A mutant of L-threonine aldolase and its efficient synthesis of chloramphenicol intermediates

Abstract

The present invention discloses a strain of L-threonine transaldolase mutant and its efficient synthesis of chloramphenicol intermediate, belonging to the field of biocatalytic engineering. The present invention obtains L-threonine transaldolase mutant F70C / N268S with improved catalytic ability. The mutant shows excellent conversion rate and diastereomeric selectivity for L-threo-p-nitrophenylserine. The present invention analyzes the molecular mechanism of improved catalytic performance of mutant F70C / N268S based on structural analysis, and provides a theoretical basis for the catalytic performance of mutant enzymes. Subsequently, acetaldehyde, a byproduct produced in the catalytic reaction of L-threonine transaldolase, is introduced into a byproduct elimination system, and the multi-enzyme "one-pot method" whole-cell catalytic reaction is optimized, and the final L-threo-p-nitrophenylserine production reaches 233.5mM (a total of 63.3g / L), and the de value is ≥99%, which is the highest level reported at home and abroad.

Description

Technical Field

[0001] The invention relates to an L-threonine aldolase mutant and an efficient chloramphenicol intermediate synthesized therefrom, belonging to the field of biocatalysis engineering. Background Art

[0002] β-Hydroxy-α-amino acids (βHAAs) are indispensable intermediates in the synthesis of some drugs because they contain two chiral centers, Cα and Cβ. They are widely used in natural product synthesis, peptide research, drug development, agricultural prevention, etc. For example, L-thero-p-nitrophenylserine is an important intermediate in the synthesis of chloramphenicol. Chloramphenicol binds to the 30S ribosomal subunit of bacteria, which can prevent the extension of the peptide chain and thus block protein synthesis. Chloramphenicol is a broad-spectrum antibiotic that has a bactericidal effect on a variety of bacteria and occupies an important position in the fields of medicine and agriculture. In the field of medicine, chloramphenicol is used to treat bacterial infections of the respiratory tract, urinary tract and skin. In the field of agriculture, chloramphenicol is used to prevent and treat animal infections.

[0003] At present, βHAA mainly relies on chemical synthesis. For example, chloramphenicol usually adopts the p-nitroacetophenone method. This method involves multiple steps, including nitration, oxidation, bromination, salt formation, hydrolysis, acetylation, addition, reduction, decomposition, splitting and dichloroacetylation of ethylbenzene. The synthesis steps are cumbersome, the yield is low (<30%), there are many by-products, the product stereoselectivity is poor, and it is not environmentally friendly. The biological synthesis of chloramphenicol intermediates has the advantages of high efficiency and low cost, and has gradually become a research hotspot.

[0004] The key to the bioenzymatic synthesis of βHAAs lies in the formation of CC bonds at two chiral centers. The enzymes reported so far include L-threonine aldolase (EC 4.1.2.5), L-threonine transaldolase (EC 2.2.1.2) and serine hydroxymethyltransferase (EC 2.1.2.1). These enzymes can reconstruct new chiral centers to achieve asymmetric synthesis of βHAAs. Among the three enzymes, L-threonine transaldolase has become the focus of current research due to its excellent natural stereoselectivity. L-threonine transaldolase from various sources has been characterized for its good natural stereoselectivity. Some related patents, for example, the use of an L-threonine aldolase in the synthesis of a chiral intermediate of florfenicol recorded in publication number CN 110540977A, a method for preparing chloramphenicol recorded in publication number CN 111662937 A, a method for enzymatic synthesis of a chloramphenicol intermediate recorded in publication number CN 111662937 A, and a method for preparing 3-phenyl-L-serine or its derivatives and ethyl esters recorded in publication number CN 108323173 A, report the application of L-threonine aldolase in the catalytic synthesis of βHAAs.

[0005] L-threonine aldolase uses L-threonine and various benzaldehyde derivatives as substrates to undergo aldol condensation reactions, followed by the reconstruction of CC bonds to synthesize βHAAs. The chloramphenicol intermediate L-threo-p-nitrophenylserine is produced by the condensation of L-threonine and p-nitrobenzaldehyde using L-threonine aldolase as a catalyst. During the reaction, the industrial properties of natural catalysts are not satisfactory, so improving the catalytic efficiency and stereoselectivity of the catalyst is extremely critical for the efficient synthesis of L-threo-p-nitrophenylserine. Summary of the invention

[0006] The present invention uses L-threonine aldolase from Pseudomonas sp. as a template enzyme, and obtains an excellent recombinant L-threonine aldolase strain through rational design. The enzymatic properties of the L-threonine aldolase and the conditions for the optimal catalytic reaction are characterized in detail. At the same time, the recombinant L-threonine aldolase is transformed by protein engineering technology, and the whole cell catalytic synthesis of L-threo-p-nitrophenylserine is carried out, and the conversion reaction conditions are optimized, which significantly improves the synthesis efficiency and stereoselectivity of the product βHAA. At a scale of 50L, the chloramphenicol intermediate L-threo-p-nitrophenylserine is efficiently synthesized, laying a solid foundation for the industrial and efficient preparation of βHAA.

[0007] The present invention provides an L-threonine aldolase mutant, wherein the mutant is obtained by mutating at least one of positions 70 and 268 of the L-threonine aldolase; the L-threonine aldolase is an L-threonine aldolase having an amino acid sequence as shown in SEQ ID NO.1, or an L-threonine aldolase having a sequence identity of at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, with the sequence as shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the L-threonine transaldolase mutant comprises one or more amino acid substitutions at positions 70 / 268, wherein the amino acid at position 70 is substituted with C, and the amino acid at position 268 is substituted with S.

[0009] In one embodiment of the present invention, the catalytic efficiency of the L-threonine aldolase mutant is measured by using L-threonine and p-nitrobenzaldehyde as substrates for a whole-cell reaction and detecting the product peak area by HPLC.

[0010] The present invention provides an L-threonine aldolase mutant, wherein the L-threonine aldolase mutant is obtained by simultaneously mutating the 70th and / or 268th positions of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1.

[0011] SEQ ID NO.1 sequence is:

[0012] MKQDESNVGPVIDWLAQTLGQDYKYRQDTLSLTANENYPSELVRLTSGSTAGAFYHCSFPFPLGEWHFPEPGQMNEIADDLRGLAKRMMGAQAFDWRPNGGSPAEQAL MLAACKQGEGFVHFAHRDGGHFALEQLATKMGIEIFHLPVDPQSLLIDVAKLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAILPDSCTLTYDMSHDGGLILGGVFDSP LACGADIAHGNTHKTIPGPQKGFIAFKSAQHPLLVETSLWVCPHLQSNCHAELLPSMWAAFKEMEAFGPAYAHQMVRNAKALANQLHELGLNVSGESFGFTETHQVHFAV GDLQQALSMCVDSLHAGGIRSTNIEIPGKPGMHGIRLGVQAMTRRGMKEDDFRRVAGLIADLYFKRTEPARVASKVKELLGDFPLAPLAYSFDQQIDESRRRLLERGIQR

[0013] In one embodiment of the present invention, the nucleotide sequence encoding the L-threonine aldolase parent enzyme is shown as SEQ ID NO.2.

[0014] SEQ ID NO.2:

[0015]

[0016] The invention provides an L-threonine aldolase mutant. The L-threonine aldolase mutant is a mutant obtained by mutating the L-threonine aldolase with an amino acid sequence as shown in SEQ ID NO.1: C57A, C57Q, F59A, F70A, F70H, F70T, F70N, F70C, F70D, N268S, N268T, N268C, N268Q, F59A / F70C, F59A / N268S, F70C / N268S or F59A / F70C / N268S.

[0017] In one embodiment of the present invention, the mutant is obtained by mutating the cysteine ​​at position 57 of the L-threonine transaldolase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and is named C57A;

[0018] Or the mutant is obtained by mutating the cysteine ​​at position 57 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to glutamine, and is named C57Q;

[0019] Or the mutant is obtained by mutating the phenylalanine at position 59 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and is named F59A;

[0020] Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and is named F70A;

[0021] Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to histidine, and is named F70H;

[0022] Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to threonine, and is named F70T;

[0023] Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to asparagine, and is named F70N;

[0024] Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine transaldolase with the amino acid sequence as shown in SEQ ID NO.1 to cysteine, and is named F70C;

[0025] Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence shown in SEQ ID NO.1 to aspartic acid, and is named F70D;

[0026] Or the mutant is obtained by mutating the asparagine at position 268 of the L-threonine aldolase in the amino acid sequence as shown in SEQ ID NO.1 to serine, and is named N268S;

[0027] Or the mutant is obtained by mutating the asparagine at position 268 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to threonine, and is named N268T;

[0028] Or the mutant is obtained by mutating the asparagine at position 268 of the L-threonine aldolase in the amino acid sequence as shown in SEQ ID NO.1 to cysteine, and is named N268C;

[0029] Or the mutant is obtained by mutating the asparagine at position 268 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to glutamine, and is named N268Q;

[0030] Or the mutant is obtained by mutating the phenylalanine at position 59 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and mutating the phenylalanine at position 70 to cysteine, and is named F59A / F70C;

[0031] Or the mutant is obtained by mutating the phenylalanine at position 59 of the L-threonine aldolase with the amino acid sequence shown in SEQ ID NO.1 to alanine, and mutating the asparagine at position 268 to serine, and is named F59A / N268S;

[0032] Or the mutant is obtained by mutating the phenylalanine at position 70 to cysteine ​​and the asparagine at position 268 to serine in the L-threonine transaldolase with the amino acid sequence as shown in SEQ ID NO.1, and is named F70C / N268S;

[0033] Or the mutant is obtained by mutating the phenylalanine at position 59 to alanine, the phenylalanine at position 70 to cysteine, and the asparagine at position 268 to serine of the L-threonine transaldolase with the amino acid sequence as shown in SEQ ID NO.1, and is named F59A / F70C / N268S;

[0034] The present invention also provides a gene encoding the mutant.

[0035] The present invention also provides a recombinant vector carrying the mutant or the gene.

[0036] In one embodiment of the present invention, the recombinant vector is an expression vector using pET series vectors such as pET-28a plasmid, pET-21a plasmid, pRSF series vectors such as pRSF-Duet1 plasmid, and pGEX series vectors such as pGEX-6p-1 plasmid.

[0037] In one embodiment of the present invention, the recombinant vector uses pET28a plasmid, pRSF-Duet1 plasmid, pET21a plasmid or pGEX-6P-1 plasmid as an expression vector.

[0038] The present invention also provides a recombinant cell expressing the mutant, carrying the gene, or carrying the recombinant vector.

[0039] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0040] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0041] The invention provides a recombinant vector carrying L-threonine aldolase.

[0042] In one embodiment of the present invention, the recombinant vector uses pET28a, pRSF-Duet1, pET-Duet1, pACY-Duet1, pET21a or pGEX-6P-1 as an expression vector.

[0043] The present invention also provides the enzymatic properties of the engineered L-threonine transaldehydease, including optimum temperature, pH, temperature stability, pH stability and metal ion dependence.

[0044] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned L-threonine transaldolase mutant sequence, which is any one of the following forms:

[0045] (1) culturing the recombinant expression transformant and isolating transformant cells containing the recombinase;

[0046] (2) culturing the recombinant expression transformant, isolating transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid;

[0047] (3) culturing the recombinant expression transformant, isolating transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a lyophilized enzyme powder.

[0048] The invention provides a recombinant Escherichia coli, which simultaneously over-expresses the mutant, alcohol dehydrogenase derived from Saccharomyces mikatae and formate dehydrogenase derived from Candida boidinii.

[0049] In one embodiment of the present invention, the recombinant Escherichia coli uses pRSF-Duet1 as an expression vector, and simultaneously overexpresses the above mutant, alcohol dehydrogenase from Saccharomyces mikatae, and formate dehydrogenase from Candidaboidinii.

[0050] In one embodiment of the present invention, the recombinant Escherichia coli uses pGEX-6P-1 as an expression vector to express the above mutant, and at the same time, uses pRSF-Duet1 as an expression vector to express alcohol dehydrogenase from Saccharomyces mikatae and formate dehydrogenase from Candida boidinii.

[0051] In one embodiment of the present invention, the nucleotide sequence encoding the alcohol dehydrogenase is shown as SEQ ID NO.3, and the nucleotide sequence encoding the formate dehydrogenase is shown as SEQ ID NO.4.

[0052] The present invention also provides a method for improving the enzymatic activity of L-threonine aldolase on a substrate of p-nitrobenzaldehyde, the method comprising: mutating the cysteine ​​at position 57 of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1 to glutamine; or mutating the phenylalanine at position 59 of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1 to alanine; or mutating the phenylalanine at position 70 of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1 to alanine, histidine, threonine, asparagine, cysteine ​​or aspartic acid; or mutating the asparagine at position 268 of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1 to serine, threonine, cysteine ​​or glutamine; or mutating the phenylalanine at position 59 of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1 to alanine, and mutating the phenylalanine at position 70 to cysteine; or mutating the phenylalanine at position 70 of the L-threonine aldolase of the amino acid sequence as shown in SEQ ID NO.1 to cysteine. The phenylalanine at position 59 of the L-threonine aldolase shown in NO.1 is mutated to alanine, and the asparagine at position 268 is mutated to serine; or the phenylalanine at position 70 of the L-threonine aldolase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to cysteine, and the asparagine at position 268 is mutated to serine; or the phenylalanine at position 59 of the L-threonine aldolase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, the phenylalanine at position 70 is mutated to cysteine, and the asparagine at position 268 is mutated to serine.

[0053] The present invention also provides a method for preparing L-threo-p-nitrophenylserine, wherein L-threonine is used as a donor substrate, p-nitrobenzaldehyde is used as an acceptor substrate, and L-threonine transaldehydease having an amino acid sequence as shown in SEQ ID NO.1 or the above-mentioned recombinant Escherichia coli whole cell is used for transformation to prepare the chloramphenicol intermediate L-threo-p-nitrophenylserine.

[0054] In one embodiment of the present invention, the structural formula of p-nitrobenzaldehyde is:

[0055]

[0056] In one embodiment of the present invention, the structural formula of L-threo-p-nitrophenylserine is:

[0057]

[0058] In one embodiment of the present invention, the L-threo-p-nitrophenylserine is obtained by whole-cell biocatalysis of recombinant engineered L-threonine transaldolase using L-threonine as a donor substrate and p-nitrobenzaldehyde as an acceptor substrate. The general reaction formula is as follows:

[0059]

[0060] In one embodiment of the present invention, the reaction system further contains pyridoxal phosphate PLP, NAD + , MgCl2, sodium formate.

[0061] In one embodiment of the present invention, the concentration of p-nitrobenzaldehyde is 170 mM to 250 mM.

[0062] In one embodiment of the present invention, the concentration of L-threonine is 250 mM to 750 mM.

[0063] In one embodiment of the present invention, the NAD + The concentration is 0.1mM~1.25mM.

[0064] In one embodiment of the present invention, the concentration of sodium formate is 150 mM to 350 mM.

[0065] In one embodiment of the present invention, the reaction conditions are pH 6.0-8.0 and temperature 20°C-40°C.

[0066] In one embodiment of the present invention, the whole cell catalytic system of the L-threonine transaldolase includes but is not limited to 100 mM L-threonine, 50 mM p-nitrobenzaldehyde, 1 mM magnesium chloride (MgCl2), 0.1 mM pyridoxal phosphate (PLP), 20 mg / mL whole cells, and 10% ethyl acetate as a cosolvent. The reaction buffer is 50 mM ammonium formate solution (pH 7.5), the reaction time is 8 hours, and the reaction temperature is 25 to 35°C, such as 30°C.

[0067] The present invention also provides a "one-pot" reaction system coupled with an acetaldehyde elimination system, wherein the acetaldehyde elimination system is achieved by coupling alcohol dehydrogenase and formate dehydrogenase, wherein the alcohol dehydrogenase is mainly used to eliminate the byproduct acetaldehyde in the L-threonine transaldolase reaction, and the formate dehydrogenase is mainly responsible for the cofactor NADH / NAD + The general structural formula of the reaction is as follows:

[0068]

[0069] In one embodiment of the present invention, the combination of the three enzymes is as follows: the three enzymes are constructed into a vector pRSF-Duet1 by genetic engineering technology, and then transformed into competent cells E. coli BL21 (DE3); or alcohol dehydrogenase and formate dehydrogenase are constructed into a vector pRSF-Duet1, and an engineered L-threonine transaldolase is constructed into a vector pGEX-6P-1, and then the two vectors are co-transformed into competent cells E. coli BL21 (DE3).

[0070] The present invention also provides an optimized "one-pot" reaction system for efficiently synthesizing L-threo-p-nitrophenylserine. The whole-cell catalytic system includes but is not limited to 300 mM L-threonine, 250 mM p-nitrobenzaldehyde, 1 mM magnesium chloride (MgCl2), 0.1 mM pyridoxal phosphate (PLP), NAD + 1mM, 300mM sodium formate, 10% ethyl acetate as a cosolvent, 30mg / mL whole cells. The reaction buffer is 50mM ammonium formate solution (pH 7.5), the reaction time is 6 hours, and the reaction temperature is 25-35°C, such as 35°C.

[0071] The present invention also provides an enlarged 50L scale reaction system, which comprises but is not limited to 300mM L-threonine, 250mM p-nitrobenzaldehyde, 1mM magnesium chloride (MgCl2), 0.1mM pyridoxal phosphate (PLP), and NAD + 1mM, 300mM sodium formate, 10% ethyl acetate as a cosolvent, 30mg / mL whole cells. The reaction buffer is 50mM ammonium formate solution (pH 7.5). The reaction mixture is reacted in a 100L reactor at a speed of 300rpm for 6 hours at a reaction temperature of 25-35°C, such as 35°C.

[0072] The present invention also provides the use of the above-mentioned L-threonine aldolase mutant or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned recombinant Escherichia coli, or the above-mentioned method in the preparation of the chloramphenicol intermediate L-threo-p-nitrophenylserine or a product containing the above-mentioned chloramphenicol intermediate L-threo-p-nitrophenylserine.

[0073] In one embodiment of the present invention, the product is a chemical.

[0074] Beneficial Effects

[0075] (1) The present invention uses the existing L-threonine aldolase sequence as a template to mutate the relevant mutation sites, thereby obtaining an L-threonine aldolase mutant with significantly improved catalytic efficiency and stereoselectivity.

[0076] (2) The present invention studied the enzymatic properties of L-threonine transaldolase mutants F59A, F70C, N268S or F70C / N268S, and determined the optimal temperature, pH and metal ion dependence of the enzyme.

[0077] (3) Based on homology modeling and molecular docking analysis, the present invention analyzes the molecular mechanism of improved catalytic performance of L-threonine transaldolase mutants F59A, F70C, N268S or F70C / N268S.

[0078] (4) The present invention introduces a multi-enzyme coupling system for acetaldehyde elimination reaction to construct a "one-pot" reaction system. After optimizing the multi-enzyme catalytic reaction conditions, the multi-enzyme reaction catalyzes the synthesis of the chloramphenicol intermediate L-threo-p-nitrophenylserine with further improved yield and stereoselectivity in a 50L scale reaction system.

[0079] (5) The method of the present invention is used to construct a "one-pot" reaction system to obtain L-threo-p-nitrophenylserine with a yield of 233.5 mM (a total of 63.3 g / L), and the diastereomeric selectivity (de value) is greater than 99%. This yield is the highest yield reported for enzyme catalysis so far. The whole-cell biocatalytic reaction is completed in one step, does not require cumbersome steps, does not generate unnecessary by-products, has mild reaction conditions, is environmentally friendly, and is a green and efficient method for the biosynthesis of chloramphenicol intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 : HPLC detection diagram of L-threo-p-nitrophenylserine; wherein, a is the L-threo-p-nitrophenylserine standard product; b is the detection of L-threo-p-nitrophenylserine in the conversion solution.

[0081] Figure 2 : Protein gel images of the purification of wild type and mutant F70C / N268S; wherein 1 represents the wild type and 2 represents the mutant F70C / N268S.

[0082] Figure 3 :Study on the enzymatic properties of wild-type L-threonine aldolase and engineered L-threonine aldolase F70C / N268S; where a is the optimum temperature; b is the optimum pH; c is the pH stability; d is the temperature stability; and e is the metal ion dependence.

[0083] Figure 4: Analysis of hydrogen bond interactions between L-threo and p-nitrophenylserine in the active center of PslLTTA; wherein, a is the hydrogen bond interaction analysis between L-threo and p-nitrophenylserine in the active center of wild-type PslLTTA; b is the hydrogen bond interaction analysis between L-threo and p-nitrophenylserine in the active center of mutant F70C / N268S; wherein L-threo-p-nitrophenylserine is represented by green sticks, PLP is represented by lines, and the mutation site C70 is represented by red sticks.

[0084] Figure 5 : Analysis of hydrogen bond interaction between PslLTTA active center and PLP; wherein, a is the interaction analysis between wild-type PslLTTA active center and PLP; b is the hydrogen bond interaction analysis between mutant F70C / N268S active center and PLP; wherein L-threo-p-nitrophenylserine is represented by green lines, PLP is represented by green sticks, L-threo-p-nitrophenylserine is represented by green lines, and the mutation site S268 is represented by red sticks.

[0085] Figure 6 : Optimization of conditions for the efficient catalytic synthesis of L-threo-p-nitrophenylserine by multi-enzyme "one-pot" method; where a is the concentration of p-nitrobenzaldehyde; b is the ratio of p-nitrobenzaldehyde to L-threonine; c is the NAD + concentration; d is the sodium formate concentration; e is the pH; f is the temperature. DETAILED DESCRIPTION

[0086] Technical terms:

[0087] β-Hydroxy-α-amino acids: As key synthetic building blocks for many natural products and drugs, they have high application value. For example, L-threo-phenylserine can be used as a potential intermediate in the synthesis of cyclosporin A, which has antiviral, antibiotic and antimalarial activities, especially for the treatment of tuberculosis. L-threo-3,4-dihydroxyphenylserine (Droxidopa) is currently used to treat Parkinson's disease; L-threo-p-nitrophenylserine is the main component of chloramphenicol; methylsulfonylphenylserine is a key intermediate in the synthesis of chloramphenicol methylsulfone. L-threo-2-amino-3-hydroxy-1,6-hexanedicarboxylic acid is a potential precursor for the synthesis of carbocyclic-β-lactams and nucleotides. L-threo-2-amino-3-hydroxyadipic acid can be used to synthesize the immunosuppressant lipid mycestericin D. In addition, β-hydroxy-α-amino acids are also often used as chiral auxiliary reagents in organic synthesis and asymmetric catalysis research, and are important components of some chiral ligands.

[0088] Expression: The term "expression" includes any step involved in the production of the L-threonine transaldolase mutant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0089] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding the L-threonine transaldolase mutant of the present invention and operably linked to control sequences that provide for its expression.

[0090] Fragment: The term "fragment" means a polypeptide that lacks one or more (e.g., several) amino acids at the amino and / or carboxyl terminus of the polypeptide; wherein the fragment has L-threonine transaldolase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% of the number of amino acids 1 to 440 (i.e., not including the length of the zymogen region sequence) shown in SEQ ID NO.1.

[0091] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0092] The host cell can be any cell useful in the recombinant production of L-threonine transaldolase mutants, such as a prokaryotic cell or a eukaryotic cell.

[0093] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Mycobacterium, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0094] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0095] The culture medium involved in the following examples is as follows:

[0096] LB liquid medium (g / L): peptone 10, yeast powder 5, sodium chloride 10, natural pH.

[0097] The primer sequences involved in the following examples are shown in Table 1:

[0098] Table 1: Primer design for L-threonine transaldolase mutant enzyme gene

[0099]

[0100] The detection method involved in the following embodiments: The liquid phase detection method for the production of L-threo-p-nitrophenylserine is:

[0101] The o-phthalaldehyde (OPA) / N-acetyl-L-cysteine ​​(NAC) derivatization method was used. First, 20 mg of OPA and NAC were taken and then dissolved in 4 mL of boric acid buffer (0.2 M, pH 9.8) and 1 mL of pure acetonitrile to form a derivatizing agent. The supernatant of the whole-cell catalyzed conversion solution was appropriately diluted and pre-column derivatized by a liquid phase procedure. The mobile phase was a mixture of 50 mM potassium dihydrogen phosphate buffer (pH 8.0) and acetonitrile in a ratio of 79:21. The detection conditions were set at a flow rate of 1 mL / min, a column temperature of 45 °C, and a detection wavelength of 334 nm. Figure 1 As shown, the peak appearance time of L-threo-p-nitrophenylserine is 13.6 min, while the peak appearance time of L-erythro-p-nitrophenylserine is 17.5 min. The yield is calculated by comparing the peak area of ​​L-threo-p-nitrophenylserine with the standard curve, thereby determining the yield of the product.

[0102] Product yield = product content / total amount of substrate added.

[0103] The formula for calculating the diastereomeric selectivity (de) value of L-threo-p-nitrophenylserine is:

[0104] de=(peak area of ​​L-threo-p-nitrophenylserine+peak area of ​​L-erythro-p-nitrophenylserine) / (peak area of ​​L-threo-p-nitrophenylserine-peak area of ​​L-erythro-p-nitrophenylserine)×100%.

[0105] Example 1: Acquisition of L-threonine aldolase mutants and whole-cell catalytic verification

[0106] The specific steps are as follows:

[0107] (1) Construction of pGEX-PslLTTA

[0108] The L-threonine transaldolase PslLTTA (nucleotide sequence as shown in SEQ ID NO.2) derived from Pseudomonas sp. was connected to the plasmid pGEX-6p-1 (which can be sent to the company for synthesis) and named pGEX-PslLTTA.

[0109] (2) Construction of recombinant vector containing mutants

[0110] First, pGEX-PslLTTA was used as a template and C57A-F, C57A-R, C57Q-A, C57Q-R, F59A-F, F59A-R, F70A-F, F70A-R, F70H-F, F70H-R, F70T-F, F70T-R, F70N-F, F70N-R, F70C-F, F70C-R, F70D-F, F70D-R, N268 S-F, N268S-R, N268T-F, N268T-R, N268C-F, N268C-R, N268Q-F, N268Q-R and other primers were used for amplification to obtain recombinant vectors containing single point mutants C57A, C57Q, F59A, F70A, F70H, F70T, F70N, F70C, F70D, N268S, N268T, N268C, and N268Q;

[0111] Construction of double mutants: Taking the preparation of a recombinant vector containing mutants F70C / N268S as an example, the preparation method is to use the F70C mutant as a template and amplify with primers N268S-F and N268S-R to obtain a recombinant vector containing mutants F70C / N268S.

[0112] According to the above method, a recombinant vector containing mutants F59A / F70C, F59A / N268S, or F59A / F70C / N268S was constructed.

[0113] (3) Construction of recombinant strains

[0114] The recombinant vector containing the mutant and the recombinant vector containing the wild-type L-threonine transaldolase obtained in step (2) were transformed into E. coli BL21 (DE3) competent cells for whole-cell catalysis verification after sequencing verification.

[0115] Preparation of recombinant strains:

[0116] E.coli BL21(DE3) / pGEX-PslLTTA, E.coli BL21(DE3) / pGEX-C57A, E.coli BL21(DE3) / pGEX-C57Q, E.coli BL21(DE3) / pGEX-F59A, E.coli BL21(DE3) / pGEX-F70A, E.coli BL21(DE3) / pGEX-F70H, E.coli BL21(DE3) / pGEX-F70T, E.coli BL21(DE3) / pGEX-F70N, E.coli BL21(DE3) / pGEX-F70C, E.coli BL21(DE3) / pGEX-F70D、E.coli BL21(DE3) / pGEX-N268S, E.coli BL21(DE3) / pGEX-N268T, E.coli BL21(DE3) / pGEX-N268C, E.coli BL21(DE3) / pGEX-N268Q, E.coli BL21(DE3) / pGEX-F59A / F70C, E.coli BL21(DE3) / pGEX-F59A / N268S, E.coli BL21(DE3) / pGEX-F70C / N268S, E.coli BL21(DE3) / pGEX-F59A / F70C / N268S.

[0117] (4) Whole cell validation

[0118] The recombinant strain obtained in step (1) is further subjected to whole-cell biocatalysis using p-nitrobenzaldehyde and L-threonine as substrates. The specific steps are as follows:

[0119] Preparation of whole cell catalyst:

[0120] The obtained recombinant strains were cultured in LB liquid medium containing 100 μg / mL ampicillin for 6 to 8 h. 600 When the value reaches 0.6-0.8, IPTG is added at a final concentration of 0.2 mM for induction. The induction temperature is 25°C and the induction time is 16-18 h. The recombinant cell fluid is then centrifuged at 8000ⅹg for 10 min, the supernatant is discarded, and the cell pellet obtained is the whole-cell catalyst.

[0121] Whole cell catalytic reaction:

[0122] The prepared whole cell catalyst 30 mg / mL and 10% ethyl acetate were added to the reaction system containing 100 mM L-threonine, 50 mM p-nitrobenzaldehyde, 1 mM magnesium chloride (MgCl2), and 0.1 mM pyridoxal phosphate (PLP) as a cosolvent. The reaction system also contained a buffer solution of 50 mM ammonium formate solution (pH 7.5), and the reaction was carried out in a shaking table at a speed of 200 rpm, a temperature setting of 30°C, and a reaction time of 8 h. The content and de value of the target product were detected by liquid phase, and the results are shown in Table 2 below.

[0123] Table 2: Product yields and diastereomeric selectivity of L-threonine aldolase mutants

[0124]

[0125] As shown in Table 2, the yield and diastereomeric selectivity of the mutated L-threonine transaldolase for the synthesis of L-threo-p-nitrophenylserine were improved. Among them, the single mutants F59A, F70C, and N268S at the three sites had the strongest ability to catalyze p-nitrobenzaldehyde. Therefore, the three sites were randomly combined and mutated to obtain the double mutant F70C / N268S, which further enhanced the synthesis ability. The whole cell catalysis was 8 hours, the product yield was 83.5%, and the de value was 99.3%. The mutant F70C / N268S was subsequently studied.

[0126] Example 2: Study on the enzymatic properties of the double mutant F70C / N268S

[0127] The specific steps are as follows:

[0128] 1. Expression and purification of mutant F70C / N268S

[0129] (1) The recombinant bacteria containing the mutant F70C / N268S obtained in Example 1 were inoculated into LB liquid culture medium and cultured at 37°C for 8 to 10 hours to obtain a seed solution. The obtained seed solution was inoculated into LB culture medium at an inoculum amount of 1% (v / v) for protein expression. After culturing at 25°C for 6 to 8 hours, 0.2 mM IPTG was added, and the culture was continued at 20°C for 18 to 20 hours. The bacterial solution was centrifuged at high speed at 4°C to prepare recombinant cells (wet cells) of the mutant F70C / N268S.

[0130] (2) The recombinant E. coli wet cells prepared in step (1) were resuspended in Tris-NaCl buffer (50 mM Tris-NaCl, 10% glycerol, pH 7.0); the cell suspension was disrupted by an ultrasonic disruptor (400 W, 2 seconds of disruption, 3 seconds of rest as one cycle). The supernatant of the crude enzyme solution was obtained by centrifugation at 12,000×g for 30 minutes at 4°C.

[0131] Protein purification was performed using GSH Purose 4Fast Flow (Qianxian Biotechnology Co., Ltd., China) according to the operating instructions. The GST tag was removed by prescission protease, and 160 μL (320 units) of prescission protease was mixed with 840 μL of prescission excision buffer (50 mM Tris-NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 15% glycerol, pH 7.0) in an ice bath. Then, the prescission protease mixture was placed in the column carrying the target protein and incubated overnight at 4°C. The column was rinsed with prescission excision buffer and the effluent was collected. The effluent was the pure protein with the GST tag removed. The purified protein was frozen in liquid nitrogen and stored at -80°C for later use. The purified protein was verified by staining with Coomassie Brilliant Blue and by 12.5% ​​sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a 10-250 kDa protein marker was used. The content of purified protein was determined using the Thermo BCA protein assay kit. At the same time, the purified wild-type pure enzyme solution was obtained according to the above method. The purified protein gel is shown in Figure 2 As shown, 1 represents the wild-type PslLTTA and 2 represents the mutant F70C / N268S.

[0132] (3) Detection of L-threonine transaldolase activity. Enzyme activity was determined by measuring the rate of change of NADH absorbance at 340 nm (ε=6220 / M / cm). The reaction mixture (200 μL) consisted of 5 mM p-nitrobenzaldehyde, 30 mM L-threonine, 0.05 mM PLP, 0.3 mM NADH in Tris-HCl (50 mM, pH 7.0) and incubated at 30°C for 2 minutes. The reaction was initiated by adding 10 μg of pure enzyme and 10 U of alcohol dehydrogenase (Sigma Aldrich, CAS NO: 9031-72-5) and monitored at 340 nm for 2 minutes (once every 15 seconds).

[0133] One unit (U) of transaldolase activity was defined as the amount of enzyme that catalyzes the conversion of 1 μmol of L-threo-p-nitrophenylserine per minute.

[0134] The results showed that the specific enzyme activity of the mutant F70C / N268S was 5.12U / mg, and that of the wild type WT was 2.18U / mg.

[0135] 2. Study on the enzymatic properties of the F70C / N268S mutant.

[0136] The main research focuses on its optimal reaction pH, optimal temperature, pH stability, temperature stability and metal ion dependence. The details are as follows:

[0137] (1) Optimal reaction pH detection and pH stability

[0138] The optimum pH and pH stability of the enzyme-catalyzed reaction were studied using phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 8.0-9.0), and bicarbonate buffer (pH 9.0-10.0). The pH and temperature dependence of LTTAs were determined by measuring the activity in buffers with different pH values ​​(pH 6.0-10.0). To study pH stability, the enzyme solution was incubated at 4°C for 12 hours in different pH buffers, and then the relevant activity was measured at 30°C. The relevant enzyme activity under each condition was compared with the maximum specific enzyme activity under a certain addition, and the relative enzyme activity under other conditions was calculated.

[0139] The results showed that the enzyme activities of the F70C / N268S mutant at pH 6.0, 7.0, 8.0, 9.0, and 10.0 were 37.5%, 100%, 83.5%, 36.5%, and 21.2%, respectively. The trend of the enzyme activities of WT at pH 6.0, 7.0, 8.0, 9.0, and 10.0 was consistent with that of the F70C / N268S mutant, indicating that both F70C / N268S and WT had the highest specific enzyme activity at pH 7.0.

[0140] The enzyme solution was incubated at 4°C for 12 h at pH 6.0, 7.0, 8.0, 9.0, and 10.0, respectively, and then the residual activity was measured at 30°C. The residual activities of the F70C / N268S mutant were 16.5%, 29.2%, 100%, 40.5%, and 25.1%, respectively. The trend of the enzyme activity change of WT at pH 6.0, 7.0, 8.0, 9.0, and 10.0 was consistent with that of the F70C / N268S mutant, indicating that the specific enzyme activity of both F70C / N268S and WT was the highest when incubated at pH 7.0.

[0141] (2) Thermal stability

[0142] To evaluate the thermostability of the enzyme, the enzyme solution was incubated in 50 mM Tris buffer (pH 7.0) at different temperatures (10, 20, 30, 40, 50, 60, 70, 80°C) for 1 h, and then the residual activity was measured at 30°C.

[0143] The results showed that the enzyme activities of the F70C / N268S mutant at 10, 20, 30, 40, 50, 60, 70, and 80°C were 100%, 99.5%, 98.5%, 96.4%, 89.5%, 77.4%, 21.5%, and 10.2%, respectively. The change trend of the enzyme activities of WT at 10, 20, 30, 40, 50, 60, 70, and 80°C was consistent with that of the F70C / N268S mutant, indicating that the F70C / N268S mutant maintained its high thermal stability while improving the enzyme activity.

[0144] (3) Metal ion dependence

[0145] In the presence of different metal ions, including Ca 2+ , Ba 2+ , Mn 2+ 、Co 2+ 、Zn 2+ , Fe 2+ and Mg 2+ The effects of these metal ions at 1.0 mM and 5.0 mM concentrations on the activity of LTTAs were determined. The enzyme activity without metal ions was used as a control to calculate the relative activity of the enzymes with different metal ions added.

[0146] The results showed that the F70C / N268S mutant was 2+ , Ba 2+ , Mn 2+ Mg 2+ 、Co 2+ , Fe 2+ and the enzyme activities were 151.4%, 139.6%, 115.4%, 161.2%, 98.6%, and 32.5%, respectively. 2+ The enzyme activity was completely inhibited. The F70C / N268S mutant was 2+ , Ba 2+ , Mn 2+ Mg 2+ 、Co 2+ The enzyme activities under the following conditions were 149.3%, 135.6%, 105.6%, 158.3%, and 11.35%, respectively. Among them, 5.0 mM Zn 2+ and Fe 2+ Completely inhibited enzyme activity. Compared with WT at 1.0mM and 5.0mM Ca2+ , Ba 2+ , Mn 2+ 、Co 2+ 、Zn 2+ , Fe 2+ and Mg 2 The related enzyme activities under the same conditions showed the same trend. 2+ All showed the greatest promoting effect on enzyme activity.

[0147] In summary, if Figure 3 a in Figure 3 As shown in b, the study showed that the engineered L-threonine transaldehyde enzyme F70C / N268S had stable enzyme activity at a temperature of 10-50°C and could maintain more than 70% of the enzyme activity, indicating that it had good temperature stability.

[0148] The results showed that the optimum reaction temperature was 50°C and the pH value was 7.0-7.5, with stable and high activity ( Figure 3 c in Figure 3 d in; the optimum pH is 7.5, and Figure 3 As shown in e, Ca 2+ With Mg 2+ The addition of has a significant promoting effect on enzyme activity. By studying the enzymatic properties of engineered L-threonine transaldolase F70C / N268S, we can better explore the reaction conditions for the synthesis of L-threo-p-nitrophenylserine.

[0149] Example 3: Analysis of the molecular mechanism of improved catalytic performance of mutants based on homology modeling and molecular docking

[0150] In order to explore the key molecular mechanism of the improved catalytic performance of mutant F70C / N268, homology modeling and molecular docking techniques were used to analyze the interaction between the key amino sites in the enzyme active center and the substrate.

[0151] First, the amino acids of wild-type PslLTTA and mutant F70C / N268 were used as targets, and Pseudomonas fluorescens (ObiH) LTTA (PDB: 7k34) was used as a template to model the three-dimensional protein structures of the two using Swiss Model homology. Subsequently, the coenzyme PLP and the product L-threo-p-nitrophenylserine were docked to the active centers of PslLTTA and mutant F70C / N268 respectively using the induced fit docking in the Schrödinger system to obtain the protein-ligand complex, and the energy was minimized and then related analysis was performed. The hydrogen bond interaction in the active center of the protein plays an important role in the normal function of the protein. Therefore, we focused on the changes in the hydrogen bonds in the active center after molecular docking to analyze the main reason for the improvement of the catalytic performance of the mutant.

[0152] like Figure 4 As shown, the hydrogen bond interactions between L-threo-p-nitrophenylserine and residues in the active center of PslLTTA were first analyzed. In the active center of wild-type PslLTTA, residues R366, H207, K234 and S180 formed a rich hydrogen bond network with the carboxyl group of L-threo-p-nitrophenylserine, which is conducive to the fixation of its configuration. Among them, the F70 site is located on the β-hydroxyl side of L-threo-p-nitrophenylserine, and the steric hindrance caused by its larger benzene ring may affect its β-hydroxyl selectivity ( Figure 4 a). Figure 4 As shown in Figure 8b, the same hydrogen bond network was observed in the active center of the mutant F70C / N268. In addition, the formation of two new hydrogen bonds was observed. One is the hydrogen bond formed between the N35 residue and the carboxyl group of L-threo-p-nitrophenylserine, and the other is the strong hydrogen bond formed between C70 and the β-hydroxyl group of L-threo-p-nitrophenylserine, which is beneficial to the fixation of L-threo-p-nitrophenylserine and the improvement of the stereoselectivity of the β carbon atom, ultimately leading to the improvement of the catalytic efficiency of the product.

[0153] At the same time, the changes in the hydrogen bond network around the coenzyme PLP in the active center of PslLTTA were studied. Figure 5 As shown in a, in the active center of wild-type PslLTTA, the phosphate group of PLP forms hydrogen bonds with residues N231 and S267, and the catalytic acid D204 forms hydrogen bonds with the pyridine nitrogen of PLP, which is conducive to the protonation of its pyridine ring. Figure 5 In b), the mutation site S268 forms an additional hydrogen bond interaction with the phosphate group of PLP, which helps to strengthen the catalytic function of PLP and improve its catalytic efficiency. The combination of double mutation sites F70C / N268S further enhances the catalytic performance of the enzyme.

[0154] Example 4: Construction of a “one-pot” system for efficiently synthesizing L-threo-p-nitrophenylserine

[0155] In the reaction catalyzed by LTTAs, acetaldehyde is generated simultaneously with the target product. Acetaldehyde is highly toxic, and its excessive accumulation not only hinders the reaction but also weakens the enzyme activity. In order to enhance the yield of L-threo-p-nitrophenylserine, alcohol dehydrogenase (SmADH) from Saccharomyces mikatae and formate dehydrogenase (CbFDH) from Candida boidinii were introduced to eliminate acetaldehyde and promote NADH / NAD + Regeneration. A one-pot cascade catalytic system was constructed by coupling the F70C / N268S mutant with an acetaldehyde elimination system. Two cascade systems were obtained by different combinations. The effects of these systems on the synthesis of L-threo-nitrophenylserine were studied using the uncoupled F70T / N268S variant (E. coli0) as a control.

[0156] The specific steps are as follows:

[0157] 1. Construction of genetically engineered bacteria

[0158] (1) Chemically synthesize the SmADH and CbFDH genes whose nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0159] SEQ ID NO.3:

[0160] atgagcatcc cgaaaaccca gaaaggcgtcatcttttacg aatccaatgg caaactggagcacaaagatatcccagtaccaaaaccgaag ccgaacgaac

[0161] tgctgatcaa cgttaaatac tctggtgtttgtcataccga tctgcatgcg tggcatggtgattggcctct gccaactaaa ctgccgctgg ttggcggcca

[0162] cgaaggcgctggtgtggttgtagcgatcgg cgacaacgtt cgtggctgga aagtgggcgatctggcaggcatcaaatggctgaacagctc ttgcatggct

[0163] tgcgaatattgcgaactgggtaacgaatctaactgcccgc atgctgatctgtctggttacactcacgacg gttcttttca gcagtatgca accgctgacg

[0164] cggtacaagcagcgcgtac ccggaaggta ctgatctggc tcaggtggct cctatcctgtgcgccggcat caccgtttac aaagccctga aatctgctaa

[0165] cctgcgtgcc ggtcattggg tagctatttc tggcgcggct ggcggtctgg gttctctggctgttcaatac gcgaaagcaa tgggctaccg cgtactgggc

[0166] atcgacggcg gtgaaggcaa agaagaactg ttcacgtctc tgggtggtga ggtgttcatcgactttacca aggaaaaaga tattgtgagc gcggtggtta

[0167] aggctaccga tggtggcgg catggtatcg tcaacgtgtc cgtttccgaa gctgctatcgaagcgagcac tcgctactgt cgtgcgaacg gtaccgttgt

[0168] tctggttggc ctgccggcgg gcgccaaatg ttgctctgac gtattcaacc acgtcgttaagtccattagc atcgtgggta gctacgttgg caaccgtgca

[0169] gacacgcgcg aagcgctgga ttttttgcg cgtggtctgg tgaaatcccc gattaaggtagtgggcctgt ccaccctgcc ggaaatttac gaaaagatgg

[0170] aaaagggtca gatcgctggc cgctacgtgg ttgacaccag caaa

[0171] SEQ ID NO.4:

[0172] atgaaaatcgttctggttctgtatgacgcgggcaaacacgcagcagatgaagaaaaactgtacggctgcaccgaaaacaaactgggcatcgctaactggctgaaag

[0173] accagggtcacgaactgatcaccaccagcgacaaagaaggtgaaacctctgaactggataaacatatcccggacgcggacatcattatcaccaccccgttccaccc

[0174] ggcgtacattaccaaagaacgtctggataaagcgaaaaaacctgaaactggttgtggtggcgggcgttggttccgaccatatcgatctggattatatcaaccagaccgg

[0175] taaaaagatctctgttctggaagttaccggtagcaacgtggtttctgttgcggaacacgttgttatgaccatgctggtgctggttcgtaactttgttccggcgcacgaacag

[0176] atcatcaaccacgattgggaagttgcggcgattgcgaaagatgcgtacgatatcgaaggtaaaaccatcgcaaccatcggcgcgggccgtatcggctaccgtgtgct

[0177] ggaacgtctgttgccgttcaacccgaaagaactgctgtactacgattaccaggcgctgccgaaagaagctgaagaaaaagttggtgctcgtcgtgttgaaaacatcga

[0178] agaactggttgcgcaggcggatatcgtgaccgttaacgctccgctgcacgctggtaccaaaggtctgatcaacaaagaactgctgtctaaattcaaaaaaggcgcttg

[0179] gctggttaacaccgcgcgcggcgcgatctgcgttgcagaggacgttgcggcggcactggaaagcggtcagctgcgtggttacggtggtgacgtttggttcccgcag

[0180] ccggcgccgaaagatcacccgtggcgtgacatgcgtaacaaatacggtgctggtaacgcgatgaccccgcactactctggcaccaccctggatgcccagacccgc

[0181] tacgcggaaggtaccaaaaacatcctggaaagcttcttcaccggtaaattcgattaccgtccgcaggatatcatcctgctgaacggtgaatacgttaccaaagcgtacg

[0182] gtaaacacgataaaaaataa

[0183] (2) The F70C / N268S mutant, SmADH and CbFDH genes were sequentially connected to the BamH I and Xho I sites of the pRSF-Duet1 vector to prepare the recombinant vector pRSF-F70C / N268S / SmADH / CbFDH;

[0184] The SmADH and CbFDH gene constructs were sequentially connected to the BamH I and Xho I sites on the pRSF-Duet1 vector to prepare the recombinant vector pRSF-SmADH / CbFDH;

[0185] The recombinant vector pGEX-F70C / N268S was prepared according to the method of Example 1;

[0186] (3) Transforming the recombinant vector pRSF-F70C / N268S / SmADH / CbFDH into competent cells E. coli BL21 (DE3) to obtain recombinant cells E. coli / pRSF-F70C / N268S / SmADH / CbFDH (named E. coli1);

[0187] The recombinant vectors pGEX-F70C / N268S and pRSF-SmADH / CbFDH were co-transformed into competent cells E. coli BL21 (DE3) to obtain recombinant cells E. coli / pGEX-F70C / N268S-pRSF-SmADH / CbFDH (named E. coli2).

[0188] The recombinant vector: pGEX-F70C / N268S was transformed into competent cells E. coli BL21 (DE3) to obtain recombinant cells E. coli / pGEX-F70C / N268S (named E. coli0).

[0189] 2. Synthesis of L-threo-p-nitrophenylserine

[0190] (1) Preparation of whole-cell catalysts

[0191] The obtained recombinant strains were cultured in LB liquid medium containing 100 μg / mL ampicillin or 50 μg / mL kanamycin for 6 to 8 h. 600 When the value reaches 0.6-0.8, IPTG is added at a final concentration of 0.2 mM for induction. The induction temperature is 25°C and the induction time is 16-18 h. The recombinant cell fluid is then centrifuged at 8000ⅹg for 10 min, the supernatant is discarded, and the cell pellets obtained are the whole-cell catalysts.

[0192] (2) Synthesis of L-threo-p-nitrophenylserine

[0193] The initial whole-cell catalytic system for the synthesis of L-threonine by the "one-pot method" contains: 300 mM L-threonine, 150 mM p-nitrobenzaldehyde, 1 mM magnesium chloride (MgCl2), 0.1 mM pyridoxal phosphate (PLP), and NAD + 1mM, sodium formate 250mM, 10% ethyl acetate as cosolvent, whole cells 30mg / mL. The reaction buffer is 50mM ammonium formate solution (pH7.5), the reaction time is 6 hours, and the reaction temperature is 30°C. The reaction is carried out in a 100mL system with a shaking speed of 200rpm. After the reaction, the yield and de value of L-threo-p-nitrophenylserine are detected by high-pressure liquid chromatography by derivatization.

[0194] Table 3: Comparison of the one-pot catalytic synthesis of L-threo-p-nitrophenylserine in different systems

[0195]

[0196] The results show:

[0197] As shown in Table 3 , the L-threonine transaldolase mutant F70C / N268S, which was not coupled to the acetaldehyde elimination system, synthesized 62.2 mM of L-threo-p-nitrophenylserine when the substrate was added at 150 mM;

[0198] After coupling the acetaldehyde elimination system, especially after the three enzymes were co-constructed into one plasmid, the production efficiency of L-threo-p-nitrophenylserine was greatly improved, reaching a yield of 148.7 mM, and the yield rate reached 99.1%.

[0199] Example 5: Optimization of conditions for efficient whole-cell catalytic synthesis of L-threo-p-nitrophenylserine by “one-pot method”

[0200] The specific steps are as follows:

[0201] The initial whole-cell catalytic system contained 300 mM L-threonine, 150 mM p-nitrobenzaldehyde, 1 mM magnesium chloride (MgCl2), 0.1 mM pyridoxal phosphate (PLP), and NAD + 0.5mM, 250mM sodium formate, 10% ethyl acetate as cosolvent, 30mg / mL whole cells (E. coli1, the preparation method of whole cell catalyst is the same as Example 4). The reaction buffer is 50mM ammonium formate solution (pH 7.0), the reaction time is 6 hours, and the reaction temperature is 30°C. The reaction is carried out in a 100mL system with a shaking speed of 200rpm.

[0202] The optimized parameters include the concentration of p-nitrobenzaldehyde, the ratio of p-nitrobenzaldehyde to L-threonine, NAD + Concentration, sodium formate concentration, pH and temperature. Single factor optimization was used for optimization, and one variable was controlled each time. The titer and de value of L-threo-p-nitrophenylserine in the reaction solution were detected by HPLC. All experiments were repeated three times and the average value was taken for data analysis.

[0203] Specifically:

[0204] (1) The specific reaction is the same as above, except that the concentration of the substrate nitrobenzaldehyde is adjusted to 150 mM, 170 mM, 190 mM, 210 mM, 230 mM, 250 mM, 270 mM, and 290 mM, respectively.

[0205] The results show that: Figure 6 As shown in a, when the concentrations of substrate nitrobenzaldehyde are 170mM, 190mM, 210mM, 230mM, and 250mM, the concentrations of L-threo-p-nitrophenylserine products are 159.8mM, 171.0mM, 176.4mM, 184.0mM, and 182.1mM, respectively;

[0206] It can be seen that with the increase of p-nitrobenzaldehyde concentration, the concentration of L-threo-p-nitrophenylserine showed a trend of first increasing and then decreasing. When 250 mM p-nitrobenzaldehyde was added, the concentration of L-threo-p-nitrophenylserine was 182.1 mM, which showed the first decrease. Therefore, 250 mM p-nitrobenzaldehyde concentration was determined for the next step of optimization.

[0207] (2) The specific reaction is the same as above, except that the concentration of the substrate nitrobenzaldehyde is adjusted to 250 mM, and the concentrations of L-threonine are adjusted to 250 mM, 300 mM, 375 mM, 500 mM, and 750 mM respectively;

[0208] The results showed that the concentrations of L-threonine were 250mM, 300mM, 375mM, 500mM, and 750mM, respectively; the concentrations of L-threo-p-nitrophenylserine products were 162.5mM, 180.1mM, 180.2mM, 182.3mM, and 183.1mM, respectively;

[0209] (3) The specific reaction is the same as above, except that the concentration of the substrate nitrobenzaldehyde is adjusted to 250 mM, L-threonine is adjusted to 500 mM, and NAD + The concentrations are: 0.1mM, 0.25mM, 0.5mM, 0.75mM, 1mM, 1.25mM;

[0210] The results showed that NAD + When the concentrations were: 0.25mM, 0.5mM, 0.75mM, 1mM, 1.25mM, the concentrations of L-threo-p-nitrophenylserine product were: 156.8mM, 180.2mM, 193.5mM, 212.5mM, 213.5mM;

[0211] (4) The specific reaction is the same as above, except that the concentration of the substrate nitrobenzaldehyde is adjusted to 250 mM, L-threonine is adjusted to 500 mM, and NAD + The concentration is: 1mM; the sodium formate concentrations are adjusted to: 150mM, 200mM, 250mM, 300mM, 350mM;

[0212] The results showed that when the concentrations of sodium formate were 150mM, 200mM, 250mM, 300mM, and 350mM, the concentrations of L-threo-p-nitrophenylserine products were 147.3mM, 175.6mM, 212.5mM, 218.9mM, and 219.2mM, respectively;

[0213] (5) The specific reaction is the same as above, except that the concentration of substrate nitrobenzaldehyde is adjusted to 250 mM, L-threonine is adjusted to 500 mM, and NAD+ The concentration is: 1mM; the sodium formate concentration is adjusted to: 300mM, and the pH is adjusted to: 6.0, 6.5, 7.0, 7.5, 8.0;

[0214] The results showed that when the pH was 6.0, 6.5, 7.0, 7.5, and 8.0, the concentrations of L-threo-p-nitrophenylserine product were 160.2 mM, 187.2 mM, 200.2 mM, 218.9 mM, and 208.6 mM, respectively;

[0215] (6) The specific reaction is the same as above, except that the concentration of the substrate nitrobenzaldehyde is adjusted to 250 mM, L-threonine is 500 mM, and NAD + The concentration is: 300mM; the pH is: 7.5; the adjusted temperatures are: 20℃, 25℃, 30℃, 35℃, 40℃;

[0216] The results showed that when the temperatures were 20℃, 25℃, 30℃, 35℃, and 40℃, the concentrations of L-threo-p-nitrophenylserine products were 186.9mM, 200.2mM, 218.9mM, 233.5mM, and 234.5mM, respectively;

[0217] Finally, the optimal conditions were obtained: p-nitrobenzaldehyde 250mM, L-threonine 500mM, NAD + The concentration is 1mM, the sodium formate concentration is 300mM, the pH value is 7.5, the temperature is 35°C, 10% ethyl acetate is used as a cosolvent, and the whole cell is 30mg / mL. The reaction buffer is 50mM ammonium formate solution (pH 7.5), and the reaction time is 6 hours. Under these conditions, L-threo-p-nitrophenylserine reaches an optimal concentration of 233.5mM (a total of 63.3g / L), the substrate conversion rate is 93.4%, and the de value of L-threo-p-nitrophenylserine is as high as 99.4%. This yield is the highest level of enzyme-catalyzed synthesis of L-threo-p-nitrophenylserine reported so far.

[0218] Example 6: Scale-up reaction at 50 L scale

[0219] The reaction system is 50L, containing 250mM p-nitrobenzaldehyde, 500mM L-threonine, 0.05mM PLP, 1mM NAD +, 1mM MgCl2, 300mM sodium formate, 10% ethyl acetate, and 30mg / mL whole cell catalyst, the reaction buffer is Tris-HCl (50mM) at pH 7.5, and the reaction temperature is 35°C. A large bioreactor is used for 50L whole cell transformation, the speed is set to 300rpm, and the temperature is controlled by oil bath circulation. Samples are taken regularly during the reaction to detect the content of L-threo-p-nitrophenylserine and the de value.

[0220] The reaction results are:

[0221] The catalyst showed high catalytic efficiency in the first 4 hours, converting 250 mM of substrate into 225.9 mM of L-threo-p-nitrophenylserine, demonstrating good operational stability. The content of L-threo-p-nitrophenylserine reached a stable concentration of 233.5 mM within 5 h, with a substrate conversion rate of 93.4% and a de value of 99.4%.

[0222] Comparative Example 1

[0223] Specific implementation method The construction process of E. coli1 is the same as that of Example 4, except that the alcohol dehydrogenase is adjusted to be derived from: Hypoxylon fuscum alcohol dehydrogenase, and the nucleotide sequence encoding the alcohol dehydrogenase is shown in SEQ ID NO.5.

[0224] SEQ ID NO.5

[0225] atgggtagcg ttgatattcc gagcgaacag tgggcacagg ttgtcgaaca gactggtggtccagtgcatt acaagaaaat tccagttccg aaaccgggcc

[0226] cggatgaagt gctgatcaac gttaaatact ctggtgtgtg ccacaccgat ctgcacgcgatgaacggtga ttggccaatt ccgaccaaaa tgcctctggt

[0227] tggcggccac gaaggtgctg gtatcgttgt tgcccgtggt gagctggtga aggatgtgcagattggtgat tacgcgggta ttaagtggct gaacggttct

[0228] tgcctgtcct gtagctactg tcagcaatct gacgaaagcc tgtgcccgaa accgctgctgagcggctata ccgttgacgg tagcttccag cagtatgcta

[0229] tcggcaaggc agcacacgtg gcgcgtattc cgaaagaact gtccctggaa gctgtctccccgatcctgtg tgcgggtctg accgtctaca aagctctgaa

[0230] agaaagcggt gcacgccctg gccagcaggt agcgattgtt ggtgctggtg gcggcctgggctccctggcg atccagtacg caaaagcgat gggtctgcat

[0231] accatcgcta tcgacggcgg tgaggagaaa ggcgaaatgt gcaaaagcct gggtagcgaagactacatcg acttcaccaa aaccaaaaaac gtcgtccagg

[0232] ctgttaaaaa cgcgaccacc gatggttttg gcccacatgc tgtgctgctg ctggctgtaagcgaaggccc atttcagcag gcgagcgagt acgttcgttc

[0233] ccgtggtacc attgtttgcg tcggtctgcc ggcaaacgcg ttcctgaaag cgccagtatttgatactgtg gtgcgtatga tcaacatcaa gggttcttac

[0234] gtcggcaacc gtcaggacac ggcagaagcg atcgacttct tccgccgcgg tctgatcaaagcgccgtata aagtggttgg cctgagcaag ctgcaggatg

[0235] tctttgatct gatgaaagag ggcaaaatcg cgggccgtta cgtcgttgac acctccaag

[0236] After the experiment was carried out according to the method of Example 4, the results showed that the production efficiency of L-threo-p-nitrophenylserine was 125.2 mM, and the yield was 83.4%.

[0237] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A L-threonine transaldolase mutant, characterized in that: The mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine transaldolase with the amino acid sequence shown in SEQ ID NO.1 to cysteine; Or the mutant is obtained by mutating the phenylalanine at position 59 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, and mutating the phenylalanine at position 70 to cysteine; Or the mutant is obtained by mutating the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to cysteine, and mutating the asparagine at position 268 to serine; Or the mutant is obtained by mutating the phenylalanine at position 59 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to alanine, mutating the phenylalanine at position 70 to cysteine, and mutating the asparagine at position 268 to serine.

2. A gene encoding the mutant according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene or the recombinant vector according to claim 2.

4. The recombinant cell according to claim 3, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

5. A recombinant enzyme catalyst comprising the L-threonine transaldolase mutant sequence of claim 1, characterized in that: The recombinase catalyst is any one of the following forms: (1) culturing a recombinant expression transformant containing an L-threonine aldolase mutant, and isolating a transformant cell containing a recombinant enzyme of the L-threonine aldolase mutant; (2) culturing a recombinant expression transformant containing the L-threonine aldolase mutant, isolating transformant cells containing the L-threonine aldolase mutant recombinant enzyme, and disrupting the transformant cells containing the recombinant enzyme to obtain a cell disrupted liquid; (3) Cultivating a recombinant expression transformant containing the L-threonine transaldolase mutant, isolating transformant cells containing the L-threonine transaldolase mutant recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid containing the recombinant enzyme to obtain a lyophilized enzyme powder.

6. A recombinant Escherichia coli, characterized in that The recombinant Escherichia coli simultaneously overexpresses the mutant according to claim 1, Saccharomyces mikatae Alcohol dehydrogenase and Candida boidinii of formate dehydrogenase.

7. The recombinant Escherichia coli according to claim 6, characterized in that The recombinant Escherichia coli uses pRSF-Duet1 as an expression vector and simultaneously overexpresses the mutant described in claim 1, Saccharomyces mikatae Alcohol dehydrogenase and Candida boidinii of formate dehydrogenase.

8. The recombinant Escherichia coli according to claim 6, characterized in that The recombinant Escherichia coli uses pGEX-6P-1 as an expression vector to express the mutant described in claim 1, and at the same time, uses pRSF-Duet1 as an expression vector to express the mutant derived from Saccharomyces mikatae Alcohol dehydrogenase and Candida boidinii of formate dehydrogenase.

9. The recombinant Escherichia coli according to claim 6, characterized in that The nucleotide sequence encoding the alcohol dehydrogenase is shown in SEQ ID NO.3, and the nucleotide sequence encoding the formate dehydrogenase is shown in SEQ ID NO.

4.

10. A method for improving the enzymatic activity of L-threonine aldolase on a substrate, p-nitrobenzaldehyde, characterized in that: The method is to mutate the phenylalanine at position 70 of the L-threonine transaldolase with an amino acid sequence as shown in SEQ ID NO.1 to cysteine; Or the method is to mutate the phenylalanine at position 59 of the L-threonine aldolase with an amino acid sequence as shown in SEQ ID NO.1 to alanine, and simultaneously mutate the phenylalanine at position 70 to cysteine; Or the method is to mutate the phenylalanine at position 70 of the L-threonine aldolase with the amino acid sequence as shown in SEQ ID NO.1 to cysteine, and at the same time mutate the asparagine at position 268 to serine; Or the method is to mutate the phenylalanine at position 59 of the L-threonine transaldolase with an amino acid sequence as shown in SEQ ID NO.1 to alanine, mutate the phenylalanine at position 70 to cysteine, and mutate the asparagine at position 268 to serine.

11. A method for preparing chloramphenicol intermediate L-threo-p-nitrophenylserine using whole cells, characterized in that: The method comprises the following steps: using L-threonine as a donor substrate and p-nitrobenzaldehyde as an acceptor substrate, and adopting the recombinant cell of claim 3 or claim 4 or the recombinant Escherichia coli whole cell of claim 6 for transformation to prepare the chloramphenicol intermediate L-threo-p-nitrophenylserine.

12. The method according to claim 11, characterized in that The reaction system also contains pyridoxal phosphate PLP, NAD + , MgCl2, sodium formate.

13. The method according to claim 11, characterized in that The concentration of the p-nitrobenzaldehyde is 170 mM to 250 mM.

14. The method according to claim 11, characterized in that The concentration of the L-threonine is 250 mM to 750 mM.

15. The method according to claim 11, characterized in that When preparing the chloramphenicol intermediate L-threo-p-nitrophenylserine, the reaction conditions are pH 6.0-8.0 and temperature 20°C-40°C.

16. The method according to claim 12, characterized in that The NAD + The concentration is 0.1 mM~1.25 mM.

17. The method according to claim 12, characterized in that The concentration of sodium formate is 150 mM~350 mM.

18. Use of the L-threonine transaldolase mutant according to claim 1, or the gene or recombinant vector according to claim 2, or the recombinant cell according to claim 3 or 4, or the recombinant enzyme catalyst according to claim 5, or the recombinant Escherichia coli according to claim 6, or the method according to any one of claims 10 to 17 in the preparation of a chloramphenicol intermediate L-threo-p-nitrophenylserine or a product containing the chloramphenicol intermediate L-threo-p-nitrophenylserine.

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