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WO2020259585A1 - Bactérie génétiquement modifiée pour la production d'hydroxytyrosol - Google Patents

Bactérie génétiquement modifiée pour la production d'hydroxytyrosol 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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dehydrogenase
hydroxytyrosol
engineered bacteria
formate
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赵华
宋田青
张婷
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Maple Bio Nanjing Co Ltd
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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

L'invention concerne une bactérie génétiquement modifiée pour produire de l'hydroxytyrosol et un procédé de production d'hydroxytyrosol à l'aide de la bactérie modifiée. En utilisant la bactérie modifiée pour catalyser la tyrosine pour produire de l'hydroxytyrosol au moyen d'une voie double, un rendement élevé d'hydroxytyrosol peut être obtenu avec une productivité élevée sur la base d'une concentration de substrat supérieure. Dans le même temps, le procédé de préparation est simple, et se caractérise par des matières premières facilement disponibles avec un prix inférieur, avec de bonnes perspectives d'industrialisation.
PCT/CN2020/098114 2019-06-25 2020-06-24 Bactérie génétiquement modifiée pour la production d'hydroxytyrosol Ceased WO2020259585A1 (fr)

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