Method for synthesizing L-threo-beta-hydroxy-homophenylalanine through six-enzyme cascade biocatalysis

Abstract

The invention discloses a method for efficiently producing L-threo-beta-hydroxy-high phenylalanine, and belongs to the technical field of biological engineering. According to the invention, a new route for efficient biosynthesis of L-threo-beta-hydroxy-homophenylalanine by using cheap benzaldehyde, glycine and threonine as raw materials and using a six-enzyme cascade catalytic reaction is designed, the reaction is carried out in a 50mL reaction system, and the conversion rate is as high as 92%. Compared with the existing main method for producing the L-threo-beta-hydroxy-high phenylalanine, the method disclosed by the invention has the advantages that the production cost of the L-threo-beta-hydroxy-high phenylalanine is greatly reduced, and the method has a very good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biosynthesis of L-threo-β-hydroxy-homophenylalanine, and in particular to a method for synthesizing L-threo-β-hydroxy-homophenylalanine by six-enzyme cascade biocatalysis, belonging to the technical field of bioengineering. Background Art

[0002] L-threo-β-hydroxy-homophenylalanine is an important pharmaceutical intermediate, and its derivative L-threo-4-nitro-β-hydroxy-homophenylalanine is an important intermediate in the synthesis of the new antibiotic obafluorin.

[0003] At present, the main production method of L-threo-β-hydroxy-homophenylalanine is chemical synthesis, but this method has a low yield and high energy consumption, and it is also toxic to the environment, which does not meet the requirements of green production, safe production and sustainable development.

[0004] The preparation of L-threo-β-hydroxy-homophenylalanine by bioenzymatic method has the characteristics of stable and safe product quality, mild process conditions, high efficiency and environmental protection, which can reduce environmental and resource pressure. Therefore, there is an urgent need for an effective biological method to efficiently produce L-threo-β-hydroxy-homophenylalanine. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention designs a method for synthesizing L-threo-β-hydroxy-homophenylalanine by six-enzyme cascade biocatalysis. The specific technical scheme is as follows:

[0006] The method for synthesizing L-threo-β-hydroxy-homophenylalanine by six-enzyme cascade biocatalysis of the present invention comprises constructing a two-enzyme recombinant engineering bacterium and a four-enzyme recombinant engineering bacterium, taking benzaldehyde, glycine and threonine as substrates, and efficiently biosynthesizing L-threo-β-hydroxy-homophenylalanine by six-enzyme cascade catalysis reaction of L-threonine aldolase (L-TA for short), phenylserine dehydratase (PSDH for short), phenylpyruvate decarboxylase (PPDC for short), L-threonine transaldolase (L-TTA for short), acetaldehyde dehydrogenase (ALDH for short) and flavin reductase (FR for short).

[0007] Furthermore, the six-enzyme cascade catalytic reaction of the present invention includes a phenylpyruvate synthesis module, an L-threo-β-hydroxy-homophenylalanine synthesis module and an acetaldehyde elimination module. The pyruvate synthesis module includes two-step reactions, as follows:

[0008] 1) L-TA catalyzes the aldol condensation reaction of benzaldehyde and glycine to produce phenylserine;

[0009] 2) PSDH catalyzes phenylserine to produce phenylpyruvate.

[0010] The method for synthesizing L-threo-β-hydroxy-homophenylalanine is characterized in that the L-threo-β-hydroxy-homophenylalanine synthesis module includes two-step reactions, as follows:

[0011] 1) PPDC catalyzes the non-oxidative decarboxylation of phenylpyruvate to produce phenylacetaldehyde; in the entire six-enzyme cascade reaction process, this step is the key rate-limiting step of the reaction system.

[0012] 2) L-TTA catalyzes the transaldehyde reaction of L-threonine and phenylacetaldehyde to synthesize L-threo-β-hydroxy-homophenylalanine.

[0013] The acetaldehyde elimination module includes two steps of reaction, as follows:

[0014] 1) ALDH oxidizes acetaldehyde to acetic acid;

[0015] 2) FR catalyzes NAD + Regeneration reaction, establish a coenzyme circulation system, and improve the working efficiency of acetaldehyde elimination reaction. Furthermore, the acetic acid product generated in the module and the inorganic salt in the reaction system produce salt, thereby avoiding affecting the pH value of the reaction system.

[0016] Furthermore, the phenylpyruvate synthesis module is completed in a two-enzyme recombinant engineering bacterium, which can co-express L-TA and PSDH, and synthesize phenylpyruvate using benzaldehyde and glycine as reaction substrates;

[0017] The L-threo-β-hydroxy-homophenylalanine synthesis module and the acetaldehyde elimination module are completed in a four-enzyme recombinant engineering bacterium, which can co-express PPDC, L-TTA, ALDH and FR, wherein the L-threo-β-hydroxy-homophenylalanine synthesis module catalyzes phenylpyruvate to generate L-threo-β-hydroxy-homophenylalanine with PPDC and L-TTA; the acetaldehyde elimination module efficiently catalyzes acetaldehyde to acetic acid with ALDH and FR, eliminates the by-product acetaldehyde in the reaction system, and improves the synthesis conversion rate of L-threo-β-hydroxy-homophenylalanine.

[0018] Furthermore, the L-TA is derived from PsTA of Pseudomonas sp., the PSDH is derived from PxPSDH of Paraburkholderia xenovorans, and the PPDC is derived from PfPPDC of Pseudomonas fluorescens; the L-TTA is derived from PfTTA of Pseudomonas fluorescens; the ALDH is derived from PtALDH of Pichiaterricola; and the FR is derived from MgFR of Mycobacterium goodii.

[0019] The amino acid sequence of the PsTA is as follows:

[0020] SEQ ID NO.1

[0021] MTDQSQQFASDNYSGICPEAWAAMEKANHGHERAYGDDQWTARAADHFRKLFETDCEVFFAFNGTAANSLALSSLCQSYHSVICSETAHVETDECGAPEFFSNGSKLLTARSEGGKLTPASIREVALKRQDIHYPKPRVVTITQATEVGSVYRPDELKAISATCKELGLNLHM DGARFSNACAFLGCTPAELTWKAGIDVLCFGGTKNGMAVGEAILFFNRKLAEDFDYRCKQAGQLASKMRFLSAPWVGLLEDGAWLRHAAHANHCAQLLSSLVADIPGVELMFPVEANGVFLQMSEPALEALRNKGWRFYTFIGSGGARFMCSWDTEEARVRELAADIRAVMSA

[0022] The amino acid sequence of the PxPSDH is as follows:

[0023] SEQ ID NO.2

[0024] MSTAAPQHTDHTIDGEPIPTLDDIAAQHFALTPWVARTPVFDRLDFPSLEGTLVNFKFELLQAGGSFKARGAFTNLLALDESQRSAGVTCVSGGNHAVAVAYAAMRLGISAKVVLFRAANPARVALCRQYRADIVFAENIAEAFELVRRIEAEEGRYFVHPFNGYRTVLGSATLGYEWATQTPDLEAVIVPIGGGGLAAGVATAMRLANPNVHLYGVEPEGADVMGKSFAANHTVRMGKMHGIADSLMSPHTEEYSYELCRRHIDQLVTVSDDQLRAAMLTLFGQLKLAVEPACAAATAALLGPLREQLQGKRVGVLLCGTNTDPVTFAAHIERARHSESLFPQ

[0025] The amino acid sequence of the PfPPDC is specifically as follows:

[0026] SEQ ID NO.3

[0027] MTNLPSTHINLTSEEVSLGDLVGRVLVESGIDDLFCIPGDFTMQLSRELLTTPGLALRTMSHEYGTTLAALGYAVGKGVPGAVCFTYGVGVLNATNAIAQAYVERVPLLVFSGSPGTRERQAPLFLHHTIVDHQTQYRIMKEITVHQVCVTDPHQVLEQLREAVALAVLHSRPVYIEIPRDLFQARVRYSPARRVPLEPSTRYSQAARQAAELAYALVRKARDPVFVPGLDLKRRGLTDLAMRVCERLAMPWVATPMSRGGIPVSHPNYRGIYAGPASPSRVTRELLAKCDVLMLIGEPNSDVNMGIASHIAKGRLIHADDGKISVGRQHFNASTAEFLIAFSDVIHNAKTPLAPLTETQKDFIVPTPASLYPSEESPLTPFDIINELNRHFVTQPDTQLVVDCGDVFFMSLGMFPADVLTSPLYMSMGIAVPGAMGYQLGTGKRPIVLVGDGAFHMTGNELMRAAKFGLSPIVIVLNNQRWASLSSDAADIALTEQMPMSFSAAGQFLQVQAFTATTGRELRQHLEEALNMDRPVLIDAQVDPSKRSYLCERFFDAVKGQQHLPKA

[0028] The amino acid sequence of the PfTTA is specifically as follows:

[0029] SEQ ID NO.4

[0030] MSNVKQQTAQIVDWLSSTLGKDHQYREDSLSLTANENYPSALVRLTSGSTAGAFYHCSFPFEVPAGEWHFPEPGHMNAIADQVRDLGKTLIGAQAFDWRPNGGSTAEQALMLAACKPGEGFVHFAHRDGGHFALESLAQKMGIEIFHLPVNPTSLLIDVAKLDEMVRRNPHIRIVILDQSFKLRWQPLAEIRSVLPDSCTLTYDMSHDGGLIMGGVFDSPLSCGADIVHGNTHKTIPGPQKGYIGFKSAQHPLLVDTSLWVCPHLQSNCHAEQLPPMWVAFKEMELFGRDYAAQIVSNAKTLARHLHELGLDVTGESFGFTQTHQVHFAVGDLQKALDLCVNSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMKEKDFEVVARFIADLYFKKTEPAKVAQQIKEFLQAFPLAPLAYSFDNYLDEELLAAVYQGAQR

[0031] The amino acid sequence of the PtALDH is specifically as follows:

[0032] SEQ ID NO.5

[0033] MLRTATRTTFKSAQPSFMAAAAALRYYSHYPLSKSITLPNGKVYEQPTGLFINGEFVASQQHKTFEVINPSNEDEICHVYEARTEDVDAAVDAAYNAFHSEWSKMDPSIRGEHLMKLAELMEKNKDTLAAI ESMDNGKALFMAEIDVKLVINYLKYCAGWADKLFGKVVDTGSNYFNYIKREPIGVCGQIIPWNFPLLMWSWKVGPALAAGNTIVLKTAESTPLSALYAAKLAKEAGIPDGVINIVSGFGKITGEAISTHPK IKKLAFTGSTATGKHIMKAAAESNLKKVTLELGGKSPNIVFNDADIKKAVNNLILGIFFNSGEVCCAGSRVFIQEGVYDQVLEEFKIAAEALKVGNPFEEGVFQGAQTSQQQLTKILGYVESGKDEGATLV TGGERLGDKGYFVKPTIFADVKPNMKIYSEEIFGPFAVVTKFKTADEAIAMANDSEYGLAAGIHTTSLDTATYVANNLEAGTVWINTYNDFHHNMPFGGFKQSGIGREMGEAAFENYTQWKTVRIAINDGPQ

[0034] The amino acid sequence of the MgFR is as follows:

[0035] SEQ ID NO.6

[0036] MSATDLSPTSLREAFGHFPSGVIAIAAEVDGTRVGLAASTFVPVSLEPPLVAFCVQNSSTTWPKLKDLPSLGISVLGEAHDTAARTLAAKTGDRFAGLETESRDSGAVFINGTSVWLESAIEQLVPAGDHTIVVLRVSDIVINEAVPPIVFHRSAFRKLGA

[0037] Furthermore, when L-threo-β-hydroxy-homophenylalanine is biosynthesized by the constructed two-enzyme recombinant engineering bacteria and four-enzyme recombinant engineering bacteria, the reaction conditions are as follows:

[0038] In a 50mL reaction system, pH 7.0-8.0, reaction temperature 30°C, whole cell catalyst concentration selected 20g / L-50g / L, 20mM benzaldehyde, 200mM glycine and 20mM threonine as substrates, after a six-enzyme cascade catalytic reaction, 18.4mM L-threo-β-hydroxy-homophenylalanine was finally obtained, and the conversion rate reached 92%.

[0039] Furthermore, the reaction substrate benzaldehyde can also be a benzaldehyde derivative. By controlling the type of the derivative of the substrate benzaldehyde, the conversion of the corresponding L-threo-β-hydroxy-homophenylalanine is achieved. The specific conversion rate is shown in the following table:

[0040] Table 1 The corresponding products and conversion rates when benzaldehyde derivatives are used as substrates

[0041]

[0042]

[0043] The beneficial effects of the present invention are:

[0044] The present invention designs a six-enzyme cascade reaction to synthesize L-threo-β-hydroxy-homophenylalanine based on the transaldehyde reaction of threonine and aromatic aldehydes. This path includes multiple catalytic reactions. L-threo-β-hydroxy-homophenylalanine is synthesized from benzaldehyde, glycine and threonine through reactions such as aldol condensation, dehydration, decarboxylation, transaldehyde and coenzyme regeneration. In the process of generating L-threo-β-hydroxy-homophenylalanine, an equal amount of by-product acetaldehyde is generated. Acetaldehyde may inhibit the generation of products and cause environmental pollution. Therefore, an acetaldehyde elimination reaction is additionally introduced, and a coenzyme cycle is added here to improve the working efficiency of the elimination reaction.

[0045] The invention constructs a new route for efficiently biocatalyzing L-threo-β-hydroxy-homophenylalanine by using cheap benzaldehyde, glycine and threonine as raw materials and utilizing a six-stage enzyme method. The pathway enzyme is constructed into Escherichia coli, and the obtained recombinant Escherichia coli is utilized for catalysis. The reaction is carried out in a 50 mL reaction system, and the conversion rate of L-threo-β-hydroxy-homophenylalanine reaches 92%, thereby greatly reducing the production cost of L-threo-β-hydroxy-homophenylalanine and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the synthetic pathway diagram of L-threo-β-hydroxy-homophenylalanine.

[0047] Figure 2 This is a diagram of the functional modules of the two-enzyme cascade in the synthesis pathway of L-threo-β-hydroxy-homophenylalanine.

[0048] Figure 3 This is a diagram of the functional modules of the four-enzyme cascade in the synthesis pathway of L-threo-β-hydroxy-homophenylalanine.

[0049] Figure 4 This is a cellular schematic diagram of the synthesis pathway of L-threo-β-hydroxy-homophenylalanine.

[0050] Figure 5 This is the protein expression diagram of PsTA-PxPSDH two enzymes in engineered bacteria.

[0051] Figure 6 This is the protein expression diagram of PfPPDC-PfTTA enzyme engineering bacteria.

[0052] Figure 7 This is the protein expression diagram of the four-enzyme engineering bacteria PfPPDC-PfTTA-PtALDH-MgFR.

[0053] Figure 8 This is the reaction time detection chart of L-threo-β-hydroxy-homophenylalanine synthesis

[0054] Fig. 9 This is the NMR identification image of L-threo-β-hydroxy-homophenylalanine DETAILED DESCRIPTION

[0055] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0056] Example 1: Synthesis of L-threo-β-hydroxy-homophenylalanine

[0057] like Figure 1 As shown, the technical solution is based on the transaldehyde reaction of threonine and aromatic aldehydes, and designs a cascade reaction to synthesize L-threo-β-hydroxy-homophenylalanine. This pathway includes multi-step catalytic reactions, and L-threo-β-hydroxy-homophenylalanine is synthesized from benzaldehyde, glycine and threonine through reactions such as aldol condensation, dehydration, decarboxylation, transaldehyde and coenzyme cycle.

[0058] An equal amount of acetaldehyde is produced in the process of generating L-threo-β-hydroxy-homophenylalanine. Acetaldehyde may inhibit the formation of products and cause environmental pollution. Therefore, an additional acetaldehyde elimination reaction is introduced, and a coenzyme cycle is added here to improve the working efficiency of the elimination reaction.

[0059] The specific synthesis pathway steps of L-threo-β-hydroxy-homophenylalanine are as follows:

[0060] In the first step, L-TA catalyzes the aldol condensation reaction of benzaldehyde and glycine to form phenylserine; L-TA is a type of PLP-dependent enzyme that can catalyze the aldol condensation of glycine and various types of aromatic aldehydes to form a CC bond in one step, producing a β-hydroxy-α-amino acid with two chiral centers.

[0061] In the second step, PSDH catalyzes phenylserine to generate phenylpyruvate; PSDH can eliminate the chiral group NH2 on Cα and the chiral group OH on Cβ to generate phenylpyruvate. Figure 2 As shown, the first step and the second step are coupled together to form a two-enzyme cascade reaction system.

[0062] In the third step, PPDC catalyzes the non-oxidative decarboxylation of phenylpyruvate to produce phenylacetaldehyde, which is the key rate-limiting step of the entire six-enzyme cascade reaction system.

[0063] In the fourth step, L-TTA catalyzes the transaldehyde reaction of L-threonine and phenylacetaldehyde to synthesize L-threo-β-hydroxy-homophenylalanine. This step is crucial to the entire synthetic pathway and can produce a single-configuration L-threo-β-hydroxy-homophenylalanine and a single-configuration L-threo-β-hydroxy-4-nitro-homophenylalanine.

[0064] In the fifth step, ALDH catalyzes the oxidation of acetaldehyde to acetic acid, eliminating the by-product acetaldehyde produced by the reaction system.

[0065] In the sixth step, FR catalyzes the NAD+ regeneration reaction, establishes a coenzyme circulation system, and improves the efficiency of the acetaldehyde elimination reaction. Figure 3 As shown, the third to sixth steps are coupled to form a four-enzyme cascade reaction system.

[0066] like Figure 4 As shown, the two-enzyme cascade reaction system performs a catalytic reaction in one carrier cell, and the four-enzyme cascade reaction system performs a catalytic reaction in one carrier cell.

[0067] Example 2: Enzyme selection

[0068] (1) L-TA enzyme

[0069] The L-threonine aldolase selected was L-TA from Pseudomonas sp., which has a relatively high conversion rate and a broad substrate spectrum, and is very suitable for the constructed six-enzyme cascade reaction system.

[0070] (2) PSDH enzyme

[0071] There are few PSDHs reported in the literature that can catalyze phenylserine. Currently, the known sources include Paraburkholderiaxenovorans, Corynebacteriumglutamicum, etc. Among the PSDHs from the above sources, the PxPSDH from Paraburkholderiaxenovorans has the highest conversion rate of phenylserine, which is 99.9%, so the present invention selects PxPSDH from this source.

[0072] (3) PPDC enzyme

[0073] In the six-enzyme cascade reaction, PPDC catalyzes the non-oxidative decarboxylation of phenylpyruvate to produce phenylacetaldehyde. This step is the key rate-limiting step of the entire six-enzyme cascade reaction system, and the catalytic efficiency of this enzyme determines the effect of the reaction system.

[0074] Through literature search, the bacterial source information of PPDC was obtained, including Azospirillum brasilense, Achromobacter eurydice, Enterobacter sp., Lactococcus lactis, Saccaromycescerevisiae, Proteus mirabilis, Pseudomonas fluorescens, Pseudomonas putida, etc. After experimental screening, it was finally found that PfPPDC derived from Pseudomonas fluorescens had the highest catalytic efficiency, so the present invention preferably uses PfPPDC to synthesize phenylacetaldehyde as the third enzyme of the six-enzyme cascade reaction.

[0075] (4) TTA enzyme

[0076] At present, there are relatively few studies on L-TTA. The sources reported in the literature mainly include Pseudomonasfluorescens, Pseudomonas sp., Neptunomonas marine, etc. In the present invention, PfTTA from Pseudomonasfluorescens is selected as the research object.

[0077] (5) ALDH enzyme

[0078] In the literature research on ALDH, it was found that most acetaldehyde elimination systems use ADH to reduce acetaldehyde to ethanol, but ethanol is also toxic to cells. Therefore, the present invention uses PtALDH derived from Pichia terricola to oxidize acetaldehyde to acetic acid, which can not only pull the reaction balance, but also reduce the damage to cells and is more environmentally friendly. In addition, the PtALDH selected in the present invention can only catalyze acetaldehyde, but not phenylacetaldehyde, which is conducive to the realization of high conversion efficiency of the six-enzyme cascade reaction system of the present invention.

[0079] (6) FR enzyme

[0080] In terms of FR selection, after literature research and experimental screening, the present invention selects MgFR from Mycobacterium goodii, which has high FR activity and is beneficial to NAD + regeneration.

[0081] Table 2 Enzyme selection

[0082]

[0083] Example 3: Construction of two enzyme recombinant engineering strains

[0084] The primer sequences for PsTA gene amplification are as follows:

[0085] PsTA-F: CATGCCATGGGCATGACCGACCAGTCTCAGCAGTTC;

[0086] PsTA-R: CGGGATCCTTATCAAGCAGACATAACAGCACGGAT.

[0087] The primer sequences for PxPSDH gene amplification are as follows:

[0088] PxPSDH-F:GGGTTTCATATGTCAACTGCCGCGCCGCAAC;

[0089] PxPSDH-R:CCCTCGAGTTATTGAGGAAACAGTGACTCGCTATGCCGC.

[0090] The PsTA gene was amplified by PCR using primers PsTA-F and PsTA-R, and was constructed between the restriction sites Nco I and BamH I of pACYCDuet, pCDFDuet, pETDuet and pRSFDuet by restriction ligation to obtain the recombinant plasmids PsTA-pACYCDuet, PsTA-pCDFDuet, PsTA-pETDuet and PsTA-pRSFDuet. On this basis, the PxPSDH gene was amplified with primers PxPSDH-F and PxPSDH-R, and constructed between the restriction sites Nde I and Xho I of PsTA-pACYCDuet, PsTA-pCDFDuet, PsTA-pETDuet, and PsTA-pRSFDuet by restriction ligation to obtain the recombinant plasmids of PsTA-PxPSDH-pACYCDuet, PsTA-PxPSDH-pCDFDuet, PsTA-PxPSDH-pETDuet, and PsTA-PxPSDH-pRSFDuet.

[0091] The above recombinant plasmid was transformed into E.coli BL21(DE3) and verified by colony PCR to obtain recombinant engineered bacteria PsTA-PxPSDH-pACYCDuet-E.coli BL21(DE3), PsTA-PxPSDH-pCDFDuet-E.coli BL21(DE3), PsTA-PxPSDH-pETDuet-E.coli BL21(DE3) and PsTA-PxPSDH-pRSFDuet-E.coli BL21(DE3), as shown in Table 3.

[0092] The supernatant and precipitate after ultrasonic disruption of the engineered bacterial cells in Table 3 were selected, and the expression of PsTA and PxPSDH was detected by SDS-PAGE protein gel electrophoresis. E. coli BL21 (DE3) without the target gene was used as the blank control group. The protein expression results are shown in Figure 5 As shown, 1, 3, 5, and 7 are the precipitates of PsTA-PxPSDH; 2, 4, 6, and 8 are the supernatants of PsTA-PxPSDH; 9 is the molecular weight standard protein; 10 is the blank control; 11 is the supernatant of PxPSDH; and 12 is the supernatant of PsTA.

[0093] Compared with the control group, protein bands at around 38kDa and 37kDa appeared in the supernatant of the four engineered bacteria, which were consistent with the protein sizes of PsTA and PxPSDH, respectively, indicating that the two enzyme engineered bacteria of PsTA-PxPSDH were all successfully expressed.

[0094] Table 3 Information of engineering strains for the co-expression of two enzymes

[0095]

[0096] Example 4: Construction of four-enzyme recombinant engineering strain

[0097] The primers for amplification of the PfPPDC gene are as follows:

[0098] PfPPDC-F: CATGCCATGGCGACCAACCTGCCGAGTACCCA;

[0099] PfPPDC-R:CGCGGATCCTGCTTTAGGCAGATGCTGCT.

[0100] The primers for amplification of the PfTTA gene are as follows:

[0101] PfTTA-F: CGCCATATGATGTCTAACGTTAAACAGCAGACCGC;

[0102] PfTTA-R: CCGCTCGAGACGCTGAGCACCCTGGTAAACAG.

[0103] The amplification primers for the PtALDH gene are as follows:

[0104] PtALDH-F: TAAGAAGGAGATATACATATGCATATGAGCGATAAAATTATTCACCTG;

[0105] PtALDH-R: GGTTTCTTTACCAGACTCGAGCTGCGGACCGTCGTTGATA.

[0106] The amplification primers for the MgFR gene are as follows:

[0107] MgFR-F: CATGCCATGGGCATGTCTGCTACCGACCTGTCTCCG;

[0108] MgFR-R: CGCGGATCCAGCACCCAGTTTACGGAAAGCAGA.

[0109] The PfTTA gene was amplified by PCR using primers PfTTA-F and PfTTA-R, and constructed between the restriction sites Nco I and BamH I of pACYCDuet, pCDFDuet, pETDuet and pRSFDuet by restriction ligation to obtain the recombinant plasmids PfTTA-pACYCDuet, PfTTA-pCDFDuet, PfTTA-pETDuet and PfTTA-pRSFDuet. On this basis, the PfPPDC gene was amplified using primers PfPPDC-F and PfPPDC-R, and constructed between the restriction sites Nde I and Xho I of PfTTA-pACYCDuet, PfTTA-pCDFDuet, PfTTA-pETDuet, and PfTTA-pRSFDuett by restriction digestion and ligation to obtain the recombinant plasmids of PfPPDC-PfTTA-pACYCDuet, PfPPDC-PfTTA-pCDFDuet, PfPPDC-PfTTA-pETDuet, and PfPPDC-PfTTA-pRSFDuet.

[0110] The MgFR gene was amplified by PCR using primers MgFR-F and MgFR-R, and constructed between the restriction sites Nco I and BamH I of pACYCDuet, pCDFDuet, pETDuet and pRSFDuet by restriction ligation to obtain the recombinant plasmids of MgFR-pACYCDuet, MgFR-pCDFDuet, MgFR-pETDuet and MgFR-pRSFDuet. On this basis, the PtALDH gene was amplified with primers PtALDH-F and PtALDH-R, and constructed between the restriction sites Nde I and Xho I of MgFR-pACYCDuet, MgFR-pCDFDuet, MgFR-pETDuet and MgFR-pRSFDuet by restriction digestion and ligation to obtain the recombinant plasmids of PtALDH-MgFR-pACYCDuet, PtALDH-MgFR-pCDFDuet, PtALDH-MgFR-pETDuet and PtALDH-MgFR-pRSFDuet.

[0111] Four engineering bacteria, PtALDH-MgFR-pACYCDuet-E.coli BL21(DE3), PtALDH-MgFR-pCDFDuet-E.coliBL21(DE3), PtALDH-MgFR-pETDuet-E.coli BL21(DE3) and PtALDH-MgFR-pRSFDuet-E.coli BL21(DE3), were prepared into competent cells. The recombinant plasmids of PfPPDC-PfTTA-pACYCDuet, PfPPDC-PfTTA-pCDFDuet, PfPPDC-PfTTA-pETDuet and PfPPDC-PfTTA-pRSFDuet were transformed into the above competent cells, respectively, to obtain 12 recombinant engineering bacteria, as shown in Table 4.

[0112] The supernatant and precipitate of PfPPDC-PfTTA-pRSFDuet-E.coli BL21(DE3), PfPPDC-PfTTA-pETDuet-E.coli BL21(DE3), PfPPDC-PfTTA-pCDFDuet-E.coli BL21(DE3), and PfPPDC-PfTTA-pACYCDuet-E.coli BL21(DE3) engineered bacterial cells after ultrasonic disruption were selected, and the expression of PfPPDC and PfTTA was detected by SDSPAGE protein gel electrophoresis. E.coli BL21(DE3) without the target gene was used as the blank control group. The results are as follows Figure 6 As shown,

[0113] Among them, 1 is a blank control; M is a molecular weight standard protein; 2, 4, 6, and 8 are the precipitates of PfPPDC-PfTTA; 3, 5, 7, and 9 are the supernatants of PfPPDC-PfTTA; 10 is the supernatant of PfPPDC; and 11 is the supernatant of PfTTA.

[0114] Compared with the control group, protein bands at around 62 kDa and 48 kDa appeared in the supernatant of the four engineered bacteria, which were consistent with the protein sizes of PfPPDC and PfTTA, respectively, indicating that all four engineered bacteria of PfPPDC-PfTTA were successfully expressed.

[0115] The four recombinant plasmids of PfPPDC-PfTTA were assembled with the four recombinant plasmids of PtALDH-MgFR to obtain 12 co-expression strains of module 2b, as shown in Table 4:

[0116] Table 4 Four-enzyme recombination co-expression engineering strain information

[0117]

[0118] The expression of the 12 co-expression strains of 2b was detected by SDS-PAGE, and E. coli BL21 (DE3) without the target gene was used as the blank control group. The results are as follows Figure 7 As shown, M is the molecular weight standard protein; 1 is the blank control; 2 is the supernatant of 2b 01; 3 is the precipitate of 2b 01; 4 is the supernatant of 2b 02; 5 is the precipitate of 2b 02; 6 is the supernatant of 2b 03; 7 is the precipitate of 2b 03; 8 is the supernatant of 2b 04; 9 is the precipitate of 2b 04; 10 is the supernatant of 2b 05; 11 is the precipitate of 2b 05; 12 is the supernatant of 2b 06; 13 is the precipitate of 2b 06; 14 is the blank control; 15 is the supernatant of 2b 07; 16 is the precipitate of 2b 07; 17 is the supernatant of 2b 08; 18 is the precipitate of 2b 08; 19 is the supernatant of 2b 09; 20 is the precipitate of 2b 09; 21 is the supernatant of 2b 10; 22 is the supernatant of 2b 10 2b is the precipitate of 2b 10; 23 is the supernatant of 2b 11; 24 is the precipitate of 2b 11; 25 is the supernatant of 2b 12; 26 is the precipitate of 2b 12.

[0119] Compared with the control group, protein bands appeared at around 62kDa, 48kDa, 75kDa and 17kDa in the supernatant of the 12 engineered bacteria, which were consistent with the protein sizes of PfPPDC, PfTTA, PtALDH and MgFR, respectively, indicating that these four enzymes were successfully expressed in all 12 engineered bacteria.

[0120] Example 5: 50 mL conversion experiment of L-threo-β-hydroxy-homophenylalanine

[0121] The first stage reaction: 20mM benzaldehyde, 200mM glycine, 0.1mM PLP, 10% DMSO (v / v), 20g / LPsTA-PxPSDH two-enzyme recombinant engineering bacteria, 100mM Tris-HCl (pH 8.0), 200rpm, 30℃ conversion. After the reaction is completed, the bacteria are removed by centrifugation and the reaction liquid is collected.

[0122] Second stage reaction: Add 60mM L-threonine, 0.1mM PLP, 0.2mM ThDP, and 20g / L PfPPDC-PfTTA-PtALDH-MgFR four-enzyme engineering bacteria to the collected reaction solution, and transform at 200rpm and 30℃. After the reaction, collect the supernatant and detect the content of the product L-threo-β-hydroxyhomophenylalanine by HPLC. Figure 8As shown, the reaction system was monitored over a 13-hour period. At 1 hour, module 2a had converted 20 mM benzaldehyde into phenylpyruvate. After 10 hours, module 2b converted 20 mM phenylpyruvate into 18.9 mM L-threo-β-hydroxy-homophenylalanine and no longer increased.

[0123] In order to further identify the structure of the purified product, high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy were finally selected to detect and analyze the purified product. The theoretical molecular weight of L-threo-β-hydroxyhomophenylalanine is 195, and the product peak consistent with the theoretical molecular weight was obtained by high-resolution mass spectrometry. When using nuclear magnetic resonance to identify the product structure, D2O solution was used to dissolve the sample for nuclear magnetic resonance detection, such as Fig. 9 As shown, the final test results show that the molecular weight and structure of the product are correct, verifying the accuracy of the multi-enzyme cascade reaction constructed by the present invention.

[0124] In this reaction system, 20 mM benzaldehyde was biocatalyzed by the constructed PsTA-PxPSDH two-enzyme cascade reaction system to ultimately obtain 20 mM phenylpyruvate, which was used as the starting substrate for the PfPPDC-PfTTA-PtALDH-MgFR four-enzyme cascade reaction system to ultimately obtain 18.4 mM L-threo-β-hydroxy-homophenylalanine, with a conversion rate of 92%.

Claims

1. A method for synthesizing L-threo-β-hydroxy-homophenylalanine by six-enzyme cascade biocatalysis, characterized in that: The method constructs a two-enzyme recombinant engineering bacterium and a four-enzyme recombinant engineering bacterium, takes benzaldehyde, glycine and threonine as substrates, and efficiently biosynthesizes L-threo-β-hydroxy-homophenylalanine through a six-enzyme cascade catalytic reaction of L-TA, PSDH, PPDC, L-TTA, ALDH and FR.

2. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 1, characterized in that: The six-enzyme cascade catalytic reaction includes a phenylpyruvate synthesis module, an L-threo-β-hydroxy-homophenylalanine synthesis module and an acetaldehyde elimination module.

3. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 2, characterized in that: The phenylpyruvate synthesis module includes two-step reactions, which are as follows: 1) L-TA catalyzes aldol condensation of benzaldehyde and glycine to generate phenylserine; 2) PSDH catalyzes phenylserine to generate phenylpyruvate.

4. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 2, characterized in that: The L-threo-β-hydroxy-homophenylalanine synthesis module includes two-step reactions, which are as follows: 1) PPDC catalyzes the non-oxidative decarboxylation of phenylpyruvate to generate phenylacetaldehyde, which is the key rate-limiting step of the entire six-enzyme cascade reaction system; 2) L-TTA catalyzes the transaldehyde reaction of L-threonine and phenylacetaldehyde to synthesize L-threo-β-hydroxy-homophenylalanine.

5. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 2, characterized in that: The acetaldehyde elimination module includes two-step reactions, which are as follows: 1) ALDH oxidizes acetaldehyde to acetic acid, consuming the byproduct acetaldehyde in the reaction system; 2) FR catalyzes the NAD+ regeneration reaction, establishes a coenzyme circulation system, and improves the working efficiency of the acetaldehyde elimination reaction.

6. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 2, characterized in that: The phenylpyruvate synthesis module is completed in a two-enzyme recombinant engineering bacterium, which can co-express L-TA and PSDH, and catalyze the synthesis of phenylpyruvate using benzaldehyde and glycine as reaction substrates; the L-threo-β-hydroxy-homophenylalanine synthesis module and the acetaldehyde elimination module are completed in a four-enzyme recombinant engineering bacterium, which can co-express PPDC, L-TTA, ALDH and FR, wherein the L-threo-β-hydroxy-homophenylalanine synthesis module catalyzes phenylpyruvate to generate L-threo-β-hydroxy-homophenylalanine with PPDC and L-TTA, and the acetaldehyde elimination module efficiently catalyzes acetaldehyde to become acetic acid with ALDH and FR, eliminates the by-product acetaldehyde in the reaction system, and improves the synthesis conversion rate of L-threo-β-hydroxy-homophenylalanine.

7. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 1, characterized in that: The L-TA is derived from PsTA of Pseudomonas sp., the PSDH is derived from PxPSDH of Paraburkholderia xenovorans, the PPDC is derived from PfPPDC of Pseudomonas fluorescens; the L-TTA is derived from PfTTA of Pseudomonasfluorescens; the ALDH is derived from PtALDH of Pichia terricola; and the FR is derived from MgFR of Mycobacterium goodii.

8. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 7, characterized in that: The amino acid sequence of the PsTA is shown in SEQ ID NO.1; the amino acid sequence of the PxPSDH is shown in SEQ ID NO.2; the amino acid sequence of the PfPPDC is shown in SEQ ID NO.3; the amino acid sequence of the L-TTA is shown in SEQ ID NO.4; the amino acid sequence of the PtALDH is shown in SEQ ID NO.5; and the amino acid sequence of the MgFR is shown in SEQ ID NO.

6.

9. The method for synthesizing L-threo-β-hydroxy-homophenylalanine as claimed in claim 1, characterized in that: By constructing two-enzyme recombinant engineering bacteria and four-enzyme recombinant engineering bacteria, in a 50mL reaction system, pH 7.0-8.0, reaction temperature 30°C, whole cell catalyst concentration selected as 20g / L-50g / L, catalyzing 20mM benzaldehyde, finally 18.4mM L-threo-β-hydroxy-homophenylalanine was obtained, and the conversion rate reached 92%.

10. The method for synthesizing L-threo-β-hydroxy-homophenylalanine according to claim 1, characterized in that: The substrate benzaldehyde can also be a benzaldehyde derivative, the structural formula of the benzaldehyde derivative is: Wherein R is fluorine, chlorine, bromine, nitro and methyl substitution at the ortho, meta and para positions, and trifluoromethyl and methylsulfone substitution at the para position; when the substrate is a benzaldehyde derivative, the L-threo-β-hydroxy-R-homophenylalanine product is obtained through a six-enzyme cascade catalytic reaction.

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