CN116790455A - Genetically engineered bacterium and method for preparing chiral phenyllactic acid by converting L-phenylalanine - Google Patents
Genetically engineered bacterium and method for preparing chiral phenyllactic acid by converting L-phenylalanine Download PDFInfo
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- CN116790455A CN116790455A CN202211122255.6A CN202211122255A CN116790455A CN 116790455 A CN116790455 A CN 116790455A CN 202211122255 A CN202211122255 A CN 202211122255A CN 116790455 A CN116790455 A CN 116790455A
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- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 title claims abstract description 75
- 229960005190 phenylalanine Drugs 0.000 title claims abstract description 38
- 241000894006 Bacteria Species 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 21
- NWCHELUCVWSRRS-SECBINFHSA-N (2r)-2-hydroxy-2-phenylpropanoic acid Chemical compound OC(=O)[C@@](O)(C)C1=CC=CC=C1 NWCHELUCVWSRRS-SECBINFHSA-N 0.000 title claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 44
- VOXXWSYKYCBWHO-QMMMGPOBSA-N (S)-3-phenyllactic acid Chemical compound OC(=O)[C@@H](O)CC1=CC=CC=C1 VOXXWSYKYCBWHO-QMMMGPOBSA-N 0.000 claims abstract description 33
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 150000008575 L-amino acids Chemical class 0.000 claims abstract description 16
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims abstract description 15
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims abstract description 15
- 108010036197 NAD phosphite oxidoreductase Proteins 0.000 claims abstract description 15
- 108010001539 D-lactate dehydrogenase Proteins 0.000 claims abstract description 11
- 102100023319 Dihydrolipoyl dehydrogenase, mitochondrial Human genes 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 22
- 239000013612 plasmid Substances 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 239000013613 expression plasmid Substances 0.000 claims description 9
- 238000003259 recombinant expression Methods 0.000 claims description 9
- 150000001413 amino acids Chemical group 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 12
- 239000005515 coenzyme Substances 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 5
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 abstract description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 3
- 239000008103 glucose Substances 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 150000007513 acids Chemical class 0.000 abstract description 2
- 125000004432 carbon atom Chemical group C* 0.000 abstract description 2
- 230000002829 reductive effect Effects 0.000 abstract description 2
- 230000008929 regeneration Effects 0.000 abstract description 2
- 238000011069 regeneration method Methods 0.000 abstract description 2
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 14
- 239000005711 Benzoic acid Substances 0.000 description 10
- 239000004284 Heptyl p-hydroxybenzoate Substances 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 229960000723 ampicillin Drugs 0.000 description 9
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 9
- 229930027917 kanamycin Natural products 0.000 description 9
- 229960000318 kanamycin Drugs 0.000 description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 9
- 229930182823 kanamycin A Natural products 0.000 description 9
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 5
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 4
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- 239000000126 substance Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- BTNMPGBKDVTSJY-UHFFFAOYSA-N keto-phenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1 BTNMPGBKDVTSJY-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
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- 230000002194 synthesizing effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000011942 biocatalyst Substances 0.000 description 2
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- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
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- 238000001704 evaporation Methods 0.000 description 2
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- 230000004151 fermentation Effects 0.000 description 2
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- 230000006698 induction Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- -1 small-molecule organic acid Chemical class 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000001903 2-oxo-3-phenylpropanoic acid Substances 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000232299 Ralstonia Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- DEDGUGJNLNLJSR-UHFFFAOYSA-N alpha-hydroxycinnamic acid Natural products OC(=O)C(O)=CC1=CC=CC=C1 DEDGUGJNLNLJSR-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 229940124411 anti-hiv antiviral agent Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000005282 brightening Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000035425 carbon utilization Effects 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 229940126904 hypoglycaemic agent Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940116871 l-lactate Drugs 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 101150050602 ptxD gene Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229930189533 tanshinol Natural products 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/09—Recombinant DNA-technology
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- C12P7/00—Preparation of oxygen-containing organic compounds
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- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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Abstract
The invention discloses a genetic engineering bacterium and a method for preparing chiral phenyllactic acid by converting L-phenylalanine, wherein the genetic engineering bacterium contains a coding gene of a first group of enzymes or a coding gene of a second group of enzymes; the first set of genes encoding enzymes includes genes encoding L-amino acid deaminase, phosphite dehydrogenase and L-lactate dehydrogenase; the genes encoding the second set of enzymes include genes encoding L-amino acid deaminase, phosphite dehydrogenase and D-lactate dehydrogenase. The genetically engineered bacterium provided by the invention can catalyze the L-phenylalanine to prepare two chiral phenyllactic acids, and the genetically engineered bacterium utilizes the phosphite dehydrogenase to catalyze inorganic phosphite to provide reduced coenzyme, so that the addition of organic substrate formate or glucose for coenzyme regeneration is avoided, the utilization efficiency of carbon atoms is improved, the L-phenylalanine can be efficiently converted into the L-phenyllactic acid or the D-phenyllactic acid, and the genetically engineered bacterium is a green biosynthesis technology with important industrialization prospect.
Description
Technical Field
The invention relates to a technology for preparing two chiral phenyllactic acids by catalyzing L-phenylalanine by genetically engineered microorganisms and whole cells, and belongs to the technical field of biology.
Background
Phenyllactic acid (phenyllactic acid, PLA) is a natural organic acid with high added value, exists in lactobacillus fermentation products and honey, has special biological activity, and can be widely applied to the fields of chemical industry, medicine, pesticide, food and the like as a chiral intermediate. As a small-molecule organic acid having optical activity, PLA has two enantiomers of L-phenyllactic acid and D-phenyllactic acid. Both chiral PLA have broad-spectrum antibacterial property, can be used as biological preservative to inhibit various food-borne pathogenic bacteria, and has stronger antibacterial activity and safety performance than some common chemical food preservatives. PLA can also be added into dermatological treatment drugs, and has the beauty effect of removing wrinkles and brightening skin in the cosmetic industry. In addition, phenyllactic acid is also a precursor of drugs such as anti-HIV agents and hypoglycemic agents, and for example, PLA derivatives tanshinol have the effects of inhibiting platelet aggregation and dilating coronary arteries. The current industrial method for producing phenyllactic acid is mainly a chemical synthesis method, and the method has complex process, needs high temperature and high pressure and can bring serious environmental pollution. Meanwhile, the chemical synthesis product is a mixed product of D type and L type, the product with single chiral is difficult to obtain, and the separation and the purification are difficult. The microbial fermentation method is a green production mode for synthesizing the phenyllactic acid, but generally has low product concentration and low efficiency, increases the cost of subsequent separation and refining, and is not suitable for industrial production.
With the continuous development of synthetic biology and enzyme catalysis technology, whole cell catalysis technology has been used for producing raw material substances in the fields of medicine, chemical industry, food and the like, and research on chiral phenyllactic acid biocatalysis synthesis is increasing at present. In 2018, zheng et al expressed L-amino acid deaminase, formate dehydrogenase, L-lactate dehydrogenase or D-lactate dehydrogenase in E.coli, and synthesized 59.9. 59.9 mM of L-phenyllactic acid or 60.3. 60.3 mM of D-phenyllactic acid (Zheng, Z., et al Enhanced biosynthesis of chiral phenyllactic acid from L-phenylalanine through a new whole-cell biocatalyst. Bioprocess biosystem. Eng. 2018, 41:1205-1212); in 2018, wang et al expressed L-amino acid deaminase, L-lactate dehydrogenase and formate dehydrogenase in E.coli, almost completely converting 30 g/L phenylalanine to L-phenyllactic acid (Wang, X., et al A new approach for efficient synthesis of phenyllactic acid from L-phenalane: pathway design and cofactor engineering J. Food biochem. 2018, 42:e 12584); in 2019, hou et al expressed L-amino acid deaminase, lactate dehydrogenase and formate dehydrogenase in E.coli, and 54 g/L of L-phenyllactic acid was synthesized using L-phenylalanine (Hou, Y., et al Combination of multi-enzyme expression fine-tuning and co-substrates addition improves phenyllactic acid production with an Escherichia coli whole-cell biocatalyst. Bioresource. Technique. 2019, 287: 121423); in 2022, zhang et al co-expressed L-lactate dehydrogenase and glucose dehydrogenase in Pichia pastoris, 400 mM phenylpyruvate was converted to 359.8 mM L-phenyllactic acid (Zhang, D., et al Enantioselective biosynthesis of L-phenyllactic acid from phenylpyruvic acid in vitro by L-lactate dehydrogenase coupling with glucose dehydrogenase, front, bioeng, biotechnol, 2022, 10: 846489). However, these methods generally utilize lactate dehydrogenase coupled formate dehydrogenase or glucose dehydrogenase for coenzyme regeneration, require the addition of higher concentrations of organic substrate formate or glucose to the reaction system, reduce the carbon utilization of the synthesis system and increase the cost. Therefore, there is still a need to develop a green synthesis method of chiral phenyllactic acid with high reaction efficiency, recycling of coenzyme and high atom utilization.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the invention provides a genetic engineering bacterium for producing chiral phenyllactic acid, which uses phosphite dehydrogenase to catalyze inorganic phosphite to provide reduced coenzyme, thereby realizing stable and efficient conversion of L-phenylalanine into chiral phenyllactic acid, and having important application prospect.
The technical scheme adopted by the invention is as follows:
a genetically engineered bacterium comprising a gene encoding a first set of enzymes or a gene encoding a second set of enzymes; the first set of genes encoding enzymes includes genes encoding L-amino acid deaminase, phosphite dehydrogenase and L-lactate dehydrogenase; the second set of genes encoding enzymes includes genes encoding L-amino acid deaminase, phosphite dehydrogenase and D-lactate dehydrogenase; the amino acid sequence of the L-amino acid deaminase is shown as SEQ ID NO. 1, and the amino acid sequence of the phosphite dehydrogenase is shown as SEQ ID NO. 2; the amino acid sequence of the L-lactate dehydrogenase is shown as SEQ ID NO. 3; the amino acid sequence of the D-lactate dehydrogenase is shown as SEQ ID NO. 4.
Further improvement, the nucleotide sequence of the coding gene of the coding L-amino acid deaminase is shown as SEQ ID NO. 5, and the nucleotide sequence of the coding gene of the coding phosphite dehydrogenase is shown as SEQ ID NO. 6; the nucleotide sequence of the coding gene of the coding L-lactate dehydrogenase is shown as SEQ ID NO. 7, and the nucleotide sequence of the coding gene of the coding D-lactate dehydrogenase is shown as SEQ ID NO. 8.
Further improvement, the coding gene of the L-amino acid deaminase and the coding gene of the phosphite dehydrogenase are respectively synthesized into pRSFDuet-1 plasmid to form recombinant expression plasmid pRSFDuet-laad-ptxD；The coding gene of the L-lactate dehydrogenase is synthesized into pETDuet-1 plasmid to form recombinant expression plasmid pETDuet-lldh，The coding gene of D-lactate dehydrogenase is synthesized into pETDuet-1 plasmid to form recombinant expression plasmid pETDuet-dldh。
Further improved, the genetically engineered bacterium is formed by pouring a gene encoding a first set of enzymes or a gene encoding a second set of enzymes into a host strain, the host strain being an E.coli BL21 (DE 3) strain.
A method for preparing chiral phenyllactic acid by converting L-phenylalanine comprises the following steps: l-phenylalanine solution is used as a substrate, and the genetically engineered bacterium is added to convert the L-phenylalanine into L-phenyllactic acid or D-phenyllactic acid.
Further improvement, the conditions of the transformation are: the final concentration of L-phenylalanine in the L-phenylalanine solution is 20-200 mM, the pH value is 6-8, the conversion temperature is 25-40 ℃, and the conversion time is 6-12 h.
Further improved, the conditions of the transformation are as follows: the L-phenylalanine solution contains 200 mM L-phenylalanineAcid, 200 mM Na 2 HPO 3 The added amount of the pH=8 genetically engineered bacteria is 20 g/L of final concentration, the conversion temperature is 30 ℃, the conversion time is 12h, and the stirring rotation speed during conversion is 200 rpm.
The beneficial effects of the invention are as follows: the invention provides a genetically engineered bacterium which is used for converting L-phenylalanine to prepare L-phenyllactic acid or D-phenyllactic acid. Compared with the prior chiral phenyllactic acid biosynthesis method for regenerating the coenzyme by using formate dehydrogenase or glucose dehydrogenase, the invention utilizes phosphite dehydrogenase to catalyze inorganic phosphite to regenerate the coenzyme, avoids adding organic substrate formate or glucose, improves the utilization efficiency of carbon atoms, can efficiently convert L-phenylalanine into L-phenyllactic acid or D-phenyllactic acid, and is a green biosynthesis technology with important industrialization prospect.
The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of the synthesis route of L-phenyllactic acid by converting L-phenylalanine to Lumy-E209 in example 1 of the present invention.
FIG. 2 is a schematic diagram of the synthesis route of L-phenylalanine by converting Lumy-E210 in example 2 of the present invention.
FIG. 3 is a graph showing the yields of L-phenyllactic acid produced by Lumy-E209 and D-phenyllactic acid produced by Lumy-E210 in examples 3 and 4 of the present invention.
Detailed Description
The technical contents of the present invention are further described below with reference to examples: the following examples are illustrative, not limiting, and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The strains and growth conditions used in the present invention are as follows:
cloning host DH 5. Alpha. And expression host BL21 (DE 3) were purchased from Invitrogen, all E.coli were cultivated at 37℃in LB medium containing 100mg/l ampicillin or 100mg/l kanamycin.
Wherein, the formula of the LB liquid medium is as follows: peptone 10 g/L, yeast extract 5 g/L, naCl 10 g/L, pH 7.0; LB solid culture medium 20 g/L agar is added into LB liquid culture medium; sterilizing with steam at 121deg.C under high temperature and high pressure for 20 min.
All plasmids were pETDuet-1 or pRSFDuet-1 (purchased from Novagen) derived plasmids for expression of the gene of interest.
Example 1
Constructing genetically engineered bacteria Lumy-E209 and Lumy-E210:
(1) Synthesis of L-amino acid deaminasePmLAAD) coding gene [ (]laad) Fragment, phosphite dehydrogenaseRaPPDH) coding gene [ ]ptxD) Fragment, L-lactate dehydrogenaseLpLLDH) coding genelldh) And D-lactate dehydrogenase [ ]LnDLDH) coding genedldh）：
Utilizing online software JCat to ensure that the colibacillus is produced according to the codon preference of the escherichia coliProteus myxofaciens) In (a) and (b)laadGene, ralstonia strainRalstoniasp.)ptxDGene, cheese bacillus and its preparing processLacticaseibacillus paracasei) In (a) and (b)lldhGene and Nasalidis lactobacillusLactobacillus nasalidis) In (a) and (b)dldhCodon optimization is carried out on the gene, and after optimizationlaadThe nucleotide sequence of (2) is shown as SEQ ID NO. 5,ptxDthe nucleotide sequence of (2) is shown as SEQ ID NO. 6,lldhthe nucleotide sequence of (2) is shown as SEQ ID NO. 7,dldhthe nucleotide sequence of (2) is shown as SEQ ID NO. 8. The above gene sequence was sent to the gene synthesis company (GENEWIZ),laadthe pRSFDuet-1 was obtained by synthesizing the plasmid intermediate the BamHI and HindIII cleavage sites of pRSFDuet-1laad，ptxDSynthesis into pRSFDuet-laadIntermediate NdeI and XhoI cleavage sites to obtain recombinant expression plasmid pRSFDuet-laad-ptxD；lldhAnddldhrespectively synthesizing the recombinant expression plasmid pETDuet-1 into the middle of BamHI and HindIII enzyme cutting sites of pETDuet-1 to obtain recombinant expression plasmid pETDuet-lldhAnd pETDuet-dldh。
(2) Obtaining genetically engineered bacterium Lumy-E209:
mu.L of the recombinant plasmid pRSFDuet obtained in step (1) was used for preparing a plasmid pRSFDuet-laad-ptxDAnd 5. Mu.L of the recombinant plasmid pETDuet obtained in step (1)lldhTransformation into competent cells of E.coli BL21 (DE 3) was carried out by the heat shock method. The heat-shocked bacterial liquid is coated on LB solid medium flat plate containing 100mg/L ampicillin and kanamycin, and cultured in a constant temperature incubator at 37 ℃ for 12 hours. Single colony was picked up on a plate to 5mL of LB liquid medium (containing 100mg/L ampicillin and kanamycin) and incubated in a shaker at 37 ℃ at 200 rpm; and (3) carrying out PCR amplification verification on the cultured bacterial liquid to obtain a genetic engineering strain Lumy-E209, wherein the Lumy-E209 can be used for converting phenylalanine to prepare L-phenyllactic acid as shown in figure 1.
(3) Obtaining genetically engineered bacterium Lumy-E210:
mu.L of the recombinant plasmid pRSFDuet obtained in step (1) was used for preparing a plasmid pRSFDuet-laad-ptxDAnd 5. Mu.L of the recombinant plasmid pETDuet obtained in step (1)dldhTransformation into competent cells of E.coli BL21 (DE 3) was carried out by the heat shock method. The heat-shocked bacterial liquid was spread on LB solid medium plates containing 100mg/L ampicillin and 100mg/L kanamycin, and cultured in a constant temperature incubator at 37℃for 12 hours. Single colonies were picked on plates into 5mL LB liquid medium (containing 100mg/L ampicillin and 100mg/L kanamycin) and incubated in a shaker at 37℃at 200 rpm. And (3) carrying out PCR amplification verification on the cultured bacterial liquid to obtain a genetic engineering strain Lumy-E210, wherein the Lumy-E210 can be used for converting L-phenylalanine to prepare D-phenyllactic acid as shown in figure 2.
Example 2
Preparation of L-phenyllactic acid by converting genetically engineered bacterium Lumy-E209 into L-phenylalanine
(1) Preparing a whole cell catalyst of genetically engineered bacterium Lumy-E209:
the genetically engineered bacterium Lumy-E209 obtained in the step (2) of the embodiment 1 is inoculated into 50mL of LB liquid medium (containing 100mg/L ampicillin and 100mg/L kanamycin) by an inoculating loop for strain activation, and the strain is subjected to 37 ℃ C. Strain activationCulturing at night; subsequently, the activated bacterial liquid was inoculated into 1L of LB liquid medium (containing 100mg/L ampicillin and 100mg/L kanamycin) at 37℃and 200rpm in an inoculum size of 1% (volume ratio), and cultured for 3-5 hours to OD 600 Reaching 0.6-0.8, adding 0.2 mM IPTG,16 ℃, and carrying out induction culture at 200rpm for 16h; the resulting culture broth was collected, centrifuged at 3500rpm at 4℃for 15 minutes to collect the cells, and the cells were washed with phosphate buffer for 2 times for use.
(2) Preparation of L-phenyllactic acid by conversion of L-phenylalanine from Lumy-E209
Constructing a reaction system by taking the thalli collected in the step (1) as a catalyst, adding 200 mM of L-phenylalanine and 200 mM of Na into the reaction system, wherein the addition amount of the thalli is 20 g/L 2 HPO 3 Ph=8, reaction conditions were 30 ℃, stirring speed was 200rpm, 12h. The amount of L-phenyllactic acid produced was measured by HPLC, using an Agilent1260 liquid chromatograph, a chromatography column of Agilent Zorbax SB-C18 (4.6X150 mm), a detection wavelength of 210 nm, a column temperature of 30℃and a mobile phase A of water (containing 1% trifluoroacetic acid), a mobile phase B of acetonitrile (containing 1% trifluoroacetic acid), a flow rate of 1 mL/min, and a gradient elution procedure of: 0 minutes, 90% mobile phase a+10% mobile phase B; 15 minutes, 5% mobile phase a+95% mobile phase B;18 minutes, 5% mobile phase a+95% mobile phase B; 20 minutes, 90% mobile phase A+10% mobile phase B. The flow rate was 1 mL/min. As shown in FIG. 3, L-phenylalanine is transformed by a recombinant bacterium to obtain 29.42 g/L-phenyllactic acid. Acidifying the reaction liquid, extracting with ethyl acetate, and vacuum rotary evaporating the extractive solution to obtain pure L-phenyllactic acid product with e.e. value of above 99%.
Example 3
Preparation of D-phenyllactic acid by converting genetically engineered bacterium Lumy-E210 into L-phenylalanine
(1) Preparing a whole cell catalyst of genetically engineered bacterium Lumy-E210:
inoculating the genetically engineered bacterium Lumy-E210 obtained in the step (3) in the embodiment 1 into 50mL of LB liquid medium (containing 100mg/L ampicillin and 100mg/L kanamycin) by an inoculating loop for strain activation, and culturing at 37 ℃ overnight; then, the activated bacterial liquid was inoculated into 5L LB liquid culture according to an inoculum size of 1% (volume ratio)Culturing in medium (containing 100mg/L ampicillin and 100mg/L kanamycin) at 37deg.C and 200rpm for 3-5 hr to OD 600 Reaching 0.6-0.8, adding 0.2 mM IPTG,16 ℃, and carrying out induction culture at 200rpm for 16h; the resulting culture broth was collected, centrifuged at 3500rpm at 4℃for 15 minutes to collect the cells, and the cells were washed with phosphate buffer for 2 times for use.
(2) Preparation of D-phenyllactic acid by converting Lumy-E210 into L-phenylalanine
Constructing a reaction system by taking the thalli collected in the step (1) as a catalyst, adding 200 mM of L-phenylalanine and 200 mM of Na into the reaction system, wherein the addition amount of the thalli is 20 g/L 2 HPO 3 Ph=8, reaction conditions were 30 ℃, stirring speed was 200rpm, 12h. The amount of L-phenyllactic acid produced was measured by HPLC, using an Agilent1260 liquid chromatograph, a chromatography column of Agilent Zorbax SB-C18 (4.6X150 mm), a detection wavelength of 210 nm, a column temperature of 30℃and a mobile phase A of water (containing 1% trifluoroacetic acid), a mobile phase B of acetonitrile (containing 1% trifluoroacetic acid), a flow rate of 1 mL/min, and a gradient elution procedure of: 0 minutes, 90% mobile phase a+10% mobile phase B; 15 minutes, 5% mobile phase a+95% mobile phase B;18 minutes, 5% mobile phase a+95% mobile phase B; 20 minutes, 90% mobile phase A+10% mobile phase B. The flow rate was 1 mL/min. As shown in FIG. 3, the D-phenyllactic acid of 27.32 g/L was obtained after transformation of L-phenylalanine by a recombinant bacterium. Acidifying the reaction liquid, extracting with ethyl acetate, and vacuum rotary evaporating the extractive solution to obtain pure D-phenyllactic acid product with e.e. value of above 99%.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (7)
1. A genetically engineered bacterium, comprising a gene encoding a first set of enzymes or a gene encoding a second set of enzymes; the first set of genes encoding enzymes includes genes encoding L-amino acid deaminase, phosphite dehydrogenase and L-lactate dehydrogenase; the second set of genes encoding enzymes includes genes encoding L-amino acid deaminase, phosphite dehydrogenase and D-lactate dehydrogenase; the amino acid sequence of the L-amino acid deaminase is shown as SEQ ID NO. 1, and the amino acid sequence of the phosphite dehydrogenase is shown as SEQ ID NO. 2; the amino acid sequence of the L-lactate dehydrogenase is shown as SEQ ID NO. 3; the amino acid sequence of the D-lactate dehydrogenase is shown as SEQ ID NO. 4.
2. The genetically engineered bacterium of claim 1, wherein the nucleotide sequence of the coding gene encoding an L-amino acid deaminase is shown in SEQ ID No. 5 and the nucleotide sequence of the coding gene encoding a phosphite dehydrogenase is shown in SEQ ID No. 6; the nucleotide sequence of the coding gene of the coding L-lactate dehydrogenase is shown as SEQ ID NO. 7, and the nucleotide sequence of the coding gene of the coding D-lactate dehydrogenase is shown as SEQ ID NO. 8.
3. The genetically engineered bacterium of claim 1, wherein the gene encoding an L-amino acid deaminase and the gene encoding a phosphite dehydrogenase are synthesized into pRSFDuet-1 plasmid to form recombinant expression plasmid pRSFDuet-laad-ptxD；The coding gene of the L-lactate dehydrogenase is synthesized into pETDuet-1 plasmid to form recombinant expression plasmid pETDuet-lldh，The coding gene of D-lactate dehydrogenase is synthesized into pETDuet-1 plasmid to form recombinant expression plasmid pETDuet-dldh。
4. The method of claim 1, wherein the genetically engineered bacterium is formed by pouring a gene encoding a first set of enzymes or a gene encoding a second set of enzymes into a host strain, the host strain being E.coli BL21 (DE 3) strain.
5. The method for preparing chiral phenyllactic acid by converting L-phenylalanine is characterized by comprising the following steps: the method comprises the steps of using an L-phenylalanine solution as a substrate, adding the genetically engineered bacterium of any one of claims 1-4, and converting the L-phenylalanine into L-phenyllactic acid or D-phenyllactic acid.
6. The method for preparing chiral phenyllactic acid by converting L-phenylalanine according to claim 5, wherein the conditions for the conversion are: the final concentration of L-phenylalanine in the L-phenylalanine solution is 20-200 mM, the pH value is 6-8, the conversion temperature is 25-40 ℃, and the conversion time is 6-12 h.
7. The method for preparing chiral phenyllactic acid by converting L-phenylalanine according to claim 6, wherein the conditions for the conversion are as follows: the L-phenylalanine solution contains 200 mM Na and 200 mM L-phenylalanine of 200 mM 2 HPO 3 The added amount of the pH=8 genetically engineered bacteria is 20 g/L of final concentration, the conversion temperature is 30 ℃, the conversion time is 12h, and the stirring rotation speed during conversion is 200 rpm.
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