, collecting thalli, reacting at 35 ℃ for 12 hours in a reaction volume of 100ml with the wet weight of the cells of 40 g/L, the concentration of levodopa of 40 g/L-glutamic acid of 30 g/L and the pH of 8.0, and determining the yield and the configuration of the tanshinol by liquid chromatography after the conversion is finished.
TABLE 4 comparison of various recombinant bacteria
Example 4
A medium expression strength constitutive Promoter (PG) in front of a corresponding gene on an Escherichia coli HM genome is increased by adopting a method described in document L image scale identification of an effective CRISPR/Cas-based multigene encoding protocol in Escherichia coli, microbiological Cell Factories,2017,16(1):68, and the sequence is shown as SEQ ID NO: 8.
When the gltS expression of the gene is enhanced, an Escherichia coli HM genome is used as a template, primers gltS-FF/gltS-FR, gltS-gpdA-F/gltS-gpdA-R, gltS-RF/gltS-RR are used for amplifying an upstream sequence, a promoter and a downstream sequence, and the gltS-FF and the gltS-RR are used as primers to fuse into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing gltS sgRNA), Cas9/sgRNA induces double strand break of the host at the gltS gene site, recombinase Red integrates the gpdA promoter in front of the gltS gene, and sequencing and verification are carried out.
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 5 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| gltS sgRNA | SEQ ID NO:20 |
| gltS-FF | SEQ ID NO:21 |
| gltS-FR | SEQ ID NO:22 |
| gltS-gpdA-F | SEQ ID NO:23 |
| gltS-gpdA-R | SEQ ID NO:24 |
| gltS-RF | SEQ ID NO:25 |
| gltS-RR | SEQ ID NO:26 |
The expression was induced according to the method described in example 1, and the transformation analysis was performed by collecting various cells, the results are shown in Table 6. the transformation system comprises 5 g/L-glutamic acid 1 g/L wet weight of the cells, 20 g/L levodopa 20 g/pH 8.0, 40 ℃, 250 rpm of shaker rotation speed, and 12 hours transformation time.
TABLE 6 comparison of transformation results
The most effective Escherichia coli HM (PG-gltS) was named Escherichia coli HMG.
Example 5
According to the method of example 4, the medium expression strength constitutive Promoter (PG) in Escherichia coli HMG is increased before nadA and pdxJ genes in 3-glyceraldehyde phosphate dehydrogenase gene (gpdA) of Escherichia coli, and the sequence is shown as SEQ ID NO: 8. The plasmid is then introduced.
When gene nadA expression is enhanced, an Escherichia coli HMG genome is used as a template, primers nadA-FF/nadA-FR and nadA-gpdA-F/nadA-gpdA-R, nadA-RF/nadA-RR are used to amplify upstream, promoter and downstream sequences, and nadA-FF and nadA-RR are used as primers to fuse into an expression frame containing a gpdA promoter. After being transferred into Escherichia coli HMG together with plasmid pCasRed, pCRISPR-gDNA (containing nadA sgRNA), Cas9/sgRNA induces double strand break at the nadA gene site in the host, recombinase Red integrates the gpdA promoter in front of the nadA gene, and sequencing is performed for verification.
When the expression of the gene pdxJ is enhanced, an Escherichia coli HMG genome is used as a template, primers pdxJ-FF/pdxJ-FR, pdxJ-gpdA-F/pdxJ-gpdA-R, pdxJ-RF/pdxJ-RR are used to amplify an upstream sequence, a promoter and a downstream sequence, and pdxJ-FF and pdxJ-RR are used as primers to fuse into an expression frame containing a gpdA promoter. After being transformed into Escherichia coli HMG together with plasmid pCasRed, pCRISPR-gDNA (containing pdxJ sgRNA), Cas9/sgRNA induces double strand break in the host at the pdxJ gene site, recombinase Red integrates the gpdA promoter in front of the pdxJ gene, and sequencing is performed for verification.
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 7 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| 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 |
After the gene transformation, the co-expression plasmid was introduced, the cells were induced according to the method described in example 1, and transformation analysis was performed on the cells, the results are shown in Table 8. the whole cell transformation system in the transformation system was 20 g/L-glutamic acid 200 g/L in wet weight, levodopa 120 g/L in pH 9.0, 30 ℃ in temperature, 250 rpm in shaker speed, and 24 hours in transformation time.
TABLE 8 comparison of transformation results
The best Escherichia coli HMG (PG-nadA, PG-pdxJ) was named Escherichia coli HNP.
Example 6
According to the inducible expression method described in example 1, Escherichia coli HNP/pCO L ADuet-1-efmddh-bsgdh-lct is subjected to inducible expression, then thallus is collected, and in a 100ml reaction system, the wet weight of the cell is1 g/L-glutamic acid 1 g/L, levodopa 1 g/L, pH is 6.0, the temperature is 15 ℃, the rotation speed of a shaking table is 250 revolutions per minute, the transformation time is1 hour, and the measurement result shows that the concentration of S-tanshinol is 93 mg/L, and the e.e% is more than 99.9.
Example 7
According to the inducible expression method described in example 1, after the strains in Table 8 are induced to express, the strains are collected, and in a 100ml reaction system, the wet weight of the cells is 200 g/L-glutamic acid 20 g/L, the levodopa is 200 g/L, the pH value is 8.5, the temperature is 40 ℃, the rotation speed of a shaking table is 250 revolutions per minute, the conversion time is 48 hours, and the result is measured after all precipitates are diluted and dissolved.
TABLE 9 comparison of transformation results
The modification and construction of the enzyme and its co-expressed genetically engineered bacteria, the culture medium composition and culture method of the bacteria, and the whole cell biotransformation described above are only preferred embodiments of the present invention, and are not intended to limit the present invention, and theoretically, other bacteria, filamentous fungi, actinomycetes, and animal cells can be used for genome modification and whole cell catalysis of multigene co-expression. Any modification, equivalent replacement, made within the principle and spirit of the present invention.
Sequence listing
<110> university of south of the Yangtze river
<120> engineering bacterium and application thereof in production of tanshinol
<130>2018.3.15
<160>26
<170>PatentIn version 3.3
<210>1
<211>20
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<213> Artificial sequence
<400>1
ttaacggcgt cggcttcggg 20
<210>2
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<213> Artificial sequence
<400>2
tcgaatcctg cacgacccac cacta 25
<210>3
<211>50
<212>DNA
<213> Artificial sequence
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tcggccactc atcaacatga ttcatcgaca ttagcgtaat attcgctgtt 50
<210>4
<211>50
<212>DNA
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<400>4
aacagcgaat attacgctaa tgtcgatgaa tcatgttgat gagtggccga 50
<210>5
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<212>DNA
<213> Artificial sequence
<400>5
tgtctggatc aaacattacg ctcatggttt tctcctgtca ggaacgttcg 50
<210>6
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<213> Artificial sequence
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cgaacgttcc tgacaggaga aaaccatgag cgtaatgttt gatccagaca 50
<210>7
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<212>DNA
<213> Artificial sequence
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catccacgga caatgcgcgc agctg 25
<210>8
<211>1100
<212>DNA
<213>Escherichia coli BL21(DE3)
<400>8
atgaatcatg ttgatgagtg gccgatcgct acgtgggaag aaaccacgaa actccattgc 60
gcaatacgct gcgataacca gtaaaaagac cagccagtga atgctgattt gtaaccttga 120
atatttattt tccataacat ttcctgcttt aacataattt tccgttaaca taacgggctt 180
ttctcaaaat ttcattaaat attgttcacc cgttttcagg taatgactcc aacttattga 240
tagtgtttta tgttcagata atgcccgatg actttgtcat gcagctccac cgattttgag 300
aacgacagcg acttccgtcc cagccgtgcc aggtgctgcc tcagattcag gttatgccgc 360
tcaattcgct gcgtatatcg cttgctgatt acgtgcagct ttcccttcag gcgggattca 420
tacagcggcc agccatccgt catccatatc accacgtcaa agggtgacag caggctcata 480
agacgcccca gcgtcgccat agtgcgttca ccgaatacgt gcgcaacaac cgtcttccgg 540
agcctgtcat acgcgtaaaa cagccagcgc tggcgcgatt tagccccgac atagccccac 600
tgttcgtcca tttccgcgca gacgatgacg tcactgcccg gctgtatgcg cgaggttacc 660
gactgcggcc tgagtttttt aagtgacgta aaatcgtgtt gaggccaacg cccataatgc 720
gggcagttgc ccggcatcca acgccattca tggccatatc aatgattttc tggtgcgtac 780
cgggttgaga agcggtgtaa gtgaactgca gttgccatgt tttacggcag tgagagcaga 840
gatagcgctg atgtccggcg gtgcttttgc cgttacgcac caccccgtca gtagctgaac 900
aggagggaca gctgatagaa acagaagcca ctggagcacc tcaaaaacac catcatacac 960
taaatcagta agttggcagc atcaccccgt tttcagtacg ttacgtttca ctgtgagaat 1020
ggagattgcc catcccgcca tcctggtcta agcctggaaa ggatcaattt tcatccgaac 1080
gttcctgaca ggagaaaacc 1100
<210>9
<211>20
<212>DNA
<213> Artificial sequence
<400>9
tatgcccgtc gatcgcgccc 20
<210>10
<211>120
<212>DNA
<213> Artificial sequence
<400>10
ccaagatcac gcacgtaccg tcgatgtatc tctctgaact gccagggaaa aaccacggtt 60
agatcagcaa gcgttgccgg gaaatgggcg tcgataccat tatcgttttc gacacccact 120
<210>11
<211>20
<212>DNA
<213> Artificial sequence
<400>11
tcatcgagta cctcttgcgc 20
<210>12
<211>120
<212>DNA
<213> Artificial sequence
<400>12
tagcctgata tgcacgctta tcttcactgt ctttcccact cgccgctggt gggatatgtc 60
aatggcgtga ttgccagcgc ccgcgagcgt attgcggctt tctcccctga actggtggtg 120
<210>13
<211>20
<212>DNA
<213> Artificial sequence
<400>13
cgtcgcggtc agtaatgtga 20
<210>14
<211>25
<212>DNA
<213> Artificial sequence
<400>14
gcagaagaag atggtcattg gcaac 25
<210>15
<211>50
<212>DNA
<213> Artificial sequence
<400>15
tcggccactc atcaacatga ttcattcgct taggcataaa ttgccggaac 50
<210>16
<211>50
<212>DNA
<213> Artificial sequence
<400>16
gttccggcaa tttatgccta agcgaatgaa tcatgttgat gagtggccga 50
<210>17
<211>50
<212>DNA
<213> Artificial sequence
<400>17
tgacgcctaa cagtaattca gccatggttt tctcctgtca ggaacgttcg 50
<210>18
<211>50
<212>DNA
<213> Artificial sequence
<400>18
cgaacgttcc tgacaggaga aaaccatggc tgaattactg ttaggcgtca 50
<210>19
<211>25
<212>DNA
<213> Artificial sequence
<400>19
tcacagcaaa acgcttcgcc agaaa 25
<210>20
<211>20
<212>DNA
<213> Artificial sequence
<400>20
ttatggcttc accaatgcga 20
<210>21
<211>25
<212>DNA
<213> Artificial sequence
<400>21
taagggttac gttgacggtt aagca 25
<210>22
<211>50
<212>DNA
<213> Artificial sequence
<400>22
tcggccactc atcaacatga ttcattgctt aaccgtcaac gtaaccctta 50
<210>23
<211>50
<212>DNA
<213> Artificial sequence
<400>23
taagggttac gttgacggtt aagcaatgaa tcatgttgat gagtggccga 50
<210>24
<211>50
<212>DNA
<213> Artificial sequence
<400>24
ttgctaaagt atcgagatga aacatggttt tctcctgtca ggaacgttcg 50
<210>25
<211>50
<212>DNA
<213> Artificial sequence
<400>25
cgaacgttcc tgacaggaga aaaccatgtt tcatctcgat actttagcaa 50
<210>26
<211>25
<212>DNA
<213> Artificial sequence
<400>26
agccagctcc cacagtttca gcccc 25
Claims (8)
1. A recombinant Escherichia coli, which simultaneously expresses L-glutamate dehydrogenase, α -hydroxycarboxylic acid dehydrogenase and L- α -amino acid transaminase, and knocks out any one or two combinations of protocatechuate 2, 3-dioxygenase genes hpaD and 2, 3-dihydroxylpropionate 1,2 dioxygenase genes mhpB on the basis of host Escherichia coli;
the α -hydroxycarboxylic acid dehydrogenase is D- α -hydroxycarboxylic acid dehydrogenase or L- α -hydroxycarboxylic acid dehydrogenase, the D- α -hydroxycarboxylic acid dehydrogenase amino acid sequence is a sequence of which the access NO on NCBI is WP _003643296.1, WP _002335374.1 or EEI22188.1, and the L- α -hydroxycarboxylic acid dehydrogenase amino acid sequence is a sequence of which the access NO on NCBI is WP _013858488.1, WP _003607654.1 or WP _ 035430779.1;
l-glutamate dehydrogenase amino acid sequence is NCBI on access NO for WP _000373021.1, WP _011338202.1, WP _003497202.1, WP _010886557.1 sequence;
the amino acid sequence of L- α -amino acid transaminase is the sequence of accession NO at NCBI as WP _000462687.1, WP _000486988.1, WP _003643296.1, YP _ 806114.1.
2. The recombinant Escherichia coli of claim 1, wherein the recombinant Escherichia coli further enhances expression of one or more of a glutamate transporter gene, an NAD synthetic gene, and a pyridoxal phosphate synthetic gene.
3. The recombinant Escherichia coli of claim 2, wherein the enhanced expression is achieved by adding a constitutive promoter in front of a gene to be enhanced on the genome of the host Escherichia coli.
4. The recombinant Escherichia coli of claim 1, wherein said L-glutamate dehydrogenase, α -hydroxycarboxylic acid dehydrogenase, L- α -amino acid transaminase is co-expressed by pCO L ADuet.
5. The recombinant Escherichia coli of claim 1, wherein said host bacterium is Escherichia coli B L21 (DE 3).
6. A method for producing tanshinol, which comprises using the recombinant bacterium according to any one of claims 1 to 5.
7. The method of claim 6, wherein the production of tanshinol is performed by whole cell transformation.
8. The method of claim 7, wherein the whole cell transformation production system comprises a wet cell weight of 1-200 g/L, levodopa of 1-200 g/L-glutamic acid of 1-200 g/L, pH of 6.0-9.0, and reaction at 15-40 deg.C for 1-48 hours.
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| KR1020207033050A KR102616750B1 (en) | 2018-04-19 | 2018-10-25 | Genetically modified bacteria and their application in sweet potato production |
| PCT/CN2018/111895 WO2019200874A1 (en) | 2018-04-19 | 2018-10-25 | Engineering bacteria and use thereof in production of danshensu |
| ES202090050A ES2825205B2 (en) | 2018-04-19 | 2018-10-25 | Genetically engineered strain and its application in danshensu production |
| US16/542,787 US10870870B2 (en) | 2018-04-19 | 2019-08-16 | Engineering strain and application thereof in production of Danshensu |
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