A biosynthetic method based on multi-enzyme cascade reaction

Abstract

The application discloses a biosynthesis method based on multi-enzyme cascade reaction and belongs to the field of bioengineering. The biosynthesis method based on multi-enzyme cascade reaction has the following steps: S1, adding omega-transaminase to racemic-alpha-phenethylamine until the racemic-alpha-phenethylamine is converted into phenylacetone by (S)-alpha-phenethylamine under the action of the omega-transaminase, and (R)-alpha-phenethylamine is left; S2, adding aldehyde-ketone reductase to the phenylacetone so that the phenylacetone is reduced into (R)-alpha-phenethyl alcohol under the action of the aldehyde-ketone reductase, and glutamate dehydrogenase is added in S1 and S2 to maintain the cycle of coenzyme NADPH / NADP + and the auxiliary substrate alpha-ketoglutarate / glutamate, (R)-alpha-phenethylamine and (R)-alpha-phenethyl alcohol are generated by the three-enzyme one-pot method catalysis of the racemic-alpha-phenethylamine; (R)-alpha-phenethylamine and (R)-alpha-phenethyl alcohol are generated by the three-enzyme co-expression of the catalysis of the engineering bacteria; (R)-alpha-phenethylamine and (R)-alpha-phenethyl alcohol can be synthesized simultaneously, the method can be suitable for various industrial production scenes, and has important industrial application value.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and more particularly to a biosynthesis method based on multi-enzyme cascade reaction. Background Art

[0002] Chiral amines and chiral alcohols are important building blocks for the industrial production of optically active drugs, agricultural chemicals, bioactive compounds, fine chemicals, dyes and other substances. α-Phenylethylamine is a simple, inexpensive and readily available chiral amine that has been widely used in the preparation of enantiomerically pure compounds. α-Phenylethanol, also known as styraxol, is an isomer of β-phenylethanol. It has a soft and pleasant rose aroma and is therefore widely used in various edible flavors. It is an important raw material for aromatic compounds used in spices. α-Phenylethanol, as a basic chiral compound intermediate, is widely used in chemical fields such as medicine and pesticides. It is an application A wide range of chiral intermediates are available. (R)-α-phenylethylamine can be used as a substrate for the synthesis of (R)-α-aminonitrile, N-{(S)-[cyclohexane-(S)-2-ol]}-(R)-α-methylbenzylamine, and N-{(R)-[cyclohexane-(R)-2-ol]}-(R)-α-methylbenzylamine. (R)-α-phenylethanol is an important building block in the synthesis of fine chemicals and pharmaceuticals, such as (R)-salbutamol, (R)-epinephrine, and (R)-atomoxetine. Clearly, chiral (R)-α-phenylethylamine and (R)-α-phenylethanol are of great significance in the synthesis of chiral compounds.

[0003] Currently, the main biopreparation methods for chiral (R)-α-phenylethylamine and (R)-α-phenylethanol include enzymatic kinetic resolution of racemates and asymmetric catalysis of prochiral substrates. However, enzymatic kinetic resolution is limited in application due to its maximum theoretical yield of 50%. Therefore, asymmetric catalysis of prochiral substrates is the most effective method for obtaining chiral (R)-α-phenylethylamine and (R)-α-phenylethanol. Studies have shown that enzymes such as ω-transaminases, lipases, imine reductases, amine oxidases, and amine dehydrogenases (Biocatalysis: enzymatic synthesis for industrial applications. Angew Chem Int Ed Engl 2002) can be used to synthesize chiral (R)-α-phenylethylamine and (R)-α-phenylethanol. 21,60(1),88-119) can convert α-phenylethylamine into acetophenone; among these enzymes, transaminase is the most promising for converting α-phenylethylamine (Transaminase biocatalysis: optimization and application. Green Chemistry 2017,19(2),333-360.); in addition, ketoreductase, perakine reductase, short-chain dehydrogenase and aldehyde-ketone reductase are used to produce chiral alcohols (Preparation of structurally diverse chiral alcohols by engineering keto reductaseCgKR1.ACSCatalysis2017,7(10),7174-7181; Enantioselectivereductionofalpha,beta-unsaturatedketonesandarylketonesbyperakinereductase.OrgLett2019,21(12),4411-4414; )-1-phenylethanolfromtea(Camellia sinensis)flowers.Food Chemistry 2019,280,27-33;Chinese invention patent 201810355748.1);However, efficient and green synthetic pathways for the simultaneous synthesis of chiral (R)-α-phenylethylamine and (R)-α-phenylethanol are still rare;Therefore, exploring ω-transaminases and aldehyde-ketoreductases with better catalytic performance to simultaneously achieve the efficient and green synthesis of (R)-α-phenylethylamine and (R)-α-phenylethanol has important research significance and practical application value.To this end,we propose a biosynthetic method based on a multi-enzyme cascade reaction. Summary of the Invention

[0004] 1. Technical problems to be solved

[0005] In view of the problems in the prior art, the purpose of the present application is to provide a biosynthesis method based on multi-enzyme cascade reaction, which can realize the simultaneous synthesis of (R)-α-phenethylamine and (R)-α-phenethyl alcohol, is suitable for various industrial production scenes, and has important industrial application value.

[0006] 2. Technical solutions

[0007] To solve the above problems, the present application adopts the following technical solutions.

[0008] A biosynthesis method based on multi-enzyme cascade reaction, comprising the following steps:

[0009] S1: adding ω-transaminase to racemic-α-phenethylamine until racemic-α-phenethylamine converts (S)-α-phenethylamine into phenylacetone under the action of ω-transaminase, leaving (R)-α-phenethylamine;

[0010] S2: adding aldehyde-ketone reductase to phenylacetone to reduce phenylacetone into (R)-α-phenethyl alcohol under the action of aldehyde-ketone reductase.

[0011] Further, glutamate dehydrogenase is added in S1 and S2 to maintain the cycle of coenzyme NADPH / NADP + and the co-substrate α-ketoglutarate / glutamate.

[0012] Further, the purification method of the ω-transaminase, aldehyde-ketone reductase and glutamate dehydrogenase comprises the following steps:

[0013] A: Related gene nucleic acid sequence acquisition, according to the NCBI database, the sequence of the gene is extracted, and primers are designed for polymerase chain reaction (PCR);

[0014] B: Construction of recombinant plasmid, using homologous recombination ligase to connect the polymerase chain reaction (PCR) product with the double enzyme cut pRSFDuet-1 plasmid, forming a transformation liquid, and the connected transformation liquid is transferred into E. coli DH5α competent cells to obtain a recombinant quality;

[0015] C: Construction and induction expression of engineering strain, the recombinant plasmid is transferred into E. coli BL21 competent cells to obtain the corresponding engineering strain, and the recombinant engineering bacteria are cultured to OD 600When the ratio of the volume of the culture medium to the volume of the bacterial cells is 0.6, the culture medium is allowed to stand and cool at 18°C for 30 minutes, and then 0.4 mM of an inducer (IPTG) is added to the culture medium, and the culture is induced at 18°C and at a rotation speed of 200 rpm for 24 hours, and then the fermentation broth is centrifuged at 4°C and at a centrifugal force of 8000 g to collect the bacterial cells, and the bacterial cells are resuspended in 20 mM of a sodium phosphate buffer for standby;

[0016] D: The recombinant protein is separated and purified by using an ultrasonic disrupter to disrupt the resuspended bacterial cells, centrifuging to collect the crude enzyme solution, filtering the obtained crude enzyme solution by using a 0.22 μm filter membrane, and selecting a HisTrap HP 1 mL affinity chromatography column for separation and purification, wherein the nickel column is first equilibrated with a binding solution, 5 mL of the crude enzyme solution is then loaded, the column is washed with six column volumes of the binding solution to elute the unbound protein, and finally the protein bound to the nickel column is linearly eluted with four column volumes of an elution solution, and the protein sample at the elution peak is collected and detected by SDS-PAGE to determine the separation and purification of each peak.

[0017] The 50 mL HiTrapTM desalting column is used to remove the high concentration of salt in the enzyme, the column is equilibrated with 20 mM of a sodium phosphate buffer at pH 7.4, 10 mL is loaded, and the protein is isocratically eluted with two column volumes of 20 mM of a sodium phosphate buffer, and the protein at the elution peak is collected to obtain the pure enzyme.

[0018] Further, the binding solution in the separation and purification of the recombinant protein is 0.5 M NaCl, 20 mM of a sodium phosphate buffer, 20 mM imidazole, and pH 7.4.

[0019] Further, the elution solution in the separation and purification of the recombinant protein is 0.5 M NaCl, 20 mM of a sodium phosphate buffer, 500 mM imidazole, and pH 7.4.

[0020] Further, the racemic-α-phenethylamine is catalytically generated into (R)-α-phenethylamine and (R)-α-phenethyl alcohol by a three-enzyme one-pot method.

[0021] Further, the racemic-α-phenethylamine is catalytically generated into (R)-α-phenethylamine and (R)-α-phenethyl alcohol by a three-enzyme co-expression engineering bacteria.

[0022] Further, the amino acid sequence of the ω-transaminase is shown in SEQ ID NO: 1.

[0023] Further, the amino acid sequence of the aldehyde-ketone reductase is shown in SEQ ID NO: 2.

[0024] Further, the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO: 3.

[0025] 3. Beneficial effects

[0026] Compared with the prior art, the present application has the advantages of:

[0027] (1) The present application obtains an omega-transaminase, an aldehyde-ketone reductase and a glutamate dehydrogenase from Proteus mirabilis, and uses genetic engineering means to obtain overexpressed enzymes or whole cells co-expressed with multiple enzymes, and uses one-pot method or whole cell catalysis to simultaneously synthesize (R)-α-phenethylamine and (R)-α-phenethyl alcohol from racemic-α-phenethylamine, which can be applied to various industrial production scenes and has important industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a schematic diagram of the multi-enzyme cascade reaction of the present application;

[0029] Figure 2 is a chiral analysis of α-phenethylamine in the sample of the present application;

[0030] Figure 3 is a chiral analysis of α-phenethyl alcohol in the sample of the present application. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application; obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application, and all other embodiments obtained by a person of ordinary skill in the art without creative labor based on the embodiments in the present application shall fall within the scope of protection of the present application.

[0032] Example 1:

[0033] Please refer to Figure 1-3 shown, a biosynthesis method based on multi-enzyme cascade reaction, comprising the following steps:

[0034] Step one: adding ω-transaminase to racemic-α-phenethylamine, until racemic-α-phenethylamine converts (S)-α-phenethylamine into phenyl ethanone under the action of ω-transaminase, leaving (R)-α-phenethylamine, and separating phenyl ethanone and (R)-α-phenethylamine;

[0035] Step two: adding aldehyde-ketone reductase to phenyl ethanone, so that phenyl ethanone is reduced to (R)-α-phenethyl alcohol under the action of aldehyde-ketone reductase;

[0036] Glutamate dehydrogenase is added in the step one and step two to maintain the cycle of coenzyme NADPH / NADP + and co-substrate α-ketoglutarate / glutamate in the whole reaction system.

[0037] Specifically, the amino acid sequences of ω-transaminase, aldehyde-keto reductase, and glutamate dehydrogenase are introduced here as follows:

[0038] The amino acid sequence of ω-transaminase is SEQ ID NO.1

[0039] MTPDDIAFDLRHIWHPYTSMSNPLPAYPIVSAKGVELTLANGKQLIDGMSSWWAAIHGYNHPELNTAVTEQLAAMSHVMFGGITHPPAVALCRKLLAITPTPLECI FLADSGSVAVEVAIKMALQYWQAKGEKRQRIVALKRGYHGDTFGAMSVCDPDNSMHSLYKGYLPNHLFVEAPKTGFYQPWDATDIDALRTTLAQHHQHIAAVMLEPI VQGAGGMRIYHPEYLTQARALCDEFNVLLIADEIATGFGRTGKLFACEHAGISPDIMCVGKALTGGYMTLSATLTTRHIADTISQGDAGCFMHGPTYMGNPLACAV ANASLSLLEQGHWVNQVAQIEDQLKTELLPLKQAKSVKDVRVLGAIGVVEMVEPVNMAKLQKYFVNEGVWIRPFGQLIYIMPPYIISPEKLTKLTQAIEKAVNLTN*

[0040] ω-transaminase gene sequence SEQ ID NO.4

[0041]

[0042] Aldoketoreductase amino acid sequence SEQ ID NO. 2

[0043] MSVPVLGLGTFRLKDDAVINSVKTALELGYRAIDTAQIYENEAAIGQAIAESGISRDALYITTKIWVENLSKDKLIPSLKQSLSDLQTDYVDLTLVHWPSPNDAVSVKEFMQELLAAKKMGLTREIGISNFTIPLMERAIAAVGVENIATNQIELSPYLQNHKVVNWAKQHGIHITSYMTLAYGKALKDETIQTIAKKHNATPAQIILAWAMNEGYSVIPSSTKRENLASNLLSVNIKLDSEDKQAIAKLDCHDRLVSPEGLAPHWD*

[0044] Aldoketoreductase gene sequence SEQ ID NO. 5

[0045] ATGAGTGTTCCCGTTTTAGGTCTTGGTACTTTTCGCTTAAAAGACGATGCGGTTATAAATTCTGTAAAAACTGCACTAGAACTAGGTTATCGTGCTATTGATACAGCACAAATTTATGAAAATGAAGCTGCTATAGGACAAGCTATTGCAGAAAGTGGTATATCTCGCGACGCGCTTTACATCACAACAAAAATTTGGGTAGAAAATTTAAGTAAAGATAAATTAATTCCTAGTTTGAAACAAAGCTTAAGTGATTTACAAACAGATTATGTTGACTTAACTCTAGTACACTGGCCATCGCCAAATGATGCAGTTTCTGTTAAAGAATTTATGCAGGAGTTATTAGCTGCGAAAAAGATGGGATTAACGCGAGAAATTGGTATTTCAAATTTTACCATTCCATTAATGGAACGAGCTATTGCTGCCGTTGGTGTTGAAAATATCGCAACAAACCAAATCGAATTATCACCTTATTTACAAAATCATAAAGTGGTAAATTGGGCTAAACAGCATGGCATTCATATTACATCTTATATGACTCTAGCTTACGGCAAAGCATTAAAAGATGAAACAATACAAACCATTGCTAAAAAACACAATGCAACACCAGCACAAATCATTTTAGCTTGGGCGATGAATGAAGGTTATTCTGTTATTCCATCTTCAACAAAACGTGAAAATTTAGCCAGTAATTTACTCAGCGTAAATATCAAATTGGATAGTGAAGATAAACAAGCGATTGCAAAGTTAGATTGTCATGATCGTTTAGTTAGCCCAGAAGGTTTAGCGCCTCATTGGGATTAA

[0046] 谷氨酸脱氢酶的氨基酸序列SEQIDNO.3

[0047] MNRSGSLSSFLEEVEKYNAHQPEYHQAVREVFTTLWPFLEKNPQYREQSLLERLVEPERIIQFRVCWLDDKGQVQVNRAWRVQFNSAIGPYKGGMRFHPSVNLSILKFLGFEQTFKNALTTLPMGGAKGGSDFNPKGRSHAEVMRFCQALMTELYRHLGADTDVPAGDIGVGAREVGFMAGmMKKLSNSNECVFTKGLSFGGSLIRPEATGYGLVYFAML KRHGMSLEGMRVAVSGAGNVAQTIEKCLELGAKVVTASDSGGTVVDEAGFTTEKLARLTEIKNNYGRIEEYAKEFGLTYLEGQQPWAVAVDIALPCATQNELDLDAAKVLIKNGVKAVAEGANMPTTIAADTAFIEAGVLFAPGKAANAGGVATSGLEMAQNAARLSWKAEKVDARLHHIMLDIHHACVNYGGEGKQTNYIQGANIAGFVKVADAQGVL*

[0048] 谷酸脱水酶的谷机地在SEQIDNO.6

[0049]

[0050] The amino acid sequence of the ω-transaminase is shown in SEQ ID NO. 1, the amino acid sequence of the aldehyde-ketone reductase is shown in SEQ ID NO. 2, and the amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO. 3;

[0051] Therefore, (R)-α-phenylethylamine and (R)-α-phenylethanol can be effectively synthesized through the above steps, and the synthesis efficiency is high.

[0052] Example 2:

[0053] Based on Example 1, a purification method of ω-transaminase, aldehyde-ketone reductase and glutamate dehydrogenase is disclosed, and the purification method comprises the following steps:

[0054] Step 1: Obtain the nucleic acid sequence of the related gene. According to the NCBI database, extract the sequence of the above-mentioned gene in P. mirabilis HI4320, and design primers for polymerase chain reaction (PCR);

[0055] The obtained primers are shown in Table 2.

[0056] Primers 5'-3' nucleotide sequence ω-AT-F TCATCATATGGAGCTCATGACACCCGATGATATTGCTTTTG ω-AT-R TAGACTGCAGGTCGACTTAACTGACAGCTGCAAAGGCTTG DkgA-F TCATCATATGGAGCTCATGAGTAATCCTAAATTGATCCAACTAT DkgA-R TAGACTGCAGGTCGACCTAGATATCAACAAAAGTATCAGGATC GDH-F TCATCATATGGAGCTCATGAATAGATCTGGCTCATTATCTTCG GDH-R TAGACTGCAGGTCGACTTACAGCACGCCTTGCGCTAACATTGC

[0057] Step 2: Construction of recombinant plasmid. Use homologous recombination ligase to connect the polymerase chain reaction (PCR) product and the double enzyme cut pRSFDuet-1 plasmid at a temperature of 50°C for 30 min. Transfer the connected transformation liquid into E. coli DH5α competent cells to obtain a recombinant plasmid.

[0058] Step 3: Construction of engineering strain and induction of expression. Transfer the recombinant plasmid into E. coli BL21 competent cells to obtain the corresponding engineering strain. Culture the recombinant engineering bacteria to OD 600 0.6, and then add an inducer (IPTG) with a final concentration of 0.4 mM to the culture medium. After that, induce the culture at a temperature of 18°C and a rotation speed of 200 rpm for 24 h. Centrifuge the fermentation liquid at a temperature of 4°C and a centrifugal force of 8000 g to collect the bacterial cells. Resuspend the bacterial cells with 20 mM sodium phosphate buffer (pH = 7.4) for standby.

[0059] The fourth step is the separation and purification of the recombinant protein. The resuspended bacteria are crushed by ultrasonic crusher, and the crude enzyme solution is collected by centrifugation (8000g, 4°C). The crude enzyme solution is filtered with a 0.22μm filter membrane and separated and purified using a HisTrapHP1mL affinity chromatography column. During purification, the nickel column is first equilibrated with a binding solution (0.5MNaCl, 20mM sodium phosphate buffer, 20mM imidazole, pH7.4); after injecting 5mL of the crude enzyme solution, the unbound protein is fully eluted with six column volumes of the binding solution, and finally the protein bound to the nickel column is linearly eluted with four times the sample volume of the eluent (0.5MNaCl, 20mM sodium phosphate buffer, 500mM imidazole, pH7.4). The protein samples at the elution peak are collected, and the separation and purification of each peak is detected by SDS-PAGE.

[0060] A 50 mL HiTrap™ desalting column was used to remove the higher concentration of salt in the enzyme. The column was equilibrated with 20 mM sodium phosphate buffer at pH 7.4, 10 mL of sample was loaded, and the protein was isocratically eluted with 2 column volumes of 20 mM phosphate buffer. The protein at the elution peak was collected to obtain pure enzyme.

[0061] Example 3:

[0062] Based on Example 1, a processing method for racemic-α-phenylethylamine is disclosed, wherein the racemic-α-phenylethylamine is catalyzed to produce (R)-α-phenylethylamine and (R)-α-phenylethanol by a three-enzyme one-pot process, specifically:

[0063] The reaction system is 10.0 mL, 20.0 mM rac-α-phenylethylamine, 0.5 mM NADP + , 0.5 mM PLP, 0.5 mM glutamate, 2.5 mg / mL ω-transaminase, 1.5 mg / mL aldehyde-keto reductase, 0.5 mg / mL glutamate dehydrogenase, 20 mM phosphate buffer, pH = 8.0, reaction temperature 40 ° C temperature conditions, the reaction was carried out for 10 h, the chirality of α-phenylethylamine and α-phenylethanol was analyzed by chiral HPLC, and the contents of (R)-α-phenylethylamine and (R)-α-phenylethanol were determined. The results are shown in Table 1.

[0064] Table 1 Enantiomeric excess values ​​of (R)-α-phenylethylamine and (R)-α-phenylethanol produced by one-pot process

[0065]

[0066] Example 4:

[0067] The difference between this embodiment and embodiment 3 is only the difference in numerical values, which are as follows:

[0068] Reaction system: 100.0 mL, 10 mM rac-α-phenethylamine, 0.5 mM NADP + , 1.0 mM PLP, 0.5 mM glutamic acid, 0.5-5.0 mg / mL ω-transaminase, 0.5-5.0 mg / mL aldehyde-ketone reductase, 0.5-2.0 mg / mL glutamate dehydrogenase, 20 mM phosphate buffer, pH = 8.0, reaction temperature 40°C, the chiralities of α-phenethylamine and α-phenethyl alcohol were analyzed by chiral HPLC, and the contents of (R)-α-phenethylamine and (R)-α-phenethyl alcohol were determined, and the results are shown in Table 2.

[0069] Table 2 Amount of (R)-α-phenethylamine and (R)-α-phenethyl alcohol produced by one-pot method under different conditions

[0070]

[0071] Example 5:

[0072] According to the method for constructing the engineering bacteria in Example 2, the following seven engineering bacteria were constructed: E. coli BL21 (pRSFDuet-at-dkga, pETDuet-gdh), E. coli BL21 (pRSFDuet-at-dkga, pCDFDuet-gdh), E. coli BL21 (pRSFDuet-at-dkga, pACYCduet-gdh), E. coli BL21 (pETDuet-at-dkga, pRSFDuet-gdh), E. coli BL21 (pCDFDuet-at-dkga, pRSFDuet-gdh), E. coli BL21 (pACYCduet-at-dkga, pRSFDuet-gdh), E. coli BL21 (pRSFDuet-dkga-at, pACYCduet-gdh). The expression of the engineering bacteria was referred to Example 1, and the whole cells of the engineering bacteria were obtained for catalyzing the reaction.

[0073] Example 6:

[0074] On the basis of the above examples, a method for whole cell catalysis of rac-α-phenethylamine to simultaneously synthesize (R)-α-phenethylamine and (R)-α-phenethyl alcohol is disclosed, which specifically is:

[0075] Reaction system: 10 mL, 20 mM rac-α-phenethylamine, 1.0 mM PLP, 1.0 mM L-glutamic acid, 20 mM phosphate buffer pH = 7.0, whole cell OD 600= 10.0, temperature 40°C, reaction time 10 h, the chiralities of α-phenethylamine and α-phenethyl alcohol were analyzed by chiral HPLC, and the results are shown in Table 2.

[0076] Table 3 Enantiomeric excess values of (R)-α-phenethylamine and (R)-α-phenethyl alcohol produced by different recombinant bacteria

[0077]

[0078] Example 7:

[0079] The difference between this example and Example 6 is only numerical difference, which is specifically:

[0080] The reaction system was 10 mL, 10-100 mM racemic-α-phenethylamine, 1.0 mM PLP, 1.0-5.0 mM L-glutamic acid, NADP + 0.5 mM, 20 mM phosphate buffer pH = 7.0, whole cell OD 600 = 5.0-25.0, temperature 40°C, the chiralities of α-phenethylamine and α-phenethyl alcohol were analyzed by chiral HPLC, and the contents of (R)-α-phenethylamine and (R)-α-phenethyl alcohol were determined, and the results are shown in Table 4.

[0081] Table 4 Amounts of (R)-α-phenethylamine and (R)-α-phenethyl alcohol produced by whole cell catalysis of racemic-α-phenethylamine

[0082]

[0083] Example 8:

[0084] The difference between this example and Example 3 is only that the racemic-α-phenethylamine is catalyzed to generate (R)-α-phenethylamine and (R)-α-phenethyl alcohol by the three-enzyme co-expression engineering bacteria, which is specifically:

[0085] The reaction system was 10 mL, 20 mM racemic-α-phenethylamine, 1.0 mM PLP, 1.0 mM L-glutamic acid, 20 mM phosphate buffer pH = 7.0, E. coli BL21 (pRSFDuet-at-dkga, pACYCduet-gdh) whole cell OD 600 = 10.0, temperature 40°C, reaction time 10 h, the chiralities of α-phenethylamine and α-phenethyl alcohol were analyzed by chiral HPLC, and the contents of (R)-α-phenethylamine and (R)-α-phenethyl alcohol were determined, and the results are shown in Table 5.

[0086] Table 5 Chiral analysis of (R)-α-phenethylamine and (R)-α-phenethyl alcohol synthesized by engineering bacteria catalysis of racemic-α-phenethylamine

[0087]

[0088] The above description is merely a preferred embodiment of the present invention; however, the scope of protection of the present invention is not limited thereto. Any person skilled in the art who, within the technical scope disclosed by the present invention, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present invention shall be covered by the scope of protection of the present invention.

Claims

1. A biosynthesis method based on a multi-enzyme cascade reaction, characterized in that: The following steps are included: S1: adding ω-transaminase to racemic-α-phenylethylamine, wherein the amino acid sequence of the ω-transaminase is shown in SEQ ID NO: 1; until the racemic-α-phenylethylamine is converted to ( S )-α-phenylethylamine is converted to acetophenone, leaving ( R )-α-phenylethylamine; S2: adding aldehyde-keto reductase to acetophenone, wherein the amino acid sequence of the aldehyde-keto reductase is shown in SEQ ID NO: 2; allowing acetophenone to be reduced to ( R )-α-phenylethanol.

2. A biosynthesis method based on a multi-enzyme cascade reaction according to claim 1, characterized in that: Glutamate dehydrogenase is added to both S1 and S2 to maintain the coenzyme NADPH / NADP + and the cycle of the cosubstrate α-ketoglutarate / glutamate.

3. A biosynthesis method based on a multi-enzyme cascade reaction according to claim 2, characterized in that: The method for purifying the ω-aminotransferase, aldehyde-keto reductase and glutamate dehydrogenase comprises the following steps: A: Obtain the nucleic acid sequence of the relevant gene. According to the NCBI database, extract the gene sequence and design primers for polymerase chain reaction; B: Construction of recombinant plasmid: Use homologous recombination ligase to ligate the polymerase chain reaction product with the double-enzyme-digested pRSFDuet-1 plasmid to form a transformation solution, which is then transformed into Escherichia coli DH5α competent cells to obtain the recombinant plasmid; C: Construction and induction of expression of engineering strains, transfer the recombinant plasmid into E. coli BL21 competent cells to obtain the corresponding engineering strains, and culture the recombinant engineering bacteria to OD 600 When the pH value is 0.6, the culture medium is allowed to cool at 18°C ​​for 30 min, an inducer is added to the culture medium at a final concentration of 0.4 mM, and then the culture medium is induced at 18°C ​​and 200 rpm for 24 h. The fermentation broth is centrifuged at 4°C and 8000 g to collect the cells, and the cells are resuspended in 20 mM sodium phosphate buffer for later use; D: Isolation and purification of recombinant protein: Use ultrasonic disruptor to disrupt the resuspended bacteria. After disruption, collect the crude enzyme solution by centrifugation. The obtained crude enzyme solution is filtered through a 0.22µm filter membrane and isolated and purified. During purification, the nickel column is first equilibrated with binding solution. After injecting 5 mL of crude enzyme solution, unbound proteins were fully eluted with six column volumes of binding buffer, and proteins bound to the nickel column were linearly eluted with four times the volume of eluent loaded. Protein samples at the elution peaks were collected and the separation and purification of each peak was detected by SDS-PAGE. Use a desalting column to remove the higher concentration of salt in the above enzyme, equilibrate the column with sodium phosphate buffer, load 10 mL of sample, and isocratically elute the protein with 2 times the column volume of 20 mM phosphate buffer, and collect the protein at the elution peak.

4. A biosynthesis method based on a multi-enzyme cascade reaction according to claim 3, characterized in that: The binding solution used in the separation and purification of the recombinant protein is 0.5 M NaCl, 20 mM sodium phosphate buffer, 20 mM imidazole, pH 7.

4.

5. The biosynthesis method based on a multi-enzyme cascade reaction according to claim 3, characterized in that: The eluent used in the separation and purification of the recombinant protein is 0.5 M NaCl, 20 mM sodium phosphate buffer, 500 mM imidazole, pH 7.

4.

6. The biosynthesis method based on a multi-enzyme cascade reaction according to claim 1, characterized in that: The racemic-α-phenylethylamine is catalyzed by a three-enzyme one-pot method ( R )-α-phenylethylamine and ( R )-α-phenylethanol.

7. The biosynthesis method based on a multi-enzyme cascade reaction according to claim 1, characterized in that: The racemic-α-phenylethylamine is catalyzed by constructing an engineering bacterium through the co-expression of three enzymes ( R )-α-phenylethylamine and ( R )-α-phenylethanol.

8. The biosynthesis method based on a multi-enzyme cascade reaction according to claim 2, characterized in that: The amino acid sequence of the glutamate dehydrogenase is shown in SEQ ID NO: 3.

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