Nicotinic acid phosphoribosyltransferase mutant, encoding gene and application

Abstract

The present invention relates to a nicotinic acid phosphoribosyltransferase mutant and a coding gene, and its application in the biosynthesis of L-phosphinothricin, D-amino acid, L-tert-leucine or L-aminobutyric acid by a multi-enzyme coupling method. The beneficial effects of the present invention are mainly reflected in: (1) the nicotinic acid phosphoribosyltransferase mutant of the present invention improves the intracellular NAD level, brings an efficient, low-cost and environmentally friendly production method for the coenzyme-dependent multi-enzyme catalytic reaction, and does not require the additional addition of coenzymes; (2) the nicotinic acid phosphoribosyltransferase mutant of the present invention has better catalytic efficiency, and when the intermediate product PPO is used as a substrate for catalytic reaction, the conversion rate is much higher than that of the original enzyme, and the L-phosphinothricin yield is also greatly improved; (3) the present invention can directly use PPO to prepare and produce L-phosphinothricin without exogenous addition of coenzymes, without the need for expensive resolution reagents, without the need for synthesizing phosphinothricin derivatives, and without the need for separation, re-racemization, and re-resolution of D-phosphinothricin.

Description

(I) Technical field

[0001] The invention relates to a nicotinic acid phosphoribosyltransferase mutant and a coding gene, and application of the mutant in biosynthesis of L-phosphinothricin, D-amino acid, L-tert-leucine or L-aminobutyric acid by a multi-enzyme coupling method. (II) Background technology

[0002] Coenzymes are non-protein compounds or metal ions that are essential for the biological functions and catalytic activities of countless enzymes in all living organisms, and can effectively promote biochemical transformations and even facilitate many thermodynamically unfavorable reactions. Among the numerous cofactors, nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are known as redox cofactors. They are present in their oxidized form (NAD + / NADP + ) and reduced forms (NADH / NADPH), which are usually associated with metabolic and synthetic reactions as electron donors or acceptors, respectively.

[0003] Reduced coenzyme I (NADH) and reduced coenzyme II (NADPH) participate in enzymatic reactions as hydrogen donors in cellular anabolism and are the two most abundant redox coenzymes in the cellular metabolic network. Under aerobic conditions, NADH participates in catabolism to provide a large amount of ATP; NADPH provides reducing power for cellular anabolism. Coenzyme II is more expensive than coenzyme I, and reduced coenzymes are more expensive than oxidized coenzymes.

[0004] Redox systems are ubiquitous in industrial applications. Redox catalytic reactions are usually accompanied by the generation, conversion, and consumption of coenzymes. In bioindustrial applications, various amino acid dehydrogenases (AADHs) such as formate dehydrogenase (FDH), leucine dehydrogenase (LeuDH), and glutamate dehydrogenase (GluDH) are the most common coenzyme-dependent enzymes. In this type of enzymatic biosynthesis, the problem of insufficient supply of NAD and NADP is often encountered, and expensive coenzymes are usually required to promote the reaction process. Insufficient supply and incomplete utilization of coenzymes have become bottlenecks for the industrialization of redox catalysis. Therefore, how to construct a coenzyme-self-sufficient strain, increase the coenzyme content in the expression system, and avoid exogenous addition of expensive cofactors is of great significance to the industrial application of redox biocatalysis.

[0005] The synthesis pathway of nicotinamide adenine coenzymes is mainly divided into de novo synthesis pathway and salvage pathway. Existing relevant scientific studies have shown that regulating key genes in metabolic pathways and constructing artificial coenzyme circulation systems can enhance the coenzyme level of chassis strains. Nicotinic acid phosphoribosyltransferase is the key rate-limiting enzyme in the NAD coenzyme synthesis pathway. Nicotinic acid phosphoribosyltransferase uses nicotinic acid as a substrate and selectively uses ATP to convert nicotinic acid into key NAD precursors such as nicotinic acid mononucleotide. The increase in NAD synthesis precursors can effectively increase the intracellular NAD synthesis level. NAD kinase catalyzes the conversion of NAD into NADP, thereby increasing the intracellular NADP synthesis level.

[0006] Chiral amino acids are widely used chiral compounds and are of great significance in active drug intermediates such as peptides and single enantiomers. The bioenzymatic method for synthesizing chiral amino acids has the advantages of simple operation, mild reaction conditions, high product yield and high optical activity value, and is currently a hot research topic in the asymmetric synthesis of chiral amino acids. (III) Summary of the invention

[0007] The present invention aims to provide a nicotinic acid phosphoribosyltransferase mutant and a coding gene, as well as the application of the mutant in the biosynthesis of L-phosphinothricin, D-amino acid, L-tert-leucine or L-aminobutyric acid by a multi-enzyme coupling method.

[0008] The technical solution adopted by the present invention is:

[0009] A nicotinic acid phosphoribosyltransferase mutant is obtained by site-directed mutagenesis of the nicotinic acid phosphoribosyltransferase shown in SEQ ID NO.1, wherein the site of the point mutation is one of the following or a combination of two or more thereof: (1) position 360, (2) position 223, and (3) position 372. The nicotinic acid phosphoribosyltransferase of the present invention is derived from Pseudomonas fluorescens, the encoding amino acid sequence is shown in SEQ ID No.1, and the encoding nucleotide sequence is shown in SEQ ID No.2.

[0010] Preferably, the amino acid sequence of the mutant is shown in one of SEQ ID NOs. 6 to 8.

[0011] The present invention also relates to a gene encoding the nicotinic acid phosphoribosyltransferase mutant.

[0012] Specifically, the nucleotide sequence of the encoding gene is shown in one of SEQ ID NOs. 3 to 5.

[0013] The invention also relates to a vector and a genetic engineering bacterium containing the coding gene.

[0014] The present invention also relates to the application of the nicotinic acid phosphoribosyltransferase mutant in the multi-enzyme coupling method for biosynthesis of L-phosphinothricin ammonium, wherein the application is: using PPO as a substrate and ammonium formate as a co-substrate, and preparing L-phosphinothricin ammonium under the catalysis of a multi-enzyme system of glutamate dehydrogenase, formate dehydrogenase and the nicotinic acid phosphoribosyltransferase mutant.

[0015] The glutamate dehydrogenase is a NADPH coenzyme-dependent dehydrogenase, and the formate dehydrogenase is a NADP coenzyme-dependent dehydrogenase. Glutamate dehydrogenase and formate dehydrogenase construct a coenzyme cycle pathway to achieve the mutual conversion of NADPH and NADP. NADP in common organisms is directly converted from NAD. The nicotinic acid phosphoribosyltransferase described in the present invention is a key rate-limiting gene in the NAD metabolic synthesis pathway, which has a significant effect on the intracellular coenzyme level, especially the intracellular NAD level; therefore, nicotinic acid phosphoribosyltransferase is used to accumulate intracellular NAD, providing a basis for the NADPH / NADP cycle.

[0016] The above enzyme system can be obtained by simultaneous expression of E. coli BL21 (DE3) genetic engineering bacteria. After condition optimization, the optimal reaction conditions for catalyzing PPO to prepare L-phosphinothricin ammonium are as follows: the addition amount of genetic engineering bacteria expressing the glutamate dehydrogenase, formate dehydrogenase and nicotinic acid phosphoribosyltransferase mutants is 20g / L, the PPO concentration is 400mM, the co-substrate ammonium formate concentration is 800mM, the reaction temperature is 35°C, and the reaction pH is 7.5.

[0017] The present invention also relates to the application of the nicotinic acid phosphoribosyltransferase mutant in the biosynthesis of D-amino acids by a multi-enzyme coupling method, wherein the application is: using pyruvate as a substrate, under the catalysis of an amino acid dehydrogenase, formate dehydrogenase and a nicotinic acid phosphoribosyltransferase mutant multi-enzyme system, D-amino acids are prepared. The present invention uses NADH-dependent amino acid dehydrogenase to asymmetric reduce pyruvate to prepare D-amino acids, uses formate dehydrogenase to construct a coenzyme circulation system, and simultaneously introduces a nicotinic acid phosphoribosyltransferase mutant to convert NA into NAMN to promote intracellular NAD accumulation, thereby providing a cosubstrate for the coenzyme circulation system.

[0018] The present invention also relates to the application of the nicotinic acid phosphoribosyltransferase mutant in the multi-enzyme coupling method for biosynthesis of L-tert-leucine, wherein the application is: using 3-methylpyruvate as a substrate, under the catalysis of a leucine dehydrogenase, formate dehydrogenase and a nicotinic acid phosphoribosyltransferase mutant multi-enzyme system, L-tert-leucine is prepared. The present invention uses NADH-dependent leucine dehydrogenase to catalyze the substrate 3-methylpyruvate to prepare L-tert-leucine; formate dehydrogenase and leucine dehydrogenase realize a coenzyme cycle, and ammonium formate is used as a cosubstrate.

[0019] The present invention also relates to the application of the nicotinic acid phosphoribosyltransferase mutant in the biosynthesis of L-aminobutyric acid by a multi-enzyme coupling method, wherein the application is: using 2-carbonylbutyric acid as a substrate, under the catalysis of a multi-enzyme system of leucine dehydrogenase, formate dehydrogenase and nicotinic acid phosphoribosyltransferase mutant, L-aminobutyric acid is prepared. The present invention uses NADPH-dependent leucine dehydrogenase to asymmetric reduce 2-carbonylbutyric acid to prepare L-aminobutyric acid, uses formate dehydrogenase to construct a coenzyme circulation system, and co-expresses a nicotinic acid phosphoribosyltransferase mutant to convert NA into NAMN to promote intracellular NAD accumulation.

[0020] The NADPH-dependent glutamate dehydrogenase used in the present invention is derived from Pseudomonas monteilii WP_060477601.1, and its nucleotide sequence is shown in SEQ ID No.9.

[0021] The NADP-dependent formate dehydrogenase used in the present invention is derived from Pseudomonas sp. 101, and its nucleotide sequence is shown in SEQ ID No.10.

[0022] The NAD-dependent formate dehydrogenase used in the present invention is derived from Yarrowia lipolytica, and its nucleotide sequence is shown in SEQ ID No.11.

[0023] The NADH-dependent amino acid dehydrogenase used in the present invention is derived from Rhodococcus erythropolis BCRC 10909, and its nucleotide sequence is shown in SEQ ID No.12.

[0024] The NADH-dependent leucine dehydrogenase used in the present invention is derived from Geobacillus stearothermophilus, and its nucleotide sequence is shown in SEQ ID No.13.

[0025] The beneficial effects of the present invention are mainly reflected in:

[0026] (1) The nicotinic acid phosphoribosyltransferase mutant of the present invention improves the intracellular NAD level, providing an efficient, low-cost and environmentally friendly production method for coenzyme-dependent multi-enzyme catalytic reactions, without the need for additional coenzymes; (2) The nicotinic acid phosphoribosyltransferase mutant of the present invention has better catalytic efficiency. When the intermediate product PPO is used as a substrate for catalytic reaction, the conversion rate is much higher than that of the original enzyme, and the yield of L-phosphinothricin ammonium is also greatly improved; (3) The present invention can directly use PPO to prepare and produce L-phosphinothricin ammonium without adding exogenous coenzymes, without the need for expensive resolution reagents, without the need to synthesize glufosinate derivatives, and without the need to separate, re-racemize, and re-resolve D-phosphinothricin ammonium. (IV) Description of the drawings

[0027] Figure 1 The invention discloses a reaction formula for preparing L-phosphinothricin ammonium by using a multi-enzyme coupling method with PPO as a substrate.

[0028] Figure 2 The multi-enzyme coupling method used in the method of the present invention is a reaction formula for preparing D-amino acids using pyruvic acid as a substrate.

[0029] Figure 3 The invention discloses a reaction formula for preparing L-tert-leucine by using a multi-enzyme coupling method with 3-methylpyruvate as a substrate.

[0030] Figure 4 The multi-enzyme coupling method used in the method of the present invention is a reaction formula for preparing L-aminobutyric acid using 2-carbonylbutyric acid as a substrate.

[0031] Figure 5 These are comparison charts of the relative NAD coenzyme content between the wild-type strain of nicotinic acid phosphoribosyltransferase and the mutant L360A strain, the mutant L360A-A223K strain, and the mutant L360A-K372D strain. (V) Specific implementation methods

[0032] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0033] The experimental methods in the present invention are all conventional methods unless otherwise specified.

[0034] Materials and reagents used in genetic engineering: plasmid extraction kits and DNA purification and recovery kits were purchased from Corning Life Sciences (Wujiang) Co., Ltd.; gene fragment amplification reagents were purchased from Nanjing Novezan Co., Ltd. MaxSuper-Fidelity DNA Polymerase; E.coli DH5α, E.coli BL21 (DE3), coenzyme detection kits were purchased from Bio-Tech, nucleic acid extraction kits were purchased from Qingke Biotech Hangzhou Branch; plasmids pETDuet-1, pRSFDuet-1, etc. were purchased from Novagen; related sequences and primers were synthesized by Qingke Biotech Hangzhou Branch. The use of the above reagents is referred to the instruction manual. Other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0035] Example 1: Construction of Nicotinic Acid Phosphoribosyltransferase Genetically Engineered Bacteria

[0036] The amino acid sequence SEQ ID No. 1 of nicotinic acid phosphoribosyltransferase (GenBank No.: ABA72292.1) from Pseudomonas fluorescens was sent to Nanjing Branch of Qingke Biotechnology for full gene synthesis after codon optimization and cloned into the recombinant expression plasmid pRSFDuet-1 between the restriction sites KpnI and XhoI. The codon-optimized nicotinic acid phosphoribosyltransferase gene sequence is shown in SEQ ID No. 2.

[0037] Example 2: Construction of a Nicotinic Acid Phosphoribosyltransferase Mutant Library

[0038] The nicotinic acid phosphoribosyltransferase strain obtained in Example 1 was streaked on a plate with corresponding resistance and cultured at 37°C for 12 hours. A single colony was picked and transferred to a 10 mL LB liquid culture medium test tube with corresponding resistance and cultured at 150 rpm for 8 hours. According to the operating instructions of the Qingke nucleic acid extraction kit, the nicotinic acid phosphoribosyltransferase plasmid was extracted and named pRSFDuet-1-NAPRT.

[0039] In the first round, the plasmid extracted above was used as a template for site-directed mutagenesis PCR. L360A-f and L360A-r in Table 1 were used as mutation primers for site-directed mutagenesis PCR. The PCR product was transformed and plated, and the L360A mutant was obtained by screening and named L360A.

[0040] In the second round, L360A was used as a template for site-directed mutagenesis PCR. A223K-f and A223K-r in Table 1 were used as mutagenesis primers for site-directed mutagenesis PCR. The PCR products were transformed and plated, and mutants with L360A mutation and A223K mutation were obtained through screening and named L360A-A223K.

[0041] In the third round, L360A was used as a template for site-directed mutagenesis PCR. K372D-f and K372D-r in Table 1 were used as mutation primers for site-directed mutagenesis PCR. The PCR products were transformed and plated, and mutants with L360A mutation and K372D mutation were obtained by screening and named L360A-K372D.

[0042] Table 1: Mutation Primers

[0043]

[0044] The PCR reaction system is as follows: 2×Phanta Max buffer: 25 μL; dNTPs: 1 μL; upstream primer: 2 μL; downstream primer: 2 μL; template: 1 μL; Phanta Super-Fidelity DNA polymerase: 1 μL; ddH2O: 18 μL.

[0045] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 59°C for 30 s, extension at 72°C for 5 min, for a total of 30 cycles; post-extension at 72°C for 10 min; storage at 4°C.

[0046] The PCR results were verified by DNA agarose gel electrophoresis, and the results showed that the amplified product was a single band with a size of about 5000 bp. The PCR product was then digested with DpnI enzyme template.

[0047] Example 3: Construction of dual-plasmid engineering bacteria

[0048] The glutamate dehydrogenase gene (SEQ ID No.9) and the formate dehydrogenase gene (SEQ ID No.10) are located in the first multiple cloning site and the second multiple cloning site on the plasmid pETDuet-1, respectively. The plasmid is named pETDuet-1-GluDH-FDH. The nicotinic acid phosphoribosyltransferase gene is located in the first multiple cloning site of the pRSFDuet-1 plasmid, named pRSFDuet-1-NAPRT. The two plasmids were extracted using a plasmid extraction kit, introduced into the same BL21 (DE3) competent cells, spread on ampicillin and kanamycin resistance plates, and cultured for 12 hours. Pick a single colony and inoculate it into 10ml LB liquid culture medium containing the corresponding resistance, and shake and culture at 37°C for 10h. Add 30% glycerol to the bacterial solution at a ratio of 1:1 and store in a -80 degree refrigerator.

[0049] Example 4: Cultivation of dual-plasmid engineered bacteria

[0050] When using engineered bacteria as catalysts for large-scale preparation of products, a certain amount of bacteria is required. Therefore, it is necessary to culture and prepare the constructed engineered bacteria on a large scale. After the glycerol tube of the preserved glutamate dehydrogenase, formate dehydrogenase and nicotinic acid phosphoribosyltransferase co-expressing engineered bacteria is activated by streaking on a plate, a single colony is picked and inoculated into 10mL LB liquid culture medium containing 50μg / mL ampicillin and kanamycin resistance, and cultured at 37°C with shaking for 12h. Transfer the inoculum to 50ml TB liquid culture medium containing 50ug / mL ampicillin and kanamycin resistance at a 2% inoculum volume, and culture at 37°C with shaking until OD 600 When the concentration reached about 0.8, IPTG was added to a final concentration of 0.1 mM, and cultured at 28°C for 16 h. After the culture was completed, the culture solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the cells were collected, and stored in a -20°C refrigerator for later use.

[0051] Example 5: Recombinant co-expressing bacteria of glutamate dehydrogenase gene, formate dehydrogenase gene and nicotinic acid phosphoribosyltransferase gene catalyze PPO reaction to produce L-phosphinothricin

[0052] A certain amount of bacterial cells were prepared and collected according to Example 4. The intermediate product PPO was used as a substrate, ammonium formate was used as a coenzyme regeneration substrate, and endogenous NADPH was used without adding exogenous NADPH or NADP. + , pH7.5 phosphate buffer was used as the reaction medium to form a 10mL reaction system. The catalyst dosage was 20g / L wet bacteria, the final concentration of PPO was 300mM, the final concentration of ammonium formate was 600mM, 35℃, 600rpm for 4h, 100ul of the reaction solution was added to a 1.5mL EP tube containing 5ul 6M hydrochloric acid to terminate the reaction. Centrifuge at 1200rmp for 1min, take 20uL of the supernatant to a new 2mL EP tube, add 980ul ultrapure water, and shake to mix for HPLC detection of PPO.

[0053] PPO concentration detection: Yuexu Technology C18 chromatographic column (5μm, 100A, 4.6mm×250mm), mobile phase is 50mM diammonium phosphate solution: acetonitrile = 88:12 (diammonium phosphate solution: weigh 5.75g diammonium phosphate and dissolve it in 800ml ultrapure water, add 1.0g tetrabutylammonium hydroxide, adjust the pH to 3.8 with phosphoric acid, and make up to 1L), flow rate is 1mL / min, column oven is 40℃, detection wavelength is 232nm. The conversion rate is 100% after 8 hours of reaction.

[0054] Example 6: Application of nicotinic acid phosphoribosyltransferase and its mutants in the preparation of L-phosphinothricin ammonium

[0055] Preparation of L-phosphinothricin by co-expression of glutamate dehydrogenase, formate dehydrogenase, nicotinic acid phosphoribosyltransferase and their mutants:

[0056] Wet cells co-expressing glutamate dehydrogenase, formate dehydrogenase, nicotinic acid phosphoribosyltransferase and their mutants were prepared by the method of Example 4. Reaction system: 400mM PPO, 800mM ammonium formate, pH 7.5. After 4h of reaction, samples were taken for liquid phase analysis. The results showed that, compared with the original nicotinic acid phosphoribosyltransferase, the yield of the L360A mutant increased by 63.98%, the yield of the L360A-A223K mutant increased by 125.04%, and the yield of the L360A-K372D mutant increased by 91.51%.

[0057] Embodiment 7:

[0058] Optimization of pH in the process of PPO producing L-phosphinothricin ammonium by engineering bacteria co-expressing glutamate dehydrogenase gene, formate dehydrogenase gene and nicotinic acid phosphoribosyltransferase gene:

[0059] A certain amount of bacterial cells were prepared and collected according to the method of Example 4. The intermediate product PPO was used as a substrate, ammonium formate was used as a coenzyme regeneration substrate, and endogenous NADPH was used without adding exogenous NADPH or NADP. + , a 10mL reaction system was formed with phosphate buffer with pH values ​​of 5.5, 6.5, 7.5, and 8.5 as the reaction medium. The catalyst dosage was 20g / L wet bacteria, the final concentration of PPO was 400mM, the final concentration of ammonium formate was 800mM, 35°C, 600 rpm for 4h, 100ul of the reaction solution was added to a 1.5mL EP tube containing 5ul 6M hydrochloric acid to terminate the reaction. Centrifuge at 1200rmp for 1min, take 20ul of the supernatant to a new 2mL EP tube, add 980ul of ultrapure water, and shake and mix for HPLC detection of PPO. The detection method refers to Example 4.

[0060] After 6 hours of reaction, the PPO conversion rate in the system with a pH of 5.5 was 65.19%; the PPO conversion rate in the system with a pH of 6.5 was 89.47%; the PPO conversion rate in the system with a pH of 7.5 was 100%; and the PPO conversion rate in the system with a pH of 8.5 was 90.98%. The optimal reaction pH was 7.5, and when the PPO substrate concentration was 400 mM, it could be completely converted to L-phosphinothricin.

[0061] Therefore, the optimal pH condition for the PPO reaction to produce L-glufosinate by the engineered bacteria co-expressing glutamate dehydrogenase gene, formate dehydrogenase gene and nicotinic acid phosphoribosyltransferase gene is 7.5.

[0062] Embodiment 8:

[0063] Temperature optimization in the process of producing L-phosphinothricin by PPO reaction catalyzed by engineered bacteria co-expressing glutamate dehydrogenase gene, formate dehydrogenase gene and nicotinic acid phosphoribosyltransferase gene:

[0064] A certain amount of bacterial cells were prepared and collected according to the method of Example 4. The intermediate product PPO was used as a substrate, ammonium formate was used as a coenzyme regeneration substrate, and endogenous NADPH was used without adding exogenous NADPH or NADP. + , phosphate buffer with reaction temperature of 25, 30, 35, 40, 45 ℃ was used as reaction medium to form a 10mL reaction system. The catalyst dosage was 20g / l wet bacteria, the final concentration of PPO was 400mM, the final concentration of ammonium formate was 800mM, the pH was 7.5, and the reaction was carried out at 600 rpm for 4h. 100ul of the reaction solution was added to a 1.5mL EP tube containing 5ul 6M hydrochloric acid to terminate the reaction. Centrifuge at 1200rmp for 1min, take 20ul of the supernatant to a new 2mL EP tube, add 980ul ultrapure water, and shake and mix for HPLC detection of PPO. The detection method is referred to Example 4.

[0065] After 4 hours of reaction, the PPO conversion rate in the 25°C system was 74.47%, in the 30°C system was 91.05%, in the 35°C system was 100%, in the 40°C system was 95.22%, and in the 45°C system was 89.41%. The optimal reaction temperature is 35°C.

[0066] Embodiment 9:

[0067] Comparison of intracellular NAD content between the wild-type strain pRSFDuet-1-NAPRT and the mutants pRSFDuet-1-L360A, pRSFDuet-1-L360A-A223K and pRSFDuet-1-L360A-K372D of nicotinic acid phosphoribosyltransferase:

[0068] The constructed mutant strain was streaked from the glycerol tube, cultured for 12 h, transferred to a test tube with the corresponding resistance, cultured at 180 rpm for 8 h, and then transferred to 100 mL of TB medium. 600 When the pH value was between 0.6 and 0.8, 0.1 mM IPTG was added for induction for 14 h at a temperature of 24°C. The fermentation broth was collected and the intracellular coenzyme levels of different mutants were detected using the Bio-Tian coenzyme kit purchased in Example 1. The detection data was processed as follows: Figure 5 The intracellular NAD of mutant L360A increased by 51.66%, that of mutant L360A-A223K increased by 78.93%, and that of mutant L360A-K372D increased by 68.25%.

[0069] Embodiment 10:

[0070] Preparation of D-amino acids by co-expression of amino acid dehydrogenase, formate dehydrogenase, nicotinic acid phosphoribosyltransferase and their mutants:

[0071] The wet cells co-expressing amino acid dehydrogenase (see SEQ ID NO.12 for the coding gene sequence), formate dehydrogenase (see SEQ ID NO.11 for the coding gene sequence) and nicotinic acid phosphoribosyltransferase or nicotinic acid phosphoribosyltransferase mutant were constructed and prepared with reference to the methods of Examples 3 and 4. 20 g / L of the co-expressed wet cells were used as biocatalysts. Reaction system: 80 mM ammonium formate, 100 mM pyruvic acid. Samples were taken after 4 hours of reaction for liquid phase analysis. The results showed that, relative to nicotinic acid phosphoribosyltransferase, the yield of the L360A mutant increased by 71.33%, the yield of the L360A-A223K mutant increased by 109.19%, and the yield of the L360A-K372D mutant increased by 87.60%.

[0072] Embodiment 11:

[0073] Preparation of L-tert-leucine by co-expression of leucine dehydrogenase, formate dehydrogenase, nicotinic acid phosphoribosyltransferase and their mutants:

[0074] The wet bacteria co-expressing leucine dehydrogenase (see SEQ ID NO.13 for the coding gene sequence), formate dehydrogenase (see SEQ ID NO.11 for the coding gene sequence) and nicotinic acid phosphoribosyltransferase or nicotinic acid phosphoribosyltransferase mutant were constructed and prepared with reference to the methods of Examples 3 and 4. Reaction system: 100 mM 3-methylpyruvic acid, 200 mM ammonium formate, pH 7. After 4 h of reaction, samples were taken for liquid phase analysis. The results showed that, relative to nicotinic acid phosphoribosyltransferase, the yield of L360A mutant increased by 80.15%, the yield of L360A-A223K mutant increased by 168.74%, and the yield of L360A-K372D mutant increased by 115.87%.

[0075] Embodiment 12:

[0076] Preparation of L-aminobutyric acid by co-expression of leucine dehydrogenase, formate dehydrogenase, nicotinic acid phosphoribosyltransferase and their mutants:

[0077] The wet bacteria co-expressing leucine dehydrogenase (see SEQ ID NO.13 for the coding gene sequence), formate dehydrogenase (see SEQ ID NO.11 for the coding gene sequence) and nicotinic acid phosphoribosyltransferase or nicotinic acid phosphoribosyltransferase mutant were constructed and prepared with reference to the methods of Examples 3 and 4. Reaction system: 1M 2-carbonylbutyric acid, 500mM ammonium formate. After 4h of reaction, samples were taken for liquid phase analysis. The results showed that, relative to nicotinic acid phosphoribosyltransferase, the yield of L360A mutant increased by 86.98%, the yield of L360A-A223K mutant increased by 167.01%, and the yield of L360A-K372D mutant increased by 115.96%.

Claims

1. A nicotinic acid phosphoribosyltransferase mutant, characterized in that The amino acid sequence of the mutant is shown in one of SEQ ID NOs. 6 to 8.

2. A gene encoding the nicotinic acid phosphoribosyltransferase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in one of SEQ ID NOs. 3 to 5.

4. A vector containing the coding gene according to claim 3.

5. A genetically engineered bacterium containing the coding gene according to claim 3.

6. Use of the nicotinic acid phosphoribosyltransferase mutant according to claim 1 in the biosynthesis of L-phosphinothricin ammonium by a multi-enzyme coupling method, wherein L-phosphinothricin ammonium is prepared by using PPO as a substrate and ammonium formate as a co-substrate under the catalysis of a multi-enzyme system of glutamate dehydrogenase, formate dehydrogenase and the nicotinic acid phosphoribosyltransferase mutant.

7. Use of the nicotinic acid phosphoribosyltransferase mutant according to claim 1 in the biosynthesis of D-amino acids by a multi-enzyme coupling method, wherein pyruvate is used as a substrate and D-amino acids are prepared under the catalysis of an amino acid dehydrogenase, formate dehydrogenase and a nicotinic acid phosphoribosyltransferase mutant multi-enzyme system.

8. Use of the nicotinic acid phosphoribosyltransferase mutant according to claim 1 in the biosynthesis of L-tert-leucine by a multi-enzyme coupling method, wherein 3-methylpyruvate is used as a substrate, and L-tert-leucine is prepared under the catalysis of a multi-enzyme system of leucine dehydrogenase, formate dehydrogenase and the nicotinic acid phosphoribosyltransferase mutant.

9. Use of the nicotinic acid phosphoribosyltransferase mutant according to claim 1 in the biosynthesis of L-aminobutyric acid by a multi-enzyme coupling method, wherein 2-carbonylbutyric acid is used as a substrate and L-aminobutyric acid is prepared under the catalysis of a multi-enzyme system of leucine dehydrogenase, formate dehydrogenase and the nicotinic acid phosphoribosyltransferase mutant.

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