Marine metagenomic-derived meso-diaminopimelate dehydrogenase mutants and their application in D-tryptophan biosynthesis

Abstract

The present invention belongs to the field of biochemical engineering technology, and specifically relates to marine metagenome-derived meso-diaminopimelate dehydrogenase mutants and their applications in D-tryptophan synthesis. The present invention uses a rapid evolution method based on Rosetta guidance to transform a meso-diaminopimelate dehydrogenase derived from a marine metagenome, and obtains a mutant with high catalytic activity for 3-indolepyruvate by screening. The meso-diaminopimelate dehydrogenase mutant disclosed in the present invention can be applied to directly catalyze the reductive amination of 3-indolepyruvate to generate chiral D-tryptophan, and can also be coupled with L-amino acid deaminase and coenzyme regeneration enzyme to achieve the configuration flip of L-tryptophan to D-tryptophan, the substrate concentration reaches 0.1 M, the product conversion rate>95%, the ee value>99%, and has a high practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to a meso-diaminopimelate dehydrogenase mutant derived from a marine metagenome and application thereof in D-tryptophan synthesis. Background Art

[0002] D-Tryptophan is a non-protein optically active amino acid with the chemical name D-2-amino-3-indolyl-1-propionic acid. D-Tryptophan has special physiological and biochemical properties and has important application value in food, medicine and agriculture. The sweetness of D-tryptophan is 35 times that of sucrose, so it can be used as a non-nutritive sweetener. In terms of food preservation, D-tryptophan is considered to be an effective food preservative that can be used to reduce or replace nitrite to improve food preservation safety. In the field of medicine, a variety of drugs with D-tryptophan as a key synthetic intermediate have been successfully launched and widely used. For example, Octreotide synthesized from D-tryptophan has a good therapeutic effect on acute pancreatitis, esophageal varicose vein bleeding caused by liver cirrhosis, gastrinoma, acromegaly, intestinal fistula, etc.; Macrilen synthesized from D-tryptophan is a growth hormone receptor agonist that can be used to diagnose adult growth hormone deficiency; the most important one is tadalafil synthesized from D-tryptophan, which is indicated for the treatment of male erectile dysfunction (ED). Therefore, considering the wide application and growing demand of D-tryptophan, it is of great significance to develop an efficient method for synthesizing D-tryptophan.

[0003] At present, chemical catalysis and enzyme catalysis are the two main methods for preparing D-tryptophan. Among them, chemical catalysis is easy to scale up, but it is usually limited by complex reaction conditions, serious environmental pollution and poor stereoselectivity. In comparison, enzyme catalysis has the advantages of mild reaction conditions, strict stereoselectivity and few side reactions, and has received much attention from academia and industry. Meso-diaminopimelate dehydrogenase (DAPDH; EC 1.4.1.16) is a NADP-containing enzyme. +DAPDH is a coenzyme oxidoreductase that can selectively catalyze the reversible reductive amination of α-keto acids to generate the corresponding D-amino acids. The reductive amination reaction catalyzed by DAPDH has strict stereoselectivity, high atom economy, and the product is easy to separate and purify. Therefore, it is regarded as one of the ideal processes for the synthesis of D-amino acids. However, the catalytic activity of wild-type DAPDH on non-natural keto acid substrates is generally low, which limits its practical application. In order to improve its catalytic ability for a variety of different substrates, researchers have carried out a large number of studies on the substrate specificity modification of DAPDH, which has improved the catalytic activity of DAPDH on some non-natural substrates to a certain extent. However, previous modifications to DAPDH have all adopted strategies that require multiple rounds of iterative screening, which is time-consuming and labor-intensive, and the catalytic activity for the D-tryptophan precursor, 3-indolepyruvate, is still low, making it impossible for the reported DAPDH-based D-tryptophan synthesis processes to obtain high substrate concentrations and ideal conversion rates (ACSSynth. Biol.. 2024, 13, 1879-1892; Catal. Sci. Technol.. 2018, 8, 4994). Therefore, in order to develop an efficient enzymatic synthesis process for D-tryptophan based on DAPDH, it is urgent to adopt a more efficient strategy to specifically modify the 3-indolepyruvate reductive amination activity of DAPDH to obtain a new mutant with high catalytic activity. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the present invention mines and obtains a new type of meso-diaminopimelate dehydrogenase from the marine seawater metagenome, and transforms it using a Rosetta-guided rapid evolution method. A plurality of mutants with high reductive amination activity for 3-indolepyruvate are obtained by screening, and an enzymatic synthesis process for D-tryptophan is established for these mutants, providing a new and efficient approach for the efficient synthesis of D-tryptophan.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention provides a meso-diaminopimelate dehydrogenase mutant, wherein the meso-diaminopimelate dehydrogenase mutant is a mutant formed by mutation of all or part of the amino acid residues at positions 93, 120, 145, 153, 170, 180 and 226 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1.

[0007] The present invention obtains a novel meso-diaminopimelate dehydrogenase (named SCSDAPDH, SEQ ID NO.1) by mining the metagenomic DNA of seawater in the South China Sea. Subsequently, D-tryptophan is used as a ligand molecule, and the substrate pocket of SCSDAPDH is designed using the enzyme design program of Rosetta software, and the mutation of the mutants is counted and a simplified mutation library is constructed. The simplified mutation library is then screened using a high-throughput screening method, and multiple mutants with high reductive amination activity for 3-indolepyruvate are successfully obtained.

[0008] Preferably, the meso-diaminopimelate dehydrogenase mutant is at least one of the following mutants:

[0009] (1) A combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 120 is mutated to methionine, the amino acid residue at position 145 is mutated to tryptophan, the amino acid residue at position 153 is mutated to leucine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to leucine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant is shown in SEQ ID NO.2;

[0010] (2) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 145 is mutated to isoleucine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to isoleucine and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant is shown in SEQ ID NO.3;

[0011] (3) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 120 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to isoleucine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant is shown in SEQ ID NO.4;

[0012] (4) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to leucine, and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO.5;

[0013] (5) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 145 is mutated to isoleucine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to methionine and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant is shown in SEQ ID NO.6;

[0014] (6) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to methionine, and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO.7;

[0015] (7) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to methionine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant being shown in SEQ ID NO.8;

[0016] (8) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to leucine, and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant being shown in SEQ ID NO.9;

[0017] (9) A combined mutant in which the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to isoleucine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant is shown in SEQ ID NO.10.

[0018] The second aspect of the present invention provides a gene encoding the meso-diaminopimelate dehydrogenase mutant described in the first aspect, wherein the gene encoding the mutant is selected from at least one of the nucleotide sequences shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22.

[0019] The third aspect of the present invention provides a biological material comprising the encoding gene described in the second aspect, wherein the biological material comprises an expression vector and a recombinant engineered bacterium.

[0020] Preferably, the expression vector is pET-28a(+) comprising the above-mentioned meso-diaminopimelate dehydrogenase mutant gene.

[0021] Preferably, the recombinant engineered bacteria is Escherichia coli BL21 (DE3) into which the above-mentioned meso-diaminopimelate dehydrogenase mutant expression vector is introduced.

[0022] The fourth aspect of the present invention provides the use of the meso-diaminopimelate dehydrogenase mutant described in the first aspect, or the encoding gene described in the second aspect, or the biomaterial described in the third aspect in synthesizing D-tryptophan.

[0023] The fifth aspect of the present invention provides a method for catalytically converting 3-indolepyruvate to synthesize chiral D-tryptophan, which comprises: first preparing an enzyme solution of the meso-diaminopimelate dehydrogenase mutant and a coenzyme regeneration enzyme as described in the first aspect, and then adding the prepared enzyme solution to a mixture containing a 3-indolepyruvate substrate, ammonium formate, superoxide dismutase (SOD) and NADP + In a mixed system, the pH of the system is controlled between 7.0 and 9.0 to carry out a reductive amination reaction to obtain chiral D-tryptophan.

[0024] Preferably, the enzyme solution is a resting cell suspension of a recombinant engineering bacterium capable of expressing the meso-diaminopimelate dehydrogenase mutant or the coenzyme regeneration enzyme, or a crude enzyme solution obtained by cell disruption, or a purified pure enzyme solution; the coenzyme regeneration enzyme is formate dehydrogenase using formic acid as the coenzyme regeneration substrate, and its amino acid sequence is as shown in SEQ ID NO.11.

[0025] Preferably, in each 1 mL reaction system, it contains 0.5 - 2 mg of the enzyme solution of the meso-diaminopimelate dehydrogenase mutant, 0.5 - 2 mg of the enzyme solution of the coenzyme regeneration enzyme, 10 - 100 mM of the 3-indolepyruvic acid substrate, 50 - 150 mM of ammonium formate, 0.1 - 1.0 mM of NADP + , and 150 - 250 U / mL of SOD.

[0026] Preferably, the reaction temperature is 25 - 50 °C.

[0027] The sixth aspect of the present invention provides a method for catalyzing the configuration inversion of L-tryptophan to D-tryptophan, specifically: first prepare the enzyme solutions of the meso-diaminopimelate dehydrogenase mutant described in the first aspect, L-amino acid deaminase shown in SEQ ID NO.12, and the coenzyme regeneration enzyme, and then add the prepared enzyme solutions into a mixed system containing the L-tryptophan substrate, ammonium formate, and NADP + , control the pH of the system to be between 7.0 - 9.0, and carry out the reaction under aerobic conditions to obtain chiral D-tryptophan.

[0028] Preferably, in each 20 mL reaction system, it contains 8 - 12 mL of the enzyme solution, 50 - 200 mM of the L-tryptophan substrate, 200 - 1000 mM of the inorganic amino donor, 0.1 - 1.0 mM of NADP + , and in the enzyme solution, the mass ratio of the enzyme solution of the meso-diaminopimelate dehydrogenase mutant, the enzyme solution of L-amino acid deaminase, and the enzyme solution of the coenzyme regeneration enzyme is 2:1:2.

[0029] Preferably, the enzyme solution is a resting cell suspension of a recombinant engineering bacterium capable of expressing the meso-diaminopimelate dehydrogenase mutant or L-amino acid deaminase or the coenzyme regeneration enzyme, or a crude enzyme solution obtained by cell disruption, or a purified pure enzyme solution; the coenzyme regeneration enzyme is formate dehydrogenase using formic acid as the coenzyme regeneration substrate, and its amino acid sequence is as shown in SEQ ID NO.11.

[0030] Preferably, the reaction temperature is 25 - 50 °C.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] The present invention adopts a rapid evolution method based on Rosetta guidance to transform a meso-diaminopimelate dehydrogenase from a marine metagenome source, and obtains a mutant with high catalytic activity for 3-indolepyruvate by screening. The present invention starts from new enzyme mining, combines the rapid evolution method guided by Rosetta, and solves the problem of low catalytic activity of wild-type meso-diaminopimelate dehydrogenase for 3-indolepyruvate. The meso-diaminopimelate dehydrogenase mutant disclosed in the present invention not only has high catalytic activity, but also has strict stereoselectivity. These meso-diaminopimelate dehydrogenase mutants can be directly used to catalyze the asymmetric reductive amination of 3-indolepyruvate to generate D-tryptophan, and can also be coupled with L-amino acid deaminase and coenzyme regeneration enzyme to achieve the configuration flip of L-tryptophan to D-tryptophan, the substrate concentration is up to 0.1 M, the product conversion rate is>95%, and the ee value is>99%, which has a high practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the method for constructing a meso-diaminopimelate dehydrogenase mutant;

[0034] Figure 2 Design catalytic constraint settings for Rosetta;

[0035] Figure 3 To calculate the mutation statistics of designed mutants;

[0036] Figure 4 To streamline the screening results of combinatorial mutation libraries;

[0037] Figure 5 This is the reaction equation for the synthesis of D-tryptophan from 3-indolepyruvate catalyzed by meso-diaminopimelate dehydrogenase;

[0038] Figure 6 This is the reaction equation for the configurational inversion of L-tryptophan to D-tryptophan catalyzed by a three-enzyme cascade. DETAILED DESCRIPTION

[0039] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0041] The experimental methods described in the following examples are conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

[0042] The DNA polymerase (2×Phanta Max Master Mix), Dpn I enzyme, recombination cloning kit and plasmid extraction kit used in the following examples were all purchased from Nanjing Novezan Biotechnology Co., Ltd.; gene synthesis, primer synthesis and gene sequencing were all completed by Qingke Zixi Biotechnology (Guangzhou) Co., Ltd., and the use of the above reagents was referred to the product instructions.

[0043] The expression vector pET-28a (+), the host Escherichia coli BL21 (DE3) used in the following examples, and the metagenomic DNA of the South China Sea seawater sample were all preserved by the Marine Natural Products Laboratory of the School of Marine Sciences, Sun Yat-sen University where the inventors are located.

[0044] The 3-indolepyruvic acid and tryptophan standards used in the following examples were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; kanamycin, ammonium chloride, superoxide dismutase (SOD), isopropyl-β-D-thiogalactopyranoside (IPTG), NADP + and NADPH were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; oxygen was purchased from Zhuhai Huaxin Gas Co., Ltd.; other commonly used reagents can be purchased from the market.

[0045] The three-letter or single-letter amino acid expressions used in the following examples all adopt the amino acid code specified by IUPAC (Eur. J. Biochem., 138:9-37, 1984).

[0046] In the following examples, the standard method for bacterial culture and protein purification is as follows: after streaking and activating the recombinant engineering strain [Escherichia coli BL21 (DE3) containing the corresponding mutant gene] constructed by the present invention from the glycerol tube, a single colony was picked and inoculated into 5 mL LB liquid culture medium containing 50 μg / mL kanamycin (Kan), and cultured at 37°C and 200 rpm for 6 h. Then, the inoculum was transferred to 250 mL of fresh TB liquid culture medium containing 50 μg / mL Kan at a 2% inoculum amount, and after 37°C and 200 rpm shaking culture for 3 h, IPTG was added to a final concentration of 0.5 mM, and induced culture was performed at 25°C for 16 h. After the culture was completed, the culture solution was centrifuged at 12000 g and 4°C for 10 min, the bacterial cells were collected, the cells were ultrasonically broken, and the protein was purified by nickel column affinity chromatography.

[0047] In the following examples, the detection system for the reductive amination activity of 3-indolepyruvate is: 0.005-0.1 mg of pure enzyme, 20 mM 3-indolepyruvate, 10 mM NADPH, 500 mM ammonium chloride, the total system is 400 μL, and the reaction medium is 0.2 M NaCl at pH = 8.5. 2 CO 3 -NaHCO 3 The reaction was carried out at 30°C and 600 rpm. After 10 min, 10 μL of 5 M NaOH solution was immediately added to terminate the reaction, followed by 400 μL of ddH 2 O for dilution, and 100 μL was taken for derivatization and subsequent HPLC analysis.

[0048] In the following examples, the definition of enzyme activity unit (U) is: the amount of enzyme required to generate 1 μmol D-tryptophan per minute under standard reaction conditions.

[0049] In the following examples, the concentration and ee value of D,L-tryptophan were analyzed by pre-column derivatization high performance liquid chromatography (HPLC), and the specific analysis method was as follows:

[0050] (1) Chromatographic conditions: Column model: Agilent ZPRBAX SB-C18, 5 μm, 4.6×250 mm; Mobile phase: 50 mM sodium acetate solution: acetonitrile = 0.84:0.16; Detection wavelength: 338 nm; Flow rate: 1 mL / min; Column temperature: 30°C.

[0051] (2) Preparation of derivatization reagent: Weigh 0.03 g of o-phthalaldehyde and 0.1 g of N-acetyl-L-cysteine ​​respectively, dissolve them in 400 μL of anhydrous ethanol, add 4 mL of 0.2 mol / L boric acid buffer (pH 9.8), shake to fully dissolve, and store in a refrigerator at 4°C until used.

[0052] (3) Derivatization reaction and determination: Take 100 μL of sample and add 150 μL of derivatization reagent. Mix well and keep at 25℃ for 5 min. Then add 1 mL of ddH 2 O for dilution, and after passing through the membrane, 20 μL was injected for reaction and determination.

[0053] Example 1: Construction and activity verification of meso-diaminopimelate dehydrogenase mutants

[0054] In this example, a rapid evolution method based on Rosetta guidance was used to transform a meso-diaminopimelate dehydrogenase from a marine metagenome to obtain a mutant with high catalytic activity for 3-indolepyruvate. Figure 1As shown in the figure, it specifically includes the following parts:

[0055] 1. Mining of meso-diaminopimelate dehydrogenase and construction of recombinant engineering bacteria

[0056] Collect the amino acid sequences of 5 reported class II DAPDHs, including StDAPDH from Symbiobacterium thermophilum (NCBI ID: BAD40410.1), PgiDAPDH from Porphyromonas gingivalis (NCBI ID: AAQ65966.1), BfaDAPDH from Bacteroides faecis (NCBI ID: ZP_09861452.1), NmDAPDH from Numidum massiliense (NCBI ID: WP_054948699.1), and TlDAPDH from Thermosyntropha lipolytica (NCBI ID: WP_073090506.1), and use Hmmer 3.1 to construct a hidden Markov model (HMM) of class II DAPDH. Based on the established HMM model, search (Hmmsearch) the metagenomic annotation data of seawater samples in the South China Sea (coordinates: 17°09.853'N, 112°13.399'E), and successfully mine a potential DAPDH gene (named SCSDAPDH). The amino acid sequence of SCSDAPDH is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.13.

[0057] Design specific primers (Table 1), use SCSDAPDH-F and SCSDAPDH-R as primers to perform PCR amplification of the SCSDAPDH gene from the metagenomic DNA of seawater samples in the South China Sea, and use 28a-F and 28a-R as primers to amplify the linearized pET-28a(+) vector.

[0058] Table 1 SCSDAPDH cloning primers

[0059]

[0060] a : The restriction enzyme sites are underlined.

[0061] The PCR reaction system and reaction conditions are as follows:

[0062] The PCR amplification system was as follows: DNA polymerase 25 µL, upstream primer (10 pmol / µL) 2.0 µL, downstream primer (10 pmol / µL) 2.0 µL, template 1.0 µL, ddH 2 O 20 µL. PCR amplification conditions were: 1) pre-denaturation: 95℃ 3 min; 2) denaturation: 95℃ 15 s; annealing: 56℃ 15 s; extension: 72℃ 0.5-2.5 min; 30 cycles in total; 3) post-extension: 72℃ 8 min; 4) storage at 4℃.

[0063] Referring to the instruction manual of the ClonExpress II One Step Cloning Kit, the two amplified fragments were digested with DpnⅠ and recovered on gel, and then the two fragments were recombined and transformed into Escherichia coli BL21 (DE3) competent cells. The plates were plated, single colonies were picked for culture, and sequencing was performed to verify the correctness of the cloned gene. The successfully constructed SCSDAPDH heterologous expression recombinant engineering bacteria were stored at -80℃ for future use.

[0064] 2. Rapid evolution of SCSDAPDH guided by Rosetta

[0065] (1) Rosetta design and streamlined combinatorial mutation library design

[0066] The 3D model of SCSDAPDH was predicted using Alphafold3 (https: / / golgi.sandbox.google.com / ), and the online tool BCL::Conf Server (http: / / carbon.structbio.vanderbilt.edu / index.php / bclconf) was used to search for small molecule conformations of D-tryptophan. The catalytic geometric constraints were set by referring to the interaction mode between the enzyme protein and small molecules in the StDAPDH crystal structure (PDB ID: 3WBF). Figure 2 , Table 2), and selected the design sites (H93, W120, F145, H153, T170, R180, H226). Subsequently, the enzyme design program of Rosetta was called to design SCSDAPDH, and the mutation types contained in each site of the obtained designed mutants were statistically analyzed ( Figure 3). Based on the statistical results, a simplified combinatorial mutation library was designed, specifically: tryptophan and methionine were randomly introduced into position 93, tryptophan, methionine and tyrosine were randomly introduced into position 120, isoleucine, phenylalanine and tryptophan were randomly introduced into position 145, methionine and isoleucine were randomly introduced into position 153, valine was introduced into position 170, leucine, isoleucine and methionine were randomly introduced into position 180, and isoleucine, methionine and isoleucine were randomly introduced into position 226.

[0067] Table 2 Distance and angle constraint setting parameters

[0068]

[0069] (2) Construction of streamlined combinatorial mutation library

[0070] The first stage: synthesize specific primers (Table 3), use the pET-28a(+)-SCSDAPDH recombinant plasmid containing the SCSDAPDH gene obtained in Example 1 as a template, and use 170_F and 170_R as primer pairs to perform whole plasmid PCR. The PCR system and conditions refer to Example 1. After the PCR is completed, the amplified product is digested with DpnⅠ digestion enzyme for 1 h to remove the template plasmid, and then the digestion product is transformed into E. coli BL21 (DE3) competent cells, plated, and cultured overnight, and then a single colony is picked to LB liquid culture, sequencing is used to verify the correctness of the mutation, and the T170V mutant single colony that shows the mutation is correct after verification is stored at -80°C for later use.

[0071] Phase II: Extract T170V mutant plasmid as the second phase PCR template. Among them, the mixed primer I (93_ATG_F:93_TGG_F=1:1) and 120_R were used as primer pairs to obtain linearized fragment I by PCR, the mixed primer II (120_TGG_F:120_TAT_F:120_ATG_F =1:1:1) and mixed primer III (145_CCA_R:145_AGW_R=1:2) were used as primer pairs to obtain linearized fragment II by PCR, and then 153_MTG_F and mixed primer IV (180_AAT_R:180_CAK_R =1:2) were used as primer pairs to obtain linearized fragment III by PCR, 180_F and mixed primer V (226_AAT_R:226_CAK_R=1:2) were used as primer pairs to obtain linearized fragment IV by PCR, and finally 226_F The linearized fragment V was obtained by PCR with 93_R as the primer pair. The PCR system and conditions refer to Example 1.

[0072] Referring to the instruction manual of the ClonExpress MultiS One Step Cloning Kit, the five fragments were subjected to subsequent DpnⅠ digestion and gel recovery, and then the five fragments were recombined in a reaction system and subsequently transformed into E. coli BL21 (DE) competent cells. These recombinant E. coli containing different mutant genes are the constructed streamlined combinatorial mutation library.

[0073] Table 3 Primers required for construction of simplified combinatorial mutation library

[0074]

[0075] (3) High-throughput screening of mutation libraries

[0076] Add 200 μL of LB medium (containing 50 μg / mL kanamycin) to a sterilized 96-deep-well plate, and then use a sterilized pipette tip to pick up a single colony to the 96-deep-well plate. Then place the deep-well plate at 37°C and 200 rpm for 6 h, which is called the primary plate. Add 400 μL of LB medium (containing 50 μg / mL kanamycin) to another sterilized 96-deep-well plate as the secondary plate, and draw 50 μL of bacterial solution from the primary plate to the secondary plate. Add 20% glycerol to the primary plate and place it in a -80°C refrigerator for long-term storage. Then place the secondary plate at 37°C for shaking culture for 3 h, add IPTG with a final concentration of 0.5 mM for induction, and then place the secondary plate at 25°C and 200 rpm for 16 h.

[0077] After centrifugation at 4000 rpm and 4°C for 20 min, collect the cells and then freeze them at -80°C for more than 24 h. Take the secondary plate out of -80°C and thaw it at room temperature for 0.5 h. Then add 400 μL of lysate (50 mM pH 7.5 phosphate buffer, 2 mg / mL lysozyme, 10 mg / L DNaseⅠ) to each well, shake to suspend the cells, and incubate them at 37°C shaker at 200 rpm for 1.5 h. After incubation, centrifuge at 4000 rpm and 4°C for 20 min, and take the supernatant for enzyme activity determination.

[0078] Add 80 μL of the activity test mixture (0.5 M pH 9.0 NH 4 CL-NH 3• H 2O buffer, 0.5 mM NADPH, 2 mM 3-indolepyruvate substrate, 500 U / mL SOD), and then aspirate 20 μL of supernatant enzyme solution and add it to the reaction plate to start the reaction. After reacting at 25℃ for 10 min, use a microplate reader to measure the absorbance at 340 nm. The lower the absorbance value, the higher the catalytic activity. The mutant strain with a lower absorbance value than the control (SCSDAPDH wild type) was selected for subsequent activity verification.

[0079] (4) Verification of the catalytic activity of 3-indolepyruvate

[0080] After screening a total of 1,000 single clones, the positive mutants obtained by screening were sequenced and their specific mutations were analyzed. Subsequently, the obtained mutants were fermented and purified, and the activity of the purified pure protein was determined using an activity standard detection system. Figure 4 As shown, through verification, a total of 9 mutants M1-M9 with significant catalytic activity for 3-indolepyruvate were obtained (amino acid sequences are shown in SEQ ID NO.2-10, and the corresponding coding genes are shown in SEQ ID NO.14-22), among which M12 had the highest catalytic activity, reaching 5.08 U / mg-protein, which is the DAPDH with the highest catalytic activity reported so far.

[0081] Specifically, mutant M1 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 120 being mutated to methionine, the amino acid residue at position 145 being mutated to tryptophan, the amino acid residue at position 153 being mutated to leucine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to leucine, and the amino acid residue at position 226 being mutated to methionine. Mutant M2 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 145 being mutated to isoleucine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to isoleucine, and the amino acid residue at position 226 being mutated to isoleucine. Mutant M3 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 120 being mutated to methionine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to isoleucine, and the amino acid residue at position 226 being mutated to methionine. Mutant M4 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to leucine, and the amino acid residue at position 226 being mutated to isoleucine. Mutant M5 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 145 being mutated to isoleucine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to methionine, and the amino acid residue at position 226 being mutated to isoleucine. Mutant M6 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to methionine, and the amino acid residue at position 226 being mutated to isoleucine.Mutant M7 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to methionine, and the amino acid residue at position 226 being mutated to methionine. Mutant M8 is a combined mutant formed by the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 being mutated to methionine, the amino acid residue at position 153 being mutated to methionine, the amino acid residue at position 170 being mutated to valine, the amino acid residue at position 180 being mutated to leucine, and the amino acid residue at position 226 being mutated to methionine. Mutant M9 is a combined mutant formed by the mutation of the 93rd amino acid residue of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 to methionine, the 153rd amino acid residue to methionine, the 170th amino acid residue to valine, the 180th amino acid residue to isoleucine and the 226th amino acid residue to methionine.

[0082] Example 2: Synthesis of D-tryptophan from 3-indolepyruvate catalyzed by meso-diaminopimelate dehydrogenase mutant

[0083] The reaction equation for the synthesis of D-tryptophan from 3-indolepyruvate catalyzed by the meso-diaminopimelate dehydrogenase mutant is as follows Figure 5 As shown, formate dehydrogenase (SEQ ID NO.11) is used to achieve NADPH regeneration.

[0084] First, the meso-diaminopimelate dehydrogenase mutant (SEQ ID NO.2-10) and formate dehydrogenase were fermented and purified according to standard methods to obtain pure enzymes. Then the reaction system was prepared: 1 mg pure SCSDAPDH mutant enzyme, 1 mg pure formate dehydrogenase enzyme, 25 mM 3-indolepyruvate, 100 mM ammonium formate, 0.5 mM NADP + , 200 U / mL SOD, the total system was 1 mL, and the reaction medium was 0.2 M phosphate buffer at pH = 8.0. The prepared system was then placed at 30°C and 800 rpm for 24 h, and 50 μL of the sample was sampled and analyzed for the concentration and ee value of D-tryptophan using pre-column derivatization HPLC.

[0085] The results are shown in Table 4. Compared with the wild type, the D-amino acid dehydrogenase mutants provided by the present invention all exhibit significantly higher catalytic efficiency. Among them, the product conversion rates of the four mutants M6, M7, M8, and M9 are all >99%, and the ee values ​​are all greater than 99%.

[0086] Table 4 Results of SCSDAPDH mutants catalyzing the synthesis of D-tryptophan from 3-indolepyruvate

[0087]

[0088] a Wild-type StDAPDH was used as a control to evaluate the effects of the mutants; b ND: No D-tryptophan product was detected in the reaction solution.

[0089] Example 3: Three-enzyme cascade catalyzes the configurational flip of L-tryptophan to D-tryptophan

[0090] The reaction equation for the configurational flip of L-tryptophan to D-tryptophan catalyzed by the three-enzyme cascade is as follows Figure 6 As shown, L-amino acid deaminase (SEQ ID NO.12) catalyzes the deamination of L-tryptophan to generate 3-indolepyruvate, and formate dehydrogenase (SEQ ID NO.11) is used to achieve NADPH regeneration. The following only takes the four mutants M6, M7, M8, and M9 as examples to show the coupling of SCSDAPDH mutants with L-amino acid deaminase and coenzyme regeneration enzyme to achieve the configuration flip from L-tryptophan to D-tryptophan:

[0091] First, the meso-diaminopimelate dehydrogenase mutant-M6 (SEQ ID NO.7) or M7 (SEQ ID NO.8) or M8 (SEQ ID NO.9) or M9 (SEQ ID NO.10), L-amino acid deaminase (SEQ ID NO.12) and formate dehydrogenase (SEQ ID NO.11) were fermented and cultured according to the standard method, and the recombinant engineered bacterial cells were collected by centrifugation. Then 1 g of meso-diaminopimelate dehydrogenase mutant wet cells, 0.5 g of L-amino acid deaminase wet cells and 1 g of formate dehydrogenase wet cells were resuspended in 10 mL pH 8.0 PBS buffer, and the cells were ultrasonically disrupted to obtain a crude enzyme solution. The crude enzyme solution was then added to a 100 mM L-tryptophan, 500 mM ammonium formate, 0.5 mM NADP + The reaction was started in a reaction solution (total volume 20 mL). The reaction conditions were 30°C, 700 rpm magnetic stirring, and the reaction medium was ddH 2 O, and the pH of the system was controlled to 8.0 by adding 2 M NaOH. Oxygen was introduced into the system every 1 h during the process. After 24 h of reaction, samples were taken and the concentration and ee value of tryptophan were analyzed by pre-column derivatization HPLC.

[0092] The test results show that for the reaction system of the M6 ​​mutant, the remaining L-tryptophan is 1.3 mM, the generated D-tryptophan is 92.1 mM, and the ee value is 97.2%. For the reaction system of the M7 mutant, the remaining L-tryptophan is 0 mM, the generated D-tryptophan is 96.7 mM, and the ee value is >99%. For the reaction system of the M8 mutant, the remaining L-tryptophan is 0 mM, the generated D-tryptophan is 98.2 mM, and the ee value is >99%. For the reaction system of the M9 mutant, the remaining L-tryptophan is 0 mM, the generated D-tryptophan is 97.4 mM, and the ee value is >99%.

[0093] In summary, the meso-diaminopimelate enzyme mutants M1-M9 (SEQ ID NO. 2-10) constructed in the present invention can be used to directly catalyze the reductive amination of 3-indolepyruvate to generate chiral D-tryptophan, and can also be coupled with L-amino acid deaminase and coenzyme regeneration enzyme to achieve the configuration flip from L-tryptophan to D-tryptophan. The substrate concentration reaches 0.1 M, the product conversion rate is >95%, and the ee value is >99%, which has a high practical application value.

[0094] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A meso-diaminopimelate dehydrogenase mutant, characterized in that: The meso-diaminopimelate dehydrogenase mutant is selected from the following 9 mutants: (1) A combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 120 is mutated to methionine, the amino acid residue at position 145 is mutated to tryptophan, the amino acid residue at position 153 is mutated to leucine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to leucine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant is shown in SEQ ID NO.2; (2) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 145 is mutated to isoleucine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to isoleucine and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant is shown in SEQ ID NO.3; (3) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 120 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to isoleucine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant is shown in SEQ ID NO.4; (4) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to leucine, and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO.5; (5) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 145 is mutated to isoleucine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to methionine and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant is shown in SEQ ID NO.6; (6) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to methionine, and the amino acid residue at position 226 is mutated to isoleucine, the amino acid sequence of the mutant being shown in SEQ ID NO.7; (7) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to methionine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant being shown in SEQ ID NO.8; (8) a combined mutant of the amino acid sequence of meso-diaminopimelate dehydrogenase shown in SEQ ID NO.1, wherein the amino acid residue at position 93 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to leucine, and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant being shown in SEQ ID NO.9; (9) A combined mutant in which the amino acid residue at position 93 of the meso-diaminopimelate dehydrogenase amino acid sequence shown in SEQ ID NO.1 is mutated to methionine, the amino acid residue at position 153 is mutated to methionine, the amino acid residue at position 170 is mutated to valine, the amino acid residue at position 180 is mutated to isoleucine and the amino acid residue at position 226 is mutated to methionine, the amino acid sequence of the mutant is shown in SEQ ID NO.

10.

2. The gene encoding the meso-diaminopimelate dehydrogenase mutant according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.

22.

3. The biological material comprising the encoding gene according to claim 2, characterized in that: The biological materials include expression vectors and recombinant engineering bacteria.

4. Use of the meso-diaminopimelate dehydrogenase mutant according to claim 1, or the encoding gene according to claim 2, or the biomaterial according to claim 3 in synthesizing D-tryptophan.

5. A method for catalytically converting 3-indolepyruvic acid to synthesize chiral D-tryptophan, characterized in that: First, prepare the enzyme solution of the meso-diaminopimelate dehydrogenase mutant and the coenzyme regeneration enzyme as claimed in claim 1, and then add the prepared enzyme solution containing 3-indolepyruvate substrate, ammonium formate, superoxide dismutase and NADP + In a mixed system, the pH of the system is controlled between 7.0 and 9.0 to carry out a reductive amination reaction to obtain chiral D-tryptophan.

6. The method for catalytically converting 3-indolepyruvic acid into chiral D-tryptophan according to claim 5, characterized in that: The enzyme solution is a resting cell suspension of a recombinant engineered bacterium capable of expressing the meso-diaminopimelate dehydrogenase mutant or the coenzyme regeneration enzyme, or a crude enzyme solution obtained by cell lysis, or a purified pure enzyme solution; the coenzyme regeneration enzyme is a formate dehydrogenase that uses formate as a coenzyme regeneration substrate, and its amino acid sequence is shown in SEQ ID NO.

11.

7. The method for catalytically converting 3-indolepyruvic acid into chiral D-tryptophan according to claim 5, characterized in that: Each 1 mL reaction system contains 0.5-2 mg of meso-diaminopimelate dehydrogenase mutant enzyme solution, 0.5-2 mg of coenzyme regeneration enzyme solution, 10-100 mM 3-indolepyruvate substrate, 50-150 mM ammonium formate, and 0.1-1.0 mM NADP. + , 150-250 U / mL superoxide dismutase.

8. A method for catalyzing the configurational reversal of L-tryptophan to D-tryptophan, characterized in that: First, an enzyme solution of the meso-diaminopimelate dehydrogenase mutant according to claim 1, the L-amino acid deaminase shown in SEQ ID NO.12 and the coenzyme regeneration enzyme is prepared, and then the prepared enzyme solution is added to a mixture containing L-tryptophan substrate, ammonium formate and NADP + In the mixed system, the pH of the system is controlled between 7.0 and 9.0, and the reaction is carried out under oxygen conditions to obtain chiral D-tryptophan.

9. A method for catalyzing the configurational reversal of L-tryptophan to D-tryptophan according to claim 8, characterized in that: Each 20 mL reaction system contains 8-12 mL enzyme solution, 50-200 mM L-tryptophan substrate, 200-1000 mM ammonium formate, 0.1-1.0 mM NADP + In the enzyme solution, the mass ratio of the meso-diaminopimelate dehydrogenase mutant enzyme solution, the L-amino acid deaminase enzyme solution and the coenzyme regeneration enzyme enzyme solution is 2:1:2.

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