WO2020259585A1 - 一种生产羟基酪醇的工程菌 - Google Patents

一种生产羟基酪醇的工程菌 Download PDF

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WO2020259585A1
WO2020259585A1 PCT/CN2020/098114 CN2020098114W WO2020259585A1 WO 2020259585 A1 WO2020259585 A1 WO 2020259585A1 CN 2020098114 W CN2020098114 W CN 2020098114W WO 2020259585 A1 WO2020259585 A1 WO 2020259585A1
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seq
dehydrogenase
hydroxytyrosol
engineered bacteria
formate
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赵华
宋田青
张婷
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枫杨生物研发(南京)有限公司
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Definitions

  • the invention belongs to the technical field of bioengineering, and particularly relates to an Escherichia coli engineered bacteria producing hydroxytyrosol.
  • HT Hydroxytyrosol
  • HT Hydroxytyrosol
  • HT is one of the most powerful antioxidants found in olive oil. It has stronger oxygen free radical absorption than resveratrol Ability to show a variety of biological activities. Studies have shown that it has pharmacological effects in anti-tumor, anti-thrombotic, regulating blood lipids and anti-arteriosclerosis, anti-pathogenic microorganisms, preventing and treating retinal macular degeneration, protecting cartilage and anti-osteoporosis. It is used in medicine, health food, and cosmetics. And food additives and other fields have broad application prospects and development potential.
  • hydroxytyrosol mainly include plant extraction, chemical synthesis, and biosynthesis.
  • HT is widely present in olive plants mainly in the form of esters. Extraction from fresh olive leaves, olive flowers or olive oil factory wastewater is the main production method currently on the market.
  • Chinese invention patent application CN201610883391.5 discloses an extraction process for olive leaf hydroxytyrosol;
  • Chinese invention patent application CN201710195462. 7 discloses a method for extracting hydroxytyrosol from olive leaves.
  • the extraction process requires the use of strong acidic steam, the purification process is cumbersome and expensive, the recovery rate is relatively low, the source of raw materials is unstable, and because hydroxytyrosol has a tendency to oxidize during the plant extraction process, the purity of the final product obtained from plant extraction is only It can reach 20%-40%.
  • Chemical synthesis is easier to obtain higher purity hydroxytyrosol products, for example, CN201110357075.1, CN201210342015.7, but currently known chemical synthesis routes involve expensive catalysts or have many reaction steps.
  • organic synthesis More organic solvents and metal oxides are involved in the process, so in terms of technology and cost, this method is not suitable for industrial production. Therefore, production by microbiological methods has received extensive attention.
  • Chinese invention patent application CN201510242626.8 discloses a monooxygenase gene cluster HpaBC derived from E. coli overexpressed in E. coli, which uses glucose as a substrate to synthesize hydroxytyrosol from scratch. Due to the toxicity of hydroxytyrosol to genes, The final yield of hydroxytyrosol was 349.05 mg/L and the yield was only 0.017 mol/mol. The effect of the de novo synthesis of hydroxytyrosol on the metabolic flux and metabolic regulation of the strain itself makes it difficult to achieve high production rates and yields. In 2012, Yasuharu Satoh et al.
  • Cisokasuene Acetate hydroxylase produces hydroxytyrosol.
  • ArAT aromatic aminotransferase
  • KDC ketoacid decarboxylase
  • ADH alcohol dehydrogenase
  • the program expresses aromatic aminotransferase (ArAT), ketoacid decarboxylase (KDC), and alcohol dehydrogenase (ADH) in the host to produce tyrosol, and then in 4-hydroxybenzene Acetate hydroxylase produces hydroxytyrosol.
  • ArAT aromatic aminotransferase
  • KDC ketoacid decarboxylase
  • ADH alcohol dehydrogenase
  • the yield of hydroxytyrosol can reach 647 ⁇ 35mg/L, when 6g/L (33mM) tyrosine is used as the substrate, the yield of hydroxytyrosol can reach 1243 ⁇ 165mg/L (8mM), and the yield is Only 0.24mol/mol.
  • the disadvantage of this scheme is the need to add a lot of expensive coenzyme pyridoxal phosphate (PLP) and reduced coenzyme I (NADH).
  • PDP coenzyme pyridoxal phosphate
  • NADH reduced coenzyme I
  • L-levodopa is produced as a by-product, making the yield of hydroxytyrosol low.
  • the present invention provides an engineered bacteria for producing hydroxytyrosol and a method for producing hydroxytyrosol by using the engineered bacteria, realizing a two-path high-efficiency production of cheap substrate tyrosine to hydroxytyrosol .
  • the present invention adopts the following technical solutions to achieve:
  • One aspect of the present invention provides an engineered bacterium for producing hydroxytyrosol.
  • the E. coli engineered bacterium simultaneously expresses 6 enzymes, namely 4-hydroxyphenylacetic acid 3-monooxygenase (HpaBC) and formate dehydrogenase ( FDH), L- ⁇ -amino acid transaminase (ArAT), L-glutamate dehydrogenase (GDH), ⁇ -keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH).
  • HpaBC 4-hydroxyphenylacetic acid 3-monooxygenase
  • FDH formate dehydrogenase
  • ArAT L- ⁇ -amino acid transaminase
  • GDH L-glutamate dehydrogenase
  • KDC ⁇ -keto acid decarboxylase
  • ADH alcohol dehydrogenase
  • the six enzymes simultaneously expressed by the engineered bacteria are achieved by co-expression of genes encoding the six enzymes through dual plasmids.
  • the dual plasmids are pRSFDuet-1 and pETDuet-1.
  • the pRSFDuet-1 is loaded with a gene encoding 4-hydroxyphenylacetic acid 3-monooxygenase and a gene encoding formate dehydrogenase; the pETDuet-1 is loaded with encoding L- ⁇ -amino acid Transaminase gene, gene encoding L-glutamate dehydrogenase, gene encoding ⁇ -keto acid decarboxylase, and gene encoding alcohol dehydrogenase.
  • the pETDuet-1 is loaded with a gene encoding 4-hydroxyphenylacetic acid 3-monooxygenase and a gene encoding formate dehydrogenase; the pRSFDuet-1 is loaded with encoding L- ⁇ -amino acid Transaminase gene, gene encoding L-glutamate dehydrogenase, gene encoding ⁇ -keto acid decarboxylase, and gene encoding alcohol dehydrogenase.
  • the engineered bacteria are obtained by transforming pRSFDuet-1 and pETDuet-1 plasmids into host Escherichia coli (E. coli) B21 competent cells.
  • the 4-hydroxyphenylacetic acid 3-monooxygenase is derived from Escherichia coli BL21 (DE3).
  • the nucleotide sequence of the 4-hydroxyphenylacetic acid 3-monooxygenase is accession NO on NCBI: NC_001136.10, REGION: complement (1234218..1236125) .
  • the formate dehydrogenase is derived from Mycobacterium intracellulare (M.iFDH).
  • amino acid sequence of the formate dehydrogenase is accession NO on NCBI WP_009957650.1.
  • the amino acid sequence of the formate dehydrogenase M.iFDH uses an online analysis tool http://www.jcat.de/ to optimize the nucleotide sequence according to E. coli codons, and the optimized The sequence is SEQ ID NO:1.
  • the L- ⁇ -amino acid transaminase is from Escherichia coli BL21 (E.cTyrB) or Saccharomyces cerevisiae BY4741 (ScARO8).
  • the amino acid sequence of the L- ⁇ -amino acid transaminase is the sequence of accession NO of NP_418478.1, NP_011313.1 on NCBI.
  • nucleotide sequence of the L- ⁇ -amino acid transaminase is accession NO on NCBI: NC_000913.3REGION: complement (4267114.. 4268307), NC_001139.9REGION: complement (116059.. 117561) sequence.
  • the L-glutamate dehydrogenase is derived from Saccharomyces cerevisiae S288C (ScGDH2).
  • nucleotide sequence of the L-glutamate dehydrogenase is accession NO on NCBI: NC_001136REGION: complement (70640..73918).
  • the amino acid sequence of the L-glutamate dehydrogenase is accession NO on NCBI is NP_010066.1.
  • the ⁇ -ketoacid decarboxylase is derived from Saccharomyces cerevisiae S288C (ScARO10).
  • nucleotide sequence of the ⁇ -keto acid decarboxylase is accession NO on NCBI: NC_001136.10, REGION: complement (1234218..1236125).
  • the amino acid sequence of the ⁇ -keto acid decarboxylase is the accession NO on NCBI is NP_010668.3.
  • the alcohol dehydrogenase is derived from anaerobic thermophilic bacillus Anoxybacillus geothermalis (BsADH) or Lactobacillus brevis (LbADH).
  • amino acid sequence of the alcohol dehydrogenase is accession NO on NCBI WP_044744228.1 and WP_107696682.
  • amino acid sequences of the alcohol dehydrogenase BsADH and LbADH are optimized using the online analysis tool http://www.jcat.de/ according to the E. coli codons, and after optimization
  • the sequence of is SEQ ID NO: 2, SEQ ID NO: 3.
  • Another aspect of the present invention provides a method for producing hydroxytyrosol, the method comprising the following steps:
  • the formate-containing compound is selected from formic acid, sodium formate, ammonium formate or calcium formate.
  • the catalytic reaction conditions are: pH 6.0-9.0, temperature 15-40°C, and time 1-48 hours.
  • Another aspect of the present invention provides the application of the engineered bacteria in the production of hydroxytyrosol.
  • yield refers to the ratio of the actual yield of the target product (hydroxytyrosol) obtained by consuming a unit amount of substrate (tyrosine) to the theoretically calculated yield of the target product during the catalytic process. For example, if 1 mM tyrosine is consumed, 1 mM hydroxytyrosol should be obtained theoretically, and 0.6 mM hydroxytyrosol should be actually obtained, and the yield is 0.6 mM/1 mM, which is 0.6 mol/mol.
  • the term "dual path" refers to starting from one substrate, through the generation of different intermediate products, and ultimately the same product.
  • tyrosine is added as the starting substrate, one path is through L- ⁇ -amino acid transaminase, L-glutamate dehydrogenase, and ⁇ -keto acid decarboxylase to generate intermediate tyrosol, and then through 4-hydroxyl Phenylacetate 3-monooxygenase and formate dehydrogenase convert tyrosol into the final product hydroxytyrosol; the other path is to first convert the substrate tyrosine into the intermediate product levodopa, and then convert levodopa to The final product is hydroxytyrosol.
  • the term "remaining amount of substrate” refers to adding a certain amount of substrate to the catalytic system. After a certain period of reaction, the amount of substrate that has not been converted in the catalytic system is detected, and this amount is the remaining amount of substrate.
  • the present invention provides an engineered bacterium for producing hydroxytyrosol.
  • the engineered bacterium can express 6 kinds of enzymes at the same time.
  • the engineered bacterium can catalyze tyrosine to produce hydroxytyrosol through a dual path, and realize the regeneration and autonomy of intracellular cofactors. Balanced, can get high yield and high yield of hydroxytyrosol on the basis of higher substrate concentration.
  • the preparation method of the invention is simple, the raw materials are easily available, the price is lower, and the industrial application prospect is good.
  • Figure 1 shows the two-path catalysis of tyrosine to produce hydroxytyrosol.
  • Figure 1a shows the first pathway.
  • Tyrosine generates 4-hydroxyphenylpyruvate through L- ⁇ -amino acid transaminase (ArAT), and then through ⁇ -ketone.
  • Acid decarboxylase (KDC) alcohol dehydrogenase (ADH) generate tyrosol
  • HpaBC 4-hydroxyphenylacetic acid 3-monooxygenase
  • L-gluten Glycine dehydrogenase (GDH) dehydrogenates L-glutamate to ⁇ -ketoglutaric acid and NADH.
  • L- ⁇ -amino acid transaminase and alcohol are L- ⁇ -amino acid transaminase and alcohol respectively.
  • the present invention can realize the self-circulation regeneration of ⁇ -ketoglutarate and NADH, and realize the self-equilibration of intracellular cofactors; formate dehydrogenase (FDH) can oxidize formate to CO 2 at the same time , Reducing NAD + to NADH;
  • Figure 1b shows the second pathway.
  • Tyrosine can generate L-dopa through 4-hydroxyphenylacetic acid 3-monooxygenase (HpaBC), and L-dopa can also pass through L- in turn.
  • HpaBC 4-hydroxyphenylacetic acid 3-monooxygenase
  • Alpha-amino acid transaminase (ArAT), alpha-keto acid decarboxylase (KDC), alcohol dehydrogenase (ADH) produce hydroxytyrosol.
  • KDC alpha-keto acid decarboxylase
  • ADH alcohol dehydrogenase
  • GDH L-glutamate dehydrogenase
  • FIG. 2 is a schematic diagram of the dual-path catalytic tyrosine production of hydroxytyrosol.
  • ArAT L- ⁇ -amino acid transaminase
  • GDH L-glutamate dehydrogenase
  • KDC ⁇ -keto acid decarboxylase
  • ADH alcohol dehydrogenase
  • HpaBC 4-hydroxyphenylacetic acid 3-monooxygenase
  • FDH formate dehydrogenase
  • ⁇ -Ketoglutaric acid ⁇ -ketoglutaric acid
  • glutamate L-glutamate.
  • the genes M.iFDH (WP_009957650.1), BsADH (WP_044744228.1), and LbADH (WP_107696682) involved in the present invention are all through the online website http://www.jcat.de/ for E. coli codon optimization, obtained artificial core
  • the nucleotide sequence (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.
  • genes involved in the present invention are ARO8 (NC_001139.9REGION: complement (116059..117561)), L-glutamate dehydrogenase ScGDH2 (NC_001136REGION: complement (70640..73918)), ⁇ -keto acid decarboxylase ARO10 (NC_001136) .10, REGION: complement (1234218..1236125)) uses the primers in Table 1 (SEQ ID NO: 8-SEQ ID NO: 13), using Saccharomyces cerevisiae S288C genome as a template for PCR amplification.
  • the gene alcohol dehydrogenase (BsADH, WP_044744228.1; LbADH, WP_107696682) and formate dehydrogenase (M.iFDH, WP_009957650.1) involved in the present invention all use the primers in Table 1 (SEQ ID NO: 14-SEQ ID NO: 20), using the plasmid containing the target gene synthesized by the company in step 1 as a template for PCR amplification.
  • MB-104-NdeI-M.iFDH-F SEQ ID NO: 19
  • MB-105-MiFDH-SacI-R SEQ ID NO: 20
  • MB-128-petDuet-M1-seq-F SEQ ID NO: 21
  • MB-129-petDuet-M1-seq-R SEQ ID NO: 22
  • MB-130-petDuet-M2-seq-F SEQ ID NO: 23
  • MB-131-T7t-seq-R SEQ ID NO: 24
  • the pETDuet-1 vector plasmid was digested with EcoRI/HindIII double restriction enzymes, and the vector gel was digested to recover; the vector digested product recovered from the gel and the TyrB fragment obtained in step 3 were used GenBuilder TM Cloning Kit from Nanjing GenScript Biotechnology Co., Ltd. Connect the kit by referring to the instruction manual of the kit; then transfer 10uL connection solution into 100uL TOP10 competent cells, ice bath for 30min and heat shock in 42°C water bath for 90s for heat shock treatment, immediately ice bath for 2min, then add 1mL of LB culture medium without antibiotics, cultured at 37°C for 1 hour to recover the bacteria.
  • the pETDuet-E.cTyrB vector plasmid was digested with HindIII single enzyme, and the vector gel was recovered by digestion;
  • the vector digested product recovered from the gel and the ScGDH2 fragment obtained in step 3 were ligated using the GenBuilder TM Cloning Kit kit.
  • the method is the same as above, and the subsequent ligation product transformation is the same as above.
  • the colony PCR primer selects MB-120-ScGDH2-F/MB-121 -ScGDH2-R (SEQ ID NO: 10, SEQ ID NO: 11) to obtain the following plasmid: recombinant plasmid pRSFDuet-E.cTyrB-ScGDH2 containing L- ⁇ -amino acid transaminase gene and L-glutamate dehydrogenase.
  • the pETDuet-E.cTyrB-ScGDH2 was digested with NdeI/KpnI, and the vector gel was digested and recovered; the Sc ARO10 gel recovery fragment and the BsADH gel recovery fragment obtained in step 3, using the primer MB-122-ScARO10-F/MB -125-BsADH-R (SEQ ID NO: 12, SEQ ID NO: 15) to perform overlap PCR, and fragment glue recovery;
  • the vector digested product recovered by the gel and the overlap PCR fragment product were ligated using the GenBuilder TM Cloning Kit kit.
  • the method is the same as above, and the subsequent ligation product transformation is the same as above.
  • the colony PCR primers are MB-122-ScARO10-F/MB-125-BsADH- R (SEQ ID NO: 12, SEQ ID NO: 15) to obtain the following plasmids: containing L- ⁇ -amino acid transaminase gene, L-glutamate dehydrogenase gene, ⁇ -keto acid decarboxylase gene, alcohol dehydrogenase Gene recombinant plasmid pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH.
  • the pETDuet-E.cTyrB-ScGDH2 was digested with NdeI/KpnI, and the vector gel was digested and recovered; the Sc ARO10 gel recovery fragment and the LbADH gel recovery fragment obtained in step 3, using the primer MB-122-ScARO10-F/MB -127-LbADH-R (SEQ ID NO: 12, SEQ ID NO: 17) to perform overlap PCR, and fragment glue recovery;
  • the vector digested product recovered by the gel and the overlap PCR fragment product were ligated using the GenBuilder TM Cloning Kit kit.
  • the method is the same as above, and the subsequent ligation product transformation is the same as above.
  • the colony PCR primer selects MB-122-ScARO10-F/MB-127-LbADH- R (SEQ ID NO: 12, SEQ ID NO: 17) to obtain the following plasmids: containing L- ⁇ -amino acid transaminase gene, L-glutamate dehydrogenase gene, ⁇ -keto acid decarboxylase gene, alcohol dehydrogenase Gene recombinant plasmid pETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH.
  • the pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH was digested with BamHI/NotI, and the vector gel was digested with the digestion;
  • the primer MB-146-A-ScGDH2-F/MB-121-ScGDH2-R (SEQ ID NO: 18, SEQ ID NO: 11) Perform PCR on ScGDH2 to obtain fragment ScGDH2-1 fragment gel recovery;
  • the vector digestion product recovered by the gel and the overlap PCR fragment product were ligated using the GenBuilder TM Cloning Kit kit.
  • the method is the same as above, and the subsequent ligation product transformation is the same as above.
  • the colony PCR primers are MB144-ARO8-F/MB-121-ScGDH2-R( SEQ ID NO:8, SEQ ID NO:11), the following plasmids were obtained: containing L- ⁇ -amino acid transaminase gene, L-glutamate dehydrogenase gene, ⁇ -keto acid decarboxylase gene, alcohol dehydrogenase gene Recombinant plasmid pETDuet-ARO8-ScGDH2-ScARO10-BsADH.
  • PETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH was digested with BamHI/NotI, and the carrier gel was recovered by digestion;
  • the vector digestion product recovered by the gel and the overlap PCR fragment product were ligated using the GenBuilder TM Cloning Kit kit.
  • the method is the same as above, and the subsequent ligation product transformation is the same as above.
  • the colony PCR primers are MB144-ARO8-F/MB-121-ScGDH2-R( SEQ ID NO:8, SEQ ID NO:11), the following plasmids were obtained: containing L- ⁇ -amino acid transaminase gene, L-glutamate dehydrogenase gene, ⁇ -keto acid decarboxylase gene, alcohol dehydrogenase gene Recombinant plasmid pETDuet-ARO8-ScGDH2-ScARO10-LbADH.
  • the pRSFDuet-1 vector plasmid was digested with SacI/SalI I, and the vector gel was recovered by digestion; the vector digested product recovered from the gel and the HpaBC gel recovered fragments obtained in step 3 were ligated using GenBuilder TM Cloning Kit, the method is the same as above
  • GenBuilder TM Cloning Kit the method is the same as above
  • the colony PCR primers are MB-128-petDuet-M1-seq-F/MB-129-petDuet-M1-seq-R (SEQ ID NO: 21, SEQ ID NO: 22) to obtain the following Plasmid: recombinant plasmid pRSFDuet-HpaBC containing 4-hydroxyphenylacetic acid 3-monooxygenase gene;
  • the pRSFDuet-HpaBC vector plasmid was digested with NdeI/XhoI double restriction enzymes, and the vector gel was recovered by enzyme digestion; the vector digested product recovered from the gel and the M.iFDH gel recovered fragment obtained in step 3 were ligated using GenBuilder TM Cloning Kit.
  • the subsequent ligation product transformation is the same as above, and the colony PCR primers are MB-130-petDuet-M2-seq-F/MB-131-T7t-seq-R (SEQ ID NO: 23, SEQ ID NO: 24) to obtain the following plasmid :
  • Induced expression method transfer the overnight cultured recombinant E. coli to 400mL TB fermentation medium at a volume ratio of 1%. When the cell OD600 reaches 0.6-0.8, add IPTG with a final concentration of 2mM and induce at 18°C Expression culture for 20h.
  • Catalytic method After the induction of expression, the cells were collected by centrifugation at 4°C, 6000 rpm, 12 minutes. In 50ml 50mM pH 7.0 phosphate buffer, the cell OD600 is 20, add levodopa or tyrosine as shown in Table 2, and react at 30°C for 24 hours. After the conversion, hydroxytyrosol was measured by liquid chromatography, and the results are shown in Table 2.
  • Two recombinant plasmids pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH and pETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH containing the alcohol dehydrogenase gene were obtained from Example 1, respectively in Escherichia coli BL21(DE3) Perform induced expression.
  • Induced expression method transfer the overnight cultured recombinant E. coli to 400mL TB fermentation medium at a volume ratio of 1%. When the cell OD600 reaches 0.6-0.8, add IPTG with a final concentration of 2mM and induce at 18°C Expression culture for 20h.
  • Catalytic method After the induction of expression, the cells were collected by centrifugation at 4°C, 6000 rpm, 12 minutes. In 50ml 50mM pH 7.0 phosphate buffer, the cell OD600 is 20, add different concentrations of tyrosine or levodopa, and react at 30°C for 24 hours. After the conversion, hydroxytyrosol was measured by liquid chromatography, and the results are shown in Table 3-1, Table 3-2 and Table 3-3.
  • the Escherichia coli transformation method of the ligation product was the same as in Example 1.
  • the transformed Escherichia coli was uniformly spread on an LB plate containing ampicillin and cultured overnight at 37°C.
  • Double plasmid strain expression Double plasmid strain expression:
  • pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH and pRSFDuet-HpaBC-M.iFDH double plasmid take 1uL plasmid pRSFDuet-HpaBC-M.iFDH into 100uL containing plasmid pETDuet used in Example 3 -E.cTyrB-ScGDH2-ScARO10-BsADH Escherichia coli BL21(DE3) competent cells, the transformation method is the same as before, and the double plasmid Escherichia coli recombinant strain E.coli BL21(DE3)/pETDuet-E containing 6 genes is obtained.
  • pETDuet-HpaBC-M.iFDH and pRSFDuet-E.cTyrB-ScGDH2-ScARO10-BsADH double plasmid combination take 1uL plasmid pETDuet-HpaBC-M.iFDH into 100uL Escherichia coli BL21(DE3) for sensing
  • the method is the same as before; take 1uL plasmid pRSFDuet-E.cTyrB-ScGDH2-ScARO10-BsADH and transfer it into 100uL competent cells containing pETDuet-HpaBC-M.iFDH plasmid constructed above to obtain a double plasmid containing 6 genes Escherichia coli recombinant strain E.coli BL21(DE3)/pETDuet-HpaBC-M.iFDH, pRSFDuet-E.cTyrB
  • the two strains constructed above were separately expressed. Using the same induction expression method as in Example 2, after induction, the cells were collected in 50ml 50mM pH 7.0 phosphate buffer, the cell OD600 was 20, the tyrosine concentration was 46mM, the ammonium formate concentration was 50mM, and the reaction was performed at 30°C. Time 24 hours. After the conversion, hydroxytyrosol was determined by liquid chromatography. The results are shown in Table 4.
  • the pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH and pRSFDuet-HpaBC-M.iFDH double plasmid system is used to produce hydroxytyrosol.
  • the substrate tyrosine is cheap and easy to obtain, and 2.5g can be obtained at a higher substrate concentration. /L Hydroxytyrosol. In the prior art, with 1 mM tyrosine as the substrate, the yield is only 0.19 (mol/mol).
  • the substrate concentration is increased to 46 mM, and the yield of hydroxytyrosol An increase of 252% solves the current problems of high substrate concentration, low yield of hydroxytyrosol and low yield.
  • SEQ ID NO: 1 nucleotide sequence encoding M.iFDH

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Abstract

一种生产羟基酪醇的工程菌以及通过该工程菌生产羟基酪醇的方法，利用该工程菌催化酪氨酸通过双路径生产羟基酪醇，可在较高底物浓度的基础上，得到高产率和高产量的羟基酪醇。同时，制备方法简单且原料易得，价格更加低廉，具有良好的工业化应用前景。

Description

一种生产羟基酪醇的工程菌 技术领域
本发明属于生物工程技术领域，特别涉及一种生产羟基酪醇的大肠杆菌工程菌。
背景技术
羟基酪醇(Hydroxytyrosol，HT)，化学名为3，4-二羟基苯乙醇，是在橄榄油中发现的最强大的抗氧化剂之一，比白藜芦醇具有更强的氧自由基吸收能力，表现出多种生物活性。已有研究表明其在抗肿瘤，抗血栓，调血脂和抗动脉硬化，抗病原微生物，防治视网膜黄斑变性，保护软骨和抗骨质疏松等多方面具有药理作用，在医药，保健食品，化妆品和食品添加剂等领域有着广阔的应用前景和开发潜力。
当前，羟基酪醇的合成方法主要包括植物提取，化学合成，以及生物合成。在自然资源中，HT主要以酯的形式广泛存在于橄榄植物中。从新鲜的橄榄叶、橄榄花或橄榄油厂废水中提取为当前市场上的主要生产方式，中国发明专利申请CN201610883391.5公开了一种橄榄叶羟基酪醇的提取工艺；中国发明专利申请CN201710195462.7公开了一种从橄榄叶中提取羟基酪醇的方法。提取过程中需要使用强酸性水蒸汽，纯化过程繁琐且费用昂贵，回收率比较低，原材料的来源不稳定，且由于羟基酪醇在植物提取过程中有氧化倾向，植物提取最终获得的产物纯度只能达到20％-40％。化学合成较易获得较高纯度的羟基酪醇产品，例如，CN201110357075.1、CN201210342015.7，但目前已知的化学合成路线中涉及价格昂贵的催化剂，或者反应的步骤较多，此外，有机合成过程中涉及较多有机溶剂和金属氧化物，因此从技术和成本考虑，该方法不适用于工业化生产。因此通过微生物法生产受到了广泛的关注。
目前已有部分报道使用微生物法生产羟基酪醇。中国发明专利申请CN201510242626.8公开了一种大肠杆菌中过表达来源于大肠杆菌的单加氧酶基因簇HpaBC，以葡萄糖为底物从头开始合成羟基酪醇，由于羟基酪醇对基因的毒性，最终羟基酪醇的产量为349.05mg/L产率仅为0.017mol/mol，从头合成的羟基酪醇产量到菌株自身代谢通量和代谢调节的影响，难以实现高的生产速率和得率。2012年，Yasuharu Satoh等人报道使用一株工程大肠杆菌，使用1mM酪氨酸为底物，依次通过酪氨酸羟化酶，L-多巴脱羧酶，酪胺氧化酶，醇脱氢酶作用下生成0.19±0.0056mM羟基酪醇，产率仅为0.19mol/mol，该方法中羟基酪醇的得率较低，中间产物积累较多，且菌株对底物的消耗速率较慢(1mM酪氨酸消耗需要将近72h)。
中国发明专利申请CN201711054680.5公开了异源代谢途径生产酪醇及羟基酪醇的方法。该方案在宿主中表达了芳香氨基转移酶(aromatic aminotransferase，ArAT)，酮酸脱羧酶(Ketoacid decarboxylase，KDC)，醇脱氢酶(Alcohol dehydrogenase，ADH)来生产酪醇，再在4-羟基苯乙酸羟化酶的作用下来生产羟基酪醇。从简单碳源出发，羟基酪醇产量能达到647±35mg/L，以6g/L(33mM)酪氨酸为底物时，羟基酪醇产量能达到1243±165mg/L(8mM)，产率仅为0.24mol/mol。该方案的缺点是需要外加许多昂贵的辅酶磷酸吡哆醛(PLP)和还原型 辅酶Ⅰ(NADH)。另外，该方案体系中除了目标化合物羟基酪醇的积累，还有L-左旋多巴作为副产物产出，使得羟基酪醇产率较低。
现有技术已知羟基酪醇的以葡萄糖为底物从头合成受到菌株自身代谢的影响导致最终产量低(低于1g/L)，耗时长(大于48h)，产率低；以酪氨酸为底物的生物转化方法，因缺乏辅因子再生体系，以及中间产物较多，催化效率随着反应持续导致辅因子不足，难以将更多的底物进行转化，最终导致催化的效率低，产率低。目前底物浓度最高做到为6g/L(33mM)，产率仅有0.24mol/mol。基于目前各种方法的缺陷，研究一种可实现高浓度底物持续性转化，可工业化生产、同时利用廉价底物可以高效生产羟基酪醇的方法非常有必要。
发明内容
基于目前各种方法的缺陷，本发明提供了一种生产羟基酪醇的工程菌以及利用该工程菌生产羟基酪醇的方法，实现了廉价底物酪氨酸到羟基酪醇的双路径高效生产。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明一方面提供了一种生产羟基酪醇的工程菌，所述大肠杆菌工程菌同时表达6种酶，分别为4-羟基苯乙酸3-单加氧酶(HpaBC)、甲酸脱氢酶(FDH)、L-α-氨基酸转氨酶(ArAT)、L-谷氨酸脱氢酶(GDH)、α-酮酸脱羧酶(KDC)和醇脱氢酶(ADH)。
在本发明的一些实施方案中，所述工程菌同时表达的6种酶是通过双质粒共表达编码所述6种酶的基因实现的。
在本发明的一些实施方案中，所述双质粒为pRSFDuet-1和pETDuet-1。
在本发明的一些实施方案中，所述pRSFDuet-1装载编码4-羟基苯乙酸3-单加氧酶的基因和编码甲酸脱氢酶的基因；所述pETDuet-1装载编码L-α-氨基酸转氨酶的基因、编码L-谷氨酸脱氢酶的基因、编码α-酮酸脱羧酶的基因和编码醇脱氢酶的基因。
在本发明的一些实施方案中，所述pETDuet-1装载编码4-羟基苯乙酸3-单加氧酶的基因和编码甲酸脱氢酶的基因；所述pRSFDuet-1装载编码L-α-氨基酸转氨酶的基因、编码L-谷氨酸脱氢酶的基因、编码α-酮酸脱羧酶的基因和编码醇脱氢酶的基因。
在本发明的一些实施方案中，所述工程菌是通过将pRSFDuet-1和pETDuet-1质粒转化到宿主Escherichia coli(大肠杆菌)B21感受态细胞中得到的。
在本发明的一些实施方案中，所述4-羟基苯乙酸3-单加氧酶(HpaBC)来自大肠杆菌Escherichia coli BL21(DE3)。
在本发明的一些实施方案中，所述4-羟基苯乙酸3-单加氧酶(HpaBC)的核苷酸序列是NCBI上accession NO为：NC_001136.10，REGION:complement(1234218..1236125)。
在本发明的一些实施方案中，所述甲酸脱氢酶来自Mycobacterium intracellulare胞内分枝杆菌(M.iFDH)。
在本发明的一些实施方案中，所述甲酸脱氢酶的氨基酸序列是NCBI上accession NO为WP_009957650.1。
在本发明的一些实施方案中，所述甲酸脱氢酶M.iFDH的氨基酸序列使用在线分析工具http://www.jcat.de/按照大肠杆菌密码子进行核苷酸序列优化，优化后的序列为SEQ ID NO:1。
在本发明的一些实施方案中，所述L-α-氨基酸转氨酶(ArAT)来自大肠杆菌Escherichia coli BL21(E.cTyrB)或酿酒酵母Saccharomyces cerevisiae BY4741(ScARO8)。
在本发明的一些实施方案中，所述L-α-氨基酸转氨酶的氨基酸序列是NCBI上accession NO为NP_418478.1、NP_011313.1的序列。
在本发明的一些实施方案中，所述L-α-氨基酸转氨酶的核苷酸序列是NCBI上accession NO为：NC_000913.3REGION:complement(4267114..4268307)、NC_001139.9REGION:complement(116059..117561)的序列。
在本发明的一些实施方案中，所述L-谷氨酸脱氢酶来自酿酒酵母Saccharomyces cerevisiae S288C(ScGDH2)。
在本发明的一些实施方案中，所述L-谷氨酸脱氢酶的核苷酸序列是NCBI上accession NO为：NC_001136REGION:complement(70640..73918)。
在本发明的一些实施方案中，所述L-谷氨酸脱氢酶的氨基酸序列是NCBI上accession NO为NP_010066.1。
在本发明的一些实施方案中，所述α-酮酸脱羧酶来自酿酒酵母Saccharomyces cerevisiae S288C(ScARO10)。
在本发明的一些实施方案中，所述α-酮酸脱羧酶的核苷酸序列是NCBI上accession NO为：NC_001136.10，REGION:complement(1234218..1236125)。
在本发明的一些实施方案中，所述α-酮酸脱羧酶的氨基酸序列是NCBI上accession NO为NP_010668.3。
在本发明的一些实施方案中，所述醇脱氢酶来自厌氧嗜热芽孢杆菌Anoxybacillus geothermalis(BsADH)或Lactobacillus brevis短乳杆菌(LbADH)。
在本发明的一些实施方案中，所述醇脱氢酶的氨基酸序列是NCBI上accession NO为WP_044744228.1和WP_107696682。
在本发明的一些实施方案中，所述醇脱氢酶BsADH和LbADH的氨基酸序列，使用在线分析工具http://www.jcat.de/按照大肠杆菌密码子进行核苷酸序列优化，优化后的序列分别为SEQ ID NO:2，SEQ ID NO:3。
本发明另一方面提供了一种生产羟基酪醇的方法，所述方法包括以下步骤：
(1)将上述实施方案中所述的工程菌放入磷酸盐缓冲液中；
(2)加入酪氨酸和含甲酸根的化合物进行催化反应。
在本发明的一些实施方案中，所述含甲酸根的化合物选自甲酸、甲酸钠、甲酸铵或甲酸钙。
在本发明的一些实施方案中，所述催化反应条件为：pH 6.0-9.0，温度15-40℃，时间1-48小时。
本发明的再一方面提供了所述工程菌在生产羟基酪醇中的应用。
术语“产率”指催化过程中，消耗单位数量的底物(酪氨酸)而实际获得的目标产物(羟基酪醇)的产量与理论计算的目标产物产量的比值。例如：消耗1mM酪氨酸，理论上应获得1mM羟基酪醇，实际获得0.6mM羟基酪醇，则产率为0.6mM/1mM，即0.6mol/mol。
术语“双路径”指以一种底物出发，通过生成不同的中间产物，最终得到相同的产物。本发明体系中，添加酪氨酸作为出发底物，一条路径通过L-α-氨基酸转氨酶、L-谷氨酸脱氢酶、α-酮酸脱羧酶生成中间体酪醇，再通过4-羟基苯乙酸3-单加氧酶和甲酸脱氢酶将酪醇转化为最终产物羟基酪醇；另一条路径则将底物酪氨酸先转化成中间产物左旋多巴，再将左旋多巴转化为最终产物羟基酪醇。
术语“底物剩余量”指添加一定量底物至催化体系，反应一定时间后，检测催化体系中未发生转化的底物量，该量为底物剩余量。
本发明的有益效果：
本发明提供了一种生产羟基酪醇的工程菌，该工程菌可以同时表达6种酶，利用该工程菌催化酪氨酸通过双路径生产羟基酪醇，并且实现胞内辅因子的再生和自平衡，可在较高底物浓度的基础上，得到高产率和高产量的羟基酪醇。同时，本发明制备方法简单且原料易得，价格更加低廉，具有良好的工业化应用前景。
附图说明
图1为双路径催化酪氨酸生产羟基酪醇，其中，图1a为第一条通路，酪氨酸通过L-α-氨基酸转氨酶(ArAT)生成4-羟基苯丙酮酸，再通过α-酮酸脱羧酶(KDC)，醇脱氢酶(ADH)生成酪醇，最后通过4-羟基苯乙酸3-单加氧酶(HpaBC)将酪醇转化为羟基酪醇；此过程中，L-谷氨酸脱氢酶(GDH)将L-谷氨酸(glutamate)脱氢生成α-酮戊二酸(α-Ketoglutaric acid)和NADH，这两种化合物又分别是L-α-氨基酸转氨酶和醇脱氢酶的辅酶，因此本发明可实现α-酮戊二酸和NADH的自循环再生，实现胞内辅因子的自平衡；甲酸脱氢酶(FDH)在将甲酸根氧化为CO 2的同时，还原NAD +到NADH；图1b为第二条通路，酪氨酸通过4-羟基苯乙酸3-单加氧酶(HpaBC)可生成L-多巴，L-多巴也可依次通过L-α-氨基酸转氨酶(ArAT)，α-酮酸脱羧酶(KDC)、醇脱氢酶(ADH)生成羟基酪醇。类似的，L-谷氨酸脱氢酶(GDH)实现α-酮戊二酸(α-Ketoglutaric acid)和NADH的再生，甲酸脱氢酶实现NADH的再生。
图2为双路径催化酪氨酸生产羟基酪醇示意图。ArAT：L-α-氨基酸转氨酶；GDH：L-谷氨酸脱氢酶；KDC：α-酮酸脱羧酶；ADH：醇脱氢酶；HpaBC：4-羟基苯乙酸3-单加氧酶；FDH：甲酸脱氢酶；α-Ketoglutaric acid：α-酮戊二酸；glutamate：L-谷氨酸。
具体实施方式
实施例1 菌株构建
a)基因合成
本发明涉及的基因M.iFDH(WP_009957650.1)、BsADH(WP_044744228.1)、LbADH(WP_107696682)均通过在线网址http://www.jcat.de/进行大肠杆菌密码子优化，获得的人工核苷酸序列(SEQ ID NO:1，SEQ ID NO:2，SEQ ID NO:3)由南京金斯瑞生物科技有限公司合成。
b)设计引物
设计引物，引物序列如表1所示
c)基因克隆
本发明涉及的基因TyrB(NC_000913.3REGION:complement(4267114..4268307))、4-羟基苯乙酸3-单加氧酶HpaBC(NC_012971REGION:4498788..4500880)使用表1中的引物(SEQ ID NO:4-SEQ ID NO:7)，以大肠杆菌Escherichia coli str.K-12基因组为模板进行PCR扩增。
本发明涉及的基因ARO8(NC_001139.9REGION:complement(116059..117561))、L-谷氨酸脱氢酶ScGDH2(NC_001136REGION:complement(70640..73918))、α-酮酸脱羧酶ARO10(NC_001136.10，REGION:complement(1234218..1236125))均使用表1中的引物(SEQ ID NO:8-SEQ ID NO:13)，以酿酒酵母Saccharomyces cerevisiae S288C基因组为模板进行PCR扩增。
本发明涉及的基因醇脱氢酶(BsADH，WP_044744228.1；LbADH，WP_107696682)、甲酸脱氢酶(M.iFDH，WP_009957650.1)，均使用表1中的引物(SEQ ID NO:14-SEQ ID NO:20)，以步骤1中公司合成的包含目标基因的质粒为模板进行PCR扩增。
设计引物，引物序列如表1.1所示
表1：扩增基因所用到的引物
名称 序列表中编号
MB-118-TyrB-F SEQ ID NO:4
MB-119-TyrB-R SEQ ID NO:5
MB-114-pETDuet-HpaBC-F-SacI SEQ ID NO:6
MB-115-pETDuet-HpaBC-R-SalI SEQ ID NO:7
MB-144-ARO8-F SEQ ID NO:8
MB-145-ARO8-R SEQ ID NO:9
MB-120-ScGDH2-F SEQ ID NO:10
MB-121-ScGDH2-R SEQ ID NO:11
MB-122-ScARO10-F SEQ ID NO:12
MB-123-ScARO10-R SEQ ID NO:13
MB-124-ARO10-BsADH-F SEQ ID NO:14
MB-125-BsADH-R SEQ ID NO:15
MB-126-ARO10-LbADH-F SEQ ID NO:16
MB-127-LbADH-R SEQ ID NO:17
MB-146-A-ScGDH2-F SEQ ID NO:18
MB-104-NdeI-M.iFDH-F SEQ ID NO:19
MB-105-MiFDH-SacI-R SEQ ID NO:20
MB-128-petDuet-M1-seq-F SEQ ID NO:21
MB-129-petDuet-M1-seq-R SEQ ID NO:22
MB-130-petDuet-M2-seq-F SEQ ID NO:23
MB-131-T7t-seq-R SEQ ID NO:24
d)重组质粒pETDuet-E.cTyrB
将pETDuet-1载体质粒采用EcoRI/HindIII双酶切，酶切载体胶回收；将胶回收的载体酶切产物与步骤3中获得的TyrB片段使用南京金斯瑞生物科技有限公司的GenBuilder TM Cloning Kit试剂盒进行连接，方法参考试剂盒的使用说明书；随后将10uL连接液转入100uL TOP10感受态细胞，冰浴30min后于42℃水浴中热激90s进行热休克处理，立即冰浴2min，然后加入1mL不含抗生素的LB培养液，37℃培养1h使菌体复苏。最后将含pRSFDuet-1重组质粒的细胞均匀涂布在含卡那霉素的LB平板上，挑单菌落，使用引物MB-118-TyrB-F/MB-119-TyrB-R(SEQ ID NO:4，SEQ ID NO:5)做菌落PCR验证其正确性，条带正确的单克隆进行DNA测序以保证其准确性，最后保存正确转化子，得到如下质粒：含有L-α-氨基酸转氨酶基因的重组质粒pETDuet-E.cTyrB。
e)重组质粒pRSFDuet-E.cTyrB-ScGDH2
将pETDuet-E.cTyrB载体质粒采用HindIII单酶切，酶切载体胶回收；
将胶回收的载体酶切产物与步骤3中获得的ScGDH2片段使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB-120-ScGDH2-F/MB-121-ScGDH2-R(SEQ ID NO:10，SEQ ID NO:11)，得到如下质粒：含有L-α-氨基酸转氨酶基因和L-谷氨酸脱氢酶的重组质粒pRSFDuet-E.cTyrB-ScGDH2。
f)重组质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH
将pETDuet-E.cTyrB-ScGDH2，采用NdeI/KpnI双酶切，酶切载体胶回收；将步骤3中获得Sc ARO10胶回收片段与BsADH胶回收片段，使用引物MB-122-ScARO10-F/MB-125-BsADH-R(SEQ ID NO:12，SEQ ID NO:15)进行overlap PCR，片段胶回收；
将胶回收的载体酶切产物与overlap PCR片段产物使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB-122-ScARO10-F/MB-125-BsADH-R(SEQ ID NO:12，SEQ ID NO:15)，得到如下质粒：含有L-α-氨基酸转氨酶基因，L-谷氨酸脱氢酶基因，α-酮酸脱羧酶基因、醇脱氢酶基因的重组质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH。
g)重组质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH
将pETDuet-E.cTyrB-ScGDH2，采用NdeI/KpnI双酶切，酶切载体胶回收；将步骤3中获得Sc ARO10胶回收片段与LbADH胶回收片段，使用引物MB-122-ScARO10-F/MB-127-LbADH-R(SEQ ID NO:12，SEQ ID NO:17)进行overlap PCR，片段胶回收；
将胶回收的载体酶切产物与overlap PCR片段产物使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB-122-ScARO10-F/MB-127-LbADH-R(SEQ ID NO:12，SEQ ID NO:17)，得到如下质粒：含有L-α-氨基酸转氨酶基因，L-谷氨酸脱氢酶基因，α-酮酸脱羧酶基因、醇脱氢酶基因的重组质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH。
h)重组质粒pETDuet-ARO8-ScGDH2-ScARO10-BsADH
将pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH采用BamHI/NotI双酶切，酶切载体胶回收；使用引物MB-146-A-ScGDH2-F/MB-121-ScGDH2-R(SEQ ID NO:18，SEQ ID NO:11)对ScGDH2进行PCR，获得片段ScGDH2-1片段胶回收；
将步骤3中获得ARO8胶回收片段与上述ScGDH2-1胶回收片段，使用引物MB144-ARO8-F/MB-121-ScGDH2-R(SEQ ID NO:8，SEQ ID NO:11)进行overlap PCR，片段胶回收；
将胶回收的载体酶切产物与overlap PCR片段产物使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB144-ARO8-F/MB-121-ScGDH2-R(SEQ ID NO:8，SEQ ID NO:11)，得到如下质粒：含有L-α-氨基酸转氨酶基因，L-谷氨酸脱氢酶基因，α-酮酸脱羧酶基因、醇脱氢酶基因的重组质粒pETDuet-ARO8-ScGDH2-ScARO10-BsADH。
i)重组质粒pETDuet-ARO8-ScGDH2-ScARO10-LbADH
将pETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH采用BamHI/NotI双酶切，酶切载体胶回收；
使用引物MB-146-A-ScGDH2-F/MB-121-ScGDH2-R(SEQ ID NO:18，SEQ ID NO:11)对ScGDH2进行PCR，获得片段ScGDH2-1片段胶回收；
将步骤3中获得ARO8胶回收片段与上述ScGDH2-1胶回收片段，使用引物MB144-ARO8-F/MB-121-ScGDH2-R(SEQ ID NO:8，SEQ ID NO:11)进行overlap PCR，片段胶回收；
将胶回收的载体酶切产物与overlap PCR片段产物使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB144-ARO8-F/MB-121-ScGDH2-R(SEQ ID NO:8，SEQ ID NO:11)，得到如下质粒：含有L-α-氨基酸转氨酶基因，L-谷氨酸脱氢酶基因，α-酮酸脱羧酶基因、醇脱氢酶基因的重组质粒pETDuet-ARO8-ScGDH2-ScARO10-LbADH。
j)重组质粒pRSFDuet-HpaBC-M.iFDH
将pRSFDuet-1载体质粒采用SacI/SalI I双酶切，酶切载体胶回收；将胶回收的载体酶切产物与步骤3中获得HpaBC胶回收片段使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB-128-petDuet-M1-seq-F/MB-129-petDuet-M1-seq-R(SEQ ID NO:21，SEQ ID NO:22)，得到如下质粒：含有4-羟基苯乙酸3-单加氧酶基因的重组质粒pRSFDuet-HpaBC；
将pRSFDuet-HpaBC载体质粒采用NdeI/XhoI双酶切，酶切载体胶回收；将胶回收的载体酶切产物与步骤3中获得M.iFDH胶回收片段使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同上，后续连接产物转化同上，菌落PCR引物选用MB-130-petDuet-M2-seq-F/MB-131-T7t-seq-R(SEQ ID NO:23，SEQ ID NO:24)，得到如下质粒：含有4-羟基苯乙酸3-单加氧酶基因，甲酸脱氢酶基因的重组质粒pRSFDuet-HpaBC-M.iFDH。
k)包含单质粒菌株构建：将上述构建的不同质粒，分别取1uL转入100uL Escherichia coli BL21(DE3)感受态细胞，转化方法同上，分别得到含有不同基因组合的大肠杆菌重组菌株。
实施例2 L-α-氨基酸转氨酶的筛选
从实施例1中获得的二种含L-α-氨基酸转氨酶基因的重组质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH和pETDuet-ARO8-ScGDH2-ScARO10-BsADH，分别在Escherichia coli BL21(DE3)中得到进行诱导表达。诱导表达方法：将过夜培养的重组大肠杆菌按体积比为1％的量转接到400mL TB发酵培养基中，当细胞OD600达到0.6-0.8后，加入终浓度为2mM的IPTG，在18℃诱导表达培养20h。催化方法：诱导表达结束后，4℃、6000rpm、12分钟离心收集细胞。于50ml 50mM pH 7.0磷酸盐缓冲液中，细胞OD600为20，加入如表2中所示的左旋多巴或者酪氨酸，于30℃反应，时间24小时。转化结束后液相色谱测定羟基酪醇，结果如表2所示。
表2：不同L-α-氨基酸转氨酶加入酪氨酸或左旋多巴的催化比较
Figure PCTCN2020098114-appb-000001
通过以上对比实验，我们发现两种来源的L-α-氨基酸转氨酶都可以实现目标化合物的高效转化，并且产率都能达到99％以上。最终我们选取了大肠杆菌自身来源的TyrB用于后续双通路合成羟基酪醇，更有利于在大肠杆菌中的表达。
实施例3 醇脱氢酶的筛选
从实施例1中获得二种含醇脱氢酶基因的重组质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH和pETDuet-E.cTyrB-ScGDH2-ScARO10-LbADH，分别在Escherichia coli BL21(DE3)中进行诱导表达。诱导表达方法：将过夜培养的重组大肠杆菌按体积比为1％的量转接到400mL TB发酵培养基中，当细胞OD600达到0.6-0.8后，加入终浓度为2mM的IPTG，在18℃诱导表达培养20h。催化方法：诱导表达结束后，4℃、6000rpm、12分钟离心收集细胞。于50ml 50mM pH 7.0磷酸盐缓冲液中，细胞OD600为20，加入不同浓度酪氨酸或者左旋多巴，于30℃反应，时间24小时。转化结束后液相色谱测定羟基酪醇，结果如表3-1、表3-2以及表3-3所示。
表3-1不同醇脱氢酶加入约1mM底物催化比较
Figure PCTCN2020098114-appb-000002
表3-2不同醇脱氢酶加入约10mM底物催化比较
Figure PCTCN2020098114-appb-000003
Figure PCTCN2020098114-appb-000004
表3-3不同醇脱氢酶加入约30mM底物催化比较
Figure PCTCN2020098114-appb-000005
从表3-1、表3-2以及表3-3的对比实验结果可以看出，加入较低浓度的底物如1mM左右时，得到目标化合物的产率达到99％以上；加入10mM左右的底物浓度时，由酪氨酸催化得到酪醇的产率达到100％，由L-多巴催化得到羟基酪醇的产率达到83％以上；加入较高的底物浓度如30mM左右时，得到目标化合物的产率仍可以达到99％以上；表明这二种工程菌在底物浓度高的时候仍可以实现和低底物浓度时相同的催化效果，解决了目前底物浓度较高，产率较低的问题。
另外，鉴于两种来源的醇脱氢酶都可以实现目标化合物的高效转化，产率和催化速率相当，考虑到厌氧嗜热芽孢杆菌来源的BsADH在较高的温度下仍然具备较好的催化活力，有利于提高菌株工艺优化的空间，最终我们选取了厌氧嗜热芽孢杆菌来源的BsADH用于后续双路径合成羟基酪醇。
实施例4 酪氨酸通过双路径合成羟基酪醇
本实验对用于表达六种酶的载体采取了两种组合方法进行比较，一种使用pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH和pRSFDuet-HpaBC-M.iFDH双质粒(质粒构建见实施例1)；另外一种则将基因对应的载体进行调换，即pETDuet-HpaBC-M.iFDH和pRSFDuet-E.cTyrB-ScGDH2-ScARO10-BsADH双质粒。
pETDuet-HpaBC-M.iFDH质粒的构建：
a)将pETDuet-1载体质粒采用SacI/XhoI双酶切，酶切载体胶回收；
b)将pRSFDuet-HpaBC-M.iFDH质粒采用SacI/XhoI双酶切，获得片段HpaBC-M.iFDH胶回收；
c)将上述获得的酶切载体与HpaBC-M.iFDH片段使用T4连接酶进行连接，方法参考T4连接酶的使用说明书；
d)连接产物的大肠杆菌转化方法同实施例1，转化后的大肠杆菌均匀涂布在含氨苄霉素的LB平板上37℃过夜培养。
e)挑单菌落，使用引物MB-128-petDuet-M1-seq-F/MB-131-T7t-seq-R(SEQ ID NO:21，SEQ ID NO:24)做菌落PCR验证其正确性，条带正确的单克隆进行DNA测序以保证其准确性，最后保存正确转化子；
pRSFDuet-E.cTyrB-ScGDH2-ScARO10-BsADH质粒的构建：
a)将pRSFDuet-1载体质粒采用NcoI/XhoI双酶切，酶切载体胶回收；
b)将pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH质粒采用XbaI/PacI双酶切，获得片段E.cTyrB-ScGDH2-ScARO10-BsADH胶回收；
c)将上述获得的酶切载体与E.cTyrB-ScGDH2-ScARO10-BsADH片段使用GenBuilder TM Cloning Kit试剂盒进行连接，方法同实施例1，后续连接产物转化与菌落筛选方法同上。
双质粒菌株表达：
对于第一种组合，pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH和pRSFDuet-HpaBC-M.iFDH双质粒，取1uL质粒pRSFDuet-HpaBC-M.iFDH转入到100uL实施例3使用的包含质粒pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH的Escherichia coli BL21(DE3)感受态细胞，转化方法同前，得到含有6种基因的双质粒大肠杆菌重组菌株E.coli BL21(DE3)/pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH，pRSFDuet-HpaBC-M.iFDH；
对于第二种组合，pETDuet-HpaBC-M.iFDH和pRSFDuet-E.cTyrB-ScGDH2-ScARO10-BsADH双质粒组合，取1uL质粒pETDuet-HpaBC-M.iFDH转入到100uL Escherichia coli BL21(DE3)感受态细胞，方法同前；取1uL质粒pRSFDuet-E.cTyrB-ScGDH2-ScARO10- BsADH转入到100uL上述构建的含pETDuet-HpaBC-M.iFDH质粒的感受态细胞，得到含有6种基因的双质粒大肠杆菌重组菌株E.coli BL21(DE3)/pETDuet-HpaBC-M.iFDH，pRSFDuet-E.cTyrB-ScGDH2-ScARO10-BsADH；
将上述构建的两种菌株分别进行表达。采取与实施例2相同的诱导表达方法，诱导后收集菌体于50ml 50mM pH 7.0磷酸盐缓冲液中，细胞OD600为20，酪氨酸浓度为46mM，甲酸铵浓度为50mM，于30℃反应，时间24小时。转化结束后液相色谱测定羟基酪醇。结果如表4所示。
表4：双路径合成羟基酪醇
Figure PCTCN2020098114-appb-000006
以上结果表明采取pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH和pRSFDuet-HpaBC-M.iFDH双质粒体系时，羟基酪醇的生产量和产率显著高于另外一种组合。
采取pETDuet-E.cTyrB-ScGDH2-ScARO10-BsADH和pRSFDuet-HpaBC-M.iFDH双质粒体系生产羟基酪醇，底物酪氨酸廉价易得，并且可在较高底物浓度下，得到2.5g/L羟基酪醇。现有技术中，以1mM酪氨酸为底物，产率仅为0.19(mol/mol)，通过本发明的双路径生产羟基酪醇，底物浓度增加到了46mM，并且羟基酪醇的产率提高了252％，解决了目前底物浓度高，羟基酪醇产率低，产量少的问题。
序列信息
SEQ ID NO:1编码M.iFDH的核苷酸序列
Figure PCTCN2020098114-appb-000007
SEQ ID NO:2编码BsADH的核苷酸序列
Figure PCTCN2020098114-appb-000008
SEQ ID NO:3编码LbADH的核苷酸序列
Figure PCTCN2020098114-appb-000009
SEQ ID NO:4 MB-118-TyrB-F
Figure PCTCN2020098114-appb-000010
SEQ ID NO: 5 MB-119-TyrB-R
Figure PCTCN2020098114-appb-000011
SEQ ID NO: 6 MB-114-pETDuet-HpaBC-F-SacI
Figure PCTCN2020098114-appb-000012
SEQ ID NO: 7 MB-115-pETDuet-HpaBC-R-SalI
Figure PCTCN2020098114-appb-000013
SEQ ID NO: 8 MB-144-ARO8-F
Figure PCTCN2020098114-appb-000014
SEQ ID NO: 9 MB-145-ARO8-R
Figure PCTCN2020098114-appb-000015
SEQ ID NO: 10 MB-120-ScGDH2-F
Figure PCTCN2020098114-appb-000016
SEQ ID NO: 11 MB-121-ScGDH2-R
Figure PCTCN2020098114-appb-000017
SEQ ID NO: 12 MB-122-ScARO10-F
Figure PCTCN2020098114-appb-000018
SEQ ID NO: 13 MB-123-ScARO10-R
Figure PCTCN2020098114-appb-000019
SEQ ID NO: 14 MB-124-ARO10-BsADH-F
Figure PCTCN2020098114-appb-000020
SEQ ID NO: 15 MB-125-BsADH-R
Figure PCTCN2020098114-appb-000021
SEQ ID NO: 16 MB-126-ARO10-LbADH-F
Figure PCTCN2020098114-appb-000022
SEQ ID NO: 17 MB-127-LbADH-R
Figure PCTCN2020098114-appb-000023
SEQ ID NO: 18 MB-146-A-ScGDH2-F
Figure PCTCN2020098114-appb-000024
SEQ ID NO: 19 MB-104-NdeI-M. iFDH-F
Figure PCTCN2020098114-appb-000025
SEQ ID NO: 20 MB-105-MiFDH-SacI-R
Figure PCTCN2020098114-appb-000026
SEQ ID NO: 21 MB-128-petDuet-M1-seq-F
Figure PCTCN2020098114-appb-000027
SEQ ID NO: 22 MB-129-petDuet-M1-seq-R
Figure PCTCN2020098114-appb-000028
SEQ ID NO: 23 MB-130-petDuet-M2-seq-F
Figure PCTCN2020098114-appb-000029
SEQ ID NO: 24 MB-131-T7t-seq-R
Figure PCTCN2020098114-appb-000030

Claims (18)

  1. 一种生产羟基酪醇的工程菌，其特征在于，所述工程菌同时表达6种酶，分别为4-羟基苯乙酸3-单加氧酶、甲酸脱氢酶、L-α-氨基酸转氨酶、L-谷氨酸脱氢酶、α-酮酸脱羧酶和醇脱氢酶。
  2. 根据权利要求1所述的工程菌，其特征在于，所述工程菌同时表达的6种酶是通过双质粒共表达编码所述6种酶的基因实现的。
  3. 根据权利要求2所述的工程菌，其特征在于，所述双质粒为pRSFDuet-1和pETDuet-1。
  4. 根据权利要求3所述的工程菌，其特征在于，所述pRSFDuet-1装载编码4-羟基苯乙酸3-单加氧酶的基因和编码甲酸脱氢酶的基因；所述pETDuet-1装载编码L-α-氨基酸转氨酶的基因、编码L-谷氨酸脱氢酶的基因、编码α-酮酸脱羧酶的基因和编码醇脱氢酶的基因。
  5. 根据权利要求3所述的工程菌，其特征在于，所述pETDuet-1装载编码4-羟基苯乙酸3-单加氧酶的基因和编码甲酸脱氢酶的基因；所述pRSFDuet-1装载编码L-α-氨基酸转氨酶的基因、编码L-谷氨酸脱氢酶的基因、编码α-酮酸脱羧酶的基因和编码醇脱氢酶的基因。
  6. 根据权利要求4或5所述的工程菌，其特征在于，所述工程菌是通过将pRSFDuet-1和pETDuet-1质粒转化到宿主大肠杆菌B21感受态细胞中得到的。
  7. 根据权利要求1所述的工程菌，其特征在于，所述4-羟基苯乙酸3-单加氧酶来自于大肠杆菌。
  8. 根据权利要求1所述的工程菌，其特征在于，所述甲酸脱氢酶来自于胞内分枝杆菌。
  9. 根据权利要求8所述的工程菌，其特征在于，所述编码甲酸脱氢酶的核苷酸序列为SEQ ID NO：1。
  10. 根据权利要求1所述的工程菌，其特征在于，所述L-α-氨基酸转氨酶来自于大肠杆菌或酿酒酵母。
  11. 根据权利要求1所述的工程菌，其特征在于，所述L-谷氨酸脱氢酶来自于酿酒酵母。
  12. 根据权利要求1所述的工程菌，其特征在于，所述α-酮酸脱羧酶来自于酿酒酵母。
  13. 根据权利要求1所述的工程菌，其特征在于，所述醇脱氢酶来自于厌氧嗜热芽孢杆菌或短乳杆菌。
  14. 根据权利要求13所述的工程菌，其特征在于，所述来自于厌氧嗜热芽孢杆菌的编码醇脱氢酶的核苷酸序列为SEQ ID NO：2；所述来自于短乳杆菌的编码醇脱氢酶的核苷酸序列为SEQ ID NO：3。
  15. 一种生产羟基酪醇的方法，包括以下步骤：
    (1)将权利要求1-14任一项所述的工程菌放入磷酸盐缓冲液中；
    (2)加入酪氨酸和含甲酸根的化合物进行催化反应。
  16. 根据权利要求15所述的生产羟基酪醇的方法，其特征在于，所述含甲酸根的化合物选自甲酸、甲酸钠、甲酸铵或甲酸钙。
  17. 根据权利要求15所述的生产羟基酪醇的方法，其特征在于，所述催化反应条件为：pH 6.0-9.0，温度15-40℃，时间1-48小时。
  18. 权利要求1-14任一项所述的工程菌在生产羟基酪醇中的应用。
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