Application of erythrose-4-phosphate dehydrogenase mutants and their encoding genes in vitamin B6 production

Abstract

This invention relates to an optimized and improved erythrose-4-phosphate dehydrogenase (Epd) mutant and its encoding gene. Based on SEQ ID NO.2, at least one amino acid is replaced at positions 13, 14, 16, 17, 31, 38, 41, 63, 67, 78, 79, 80, 83, 86, 95, 99, 109, 125, 134, 142, 143, 153, 156, 177, 179, 184, 214, 216, 218, 225, 234, 239, 243, 244, 247, 248, 281, and 318. Compared with wild-type Epd, the mutant of this invention can increase the relative yield of vitamin B6 produced by fermentation to varying degrees (4%–212%), thus showing good application prospects in the field of microbial fermentation for vitamin B6 production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to site-directed mutagenesis and recombinant DNA technology, specifically to erythrose-4-phosphate dehydrogenase mutants and their genes, engineered bacteria, and preparation methods. Background Technology

[0002] Vitamin B6, also known as pyridoxine, is an essential vitamin for humans and other animals, widely used in the pharmaceutical, food, and feed industries. Vitamin B6 exists in three natural forms: pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which are present in the body as phosphate derivatives. In its active form, pyridoxal phosphate, vitamin B6 participates in nearly a hundred enzymatic reactions, most of which are related to amino acid metabolism, such as transamination, decarboxylation, dehydration, and transsulfurization. The product form of vitamin B6 is pyridoxine hydrochloride. Currently, the main method used in the market is the total synthesis via oxazole chemical synthesis. However, the synthesis of the intermediate oxazole uses highly corrosive phosphorus oxychloride and the toxic solvent benzene, making reaction control difficult and posing significant potential safety hazards. Furthermore, the preparation process is cumbersome, energy-intensive, and generates a large amount of waste, which is detrimental to environmental protection. Microbial fermentation is currently the greenest and most environmentally friendly method for producing vitamin B6, and its widespread application is of great significance.

[0003] The de novo biosynthesis pathway of vitamin B6 mainly includes two types: the DXP-dependent pathway and the DXP-independent pathway. The DXP-dependent pathway is mainly found in γ-Proteobacteria, such as *Escherichia coli* and *Rhizobium*. The biosynthetic pathway of vitamin B6 in *E. coli* has been well elucidated. First, the substrate erythrose 4-phosphate (E4P) undergoes a two-step dehydrogenation reaction by erythrose 4-phosphate dehydrogenase (Epd) and PdxB, followed by a transamination reaction by SerC, to generate the intermediate 4-(phosphohydroxy)-L-threonine (4HTP). Simultaneously, glyceraldehyde-3-phosphate and pyruvate condense to DXP under the catalysis of DXP synthase. 4HTP and DXP condense to pyridoxine 5'-phosphate (PNP) under the catalysis of 4HTP dehydrogenase and PNP synthase. PNP can be converted to PN under the catalysis of phosphatases.

[0004] Epd is the first enzyme in the synthesis of vitamin B6 via the DXP-dependent pathway. Epd enzymes derived from *E. coli* have been used in the production of vitamin B6 by various microorganisms. However, this enzyme suffers from several problems, including poor substrate affinity, low catalytic efficiency, and poor protein stability, which seriously affect the level of vitamin B6 production in microorganisms.

[0005] Therefore, modifying the erythrose-4-phosphate dehydrogenase of Escherichia coli to improve its catalytic efficiency and constructing highly efficient engineered strains of Escherichia coli to increase vitamin B6 production is an urgent problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to modify erythrose-4-phosphate dehydrogenase from Escherichia coli, so that the modified erythrose-4-phosphate dehydrogenase mutant has higher catalytic efficiency and can produce more vitamin B6 after being expressed in microorganisms.

[0007] This invention provides wild-type erythrose-4-phosphate dehydrogenase from Escherichia coli as a template for modification, the nucleic acid sequence of which is SEQ ID NO.1 and the amino acid sequence of which are SEQ ID NO.2, respectively.

[0008] This invention employs site-directed mutagenesis to modify the erythrose-4-phosphate dehydrogenase shown in SEQ ID NO.2 to obtain an erythrose-4-phosphate dehydrogenase mutant. The mutant is obtained by performing single-point mutations at positions 13, 14, 16, 17, 31, 38, 41, 63, 67, 78, 79, 80, 83, 86, 95, 99, 109, 125, 134, 142, 143, 153, 156, 177, 179, 184, 214, 216, 218, 225, 234, 239, 243, 244, 247, 248, 281, and 318 of the erythrose-4-phosphate dehydrogenase.

[0009] Mutation at position 13: I13S; Mutation at position 14: G14A; Mutation at position 16: N16F; Mutation at position 17: V17I; Mutation at position 31: T31I, T31W, or T31Q; Mutation at position 38: L38T; Mutation at position 41: A41L; Mutation at position 63: Q63Y; Mutation at position 67: Q67M; Mutation at position 78: L78F; Mutation at position 79 Mutation at position 80: H79Q; Mutation at position 83: E80Q or E80L; Mutation at position 83: L83I; Mutation at position 86: L86I; Mutation at position 95: V95I; Mutation at position 99: C99V; Mutation at position 109: G109A; Mutation at position 125: P125Q, P125L or P125M; Mutation at position 134: V134I; Mutation at position 142: Q142 Y; Mutation at position 143: L143I; Mutation at position 153: A153G; Mutation at position 156: T156N; Mutation at position 177: T177L; 4 Mutation at position 179: T179I; Mutation at position 184: A184S or A184V; Mutation at position 214: K214R or K214H; Mutation at position 216: A216V; Mutation at position 218: G 218A or G218W; Mutation at position 225: Q225M; Mutation at position 234: A234S; Mutation at position 239: T239V; Mutation at position 243: T243V; Mutation at position 244: A244L; Mutation at position 247: L247I; Mutation at position 248: S248T; Mutation at position 281: L281F; Mutation at position 318: N318T. The original site consists of the amino acid preceding the position number, and the mutated amino acid is the one mentioned after the position number.

[0010] In a preferred embodiment, relative to the erythrose-4-phosphate dehydrogenase shown in SEQ ID NO.2, the erythrose-4-phosphate dehydrogenase mutant has at least one of the following alterations: I13S; G14A; N16F; V17I; T31I; T31W; T31Q; L38T; A41L; Q63Y; Q67M; L78F; H79Q; E80Q; E80L; L83I; L86I; V95I; C99V; G109A; P125 Q; P125L; P125M; V134I; Q142Y; L143I; A153G; T156N; T177L; T179I; A184S; A184V; K214R; K214H; A216V; G218A; G218W; Q225M; A234S; T239V; T243V; A244L; L247I; S248T; L281F; N318T. Any possible amino acid substitution at any of the mentioned positions can be combined with any possible amino acid changes at any of the remaining mentioned positions. Therefore, the erythrose-4-phosphate dehydrogenase mutant of the present invention can be a mutant having at least one of the mentioned changes or a combination of several of the changes.

[0011] Depending on the position and amino acid substituted, compared with the wild type, single-point mutants of erythrose-4-phosphate dehydrogenase can increase pyridoxine production by 4%-116% when used in Escherichia coli to produce vitamin B6. Some combinations of two-point or multi-point mutations can increase pyridoxine production by 35%-212%.

[0012] The present invention also includes a coding gene for the encoded amino acid nucleic acid sequence, which encodes the erythrose-4-phosphate dehydrogenase of the present invention having the possible alterations at one or more positions, particularly the erythrose-4-phosphate dehydrogenase based on SEQ ID NO.2 with amino acid substitutions at the above-mentioned amino acid positions.

[0013] The present invention also includes a recombinant expression vector containing the coding gene sequence of the erythrose-4-phosphate dehydrogenase mutant of the present invention.

[0014] The present invention also includes recombinant host cells containing the coding gene of the erythrose-4-phosphate dehydrogenase mutant of the present invention or a recombinant expression vector containing the erythrose-4-phosphate dehydrogenase mutant of the present invention.

[0015] The present invention also provides a recombinant strain comprising the above-mentioned erythrose-4-phosphate dehydrogenase mutant and expression vector, preferably Escherichia coli.

[0016] The present invention also provides the application of the erythrose-4-phosphate dehydrogenase mutant, its encoding gene, or recombinant strain thereof in the preparation of vitamin B6. Preferably, the method includes the steps of producing vitamin B6 through the recombinant host cell and collecting the vitamin B6.

[0017] The erythrose-4-phosphate dehydrogenase mutant of this invention is based on erythrose-4-phosphate dehydrogenase from *E. coli*. Amino acids involved in improving enzyme catalytic efficiency are selected and modified. The resulting erythrose-4-phosphate dehydrogenase mutant was overexpressed in *E. coli*. Compared with wild-type erythrose-4-phosphate dehydrogenase recombinant bacteria, the PN yield of the mutant was increased by 4%-116% after fermentation. With the superposition of some two-point or multi-point mutation combinations, the increase in PN yield compared to the wild type reached 35%-212%. The modified erythrose-4-phosphate dehydrogenase mutant of this invention can be used for the fermentation production of vitamin B6 based on prokaryotic and eukaryotic expression systems, and has great application value. Furthermore, within a certain range, it provides guidance and reference value for subsequent continuous modification of erythrose-4-phosphate dehydrogenase from other sources to increase VB6 yield through the DXP-dependent pathway. Attached Figure Description

[0018] Figure 1 The image shows the recombinant plasmid pACYCduet-1-pTac-Epd.

[0019] Figure 2 The relative yield of vitamin B6 produced by fermentation of wild-type Epd and Epd single-point mutant recombinant bacteria.

[0020] Figure 3 The relative yield of vitamin B6 produced by fermentation of wild-type Epd and multi-site superimposed Epd mutant recombinant bacteria. Detailed Implementation

[0021] The present invention will be further described below through specific embodiments to provide a better understanding of the invention, but this does not constitute a limitation thereof. The experimental materials and reagents used are as follows:

[0022] 1. Experimental Materials

[0023] (1) Strains and vectors: Escherichia coli DH5α was purchased from Invitrogen; Escherichia coli expression vector pACYCduet-1 was purchased from Novagen, with the promoter modified to Tac; Escherichia coli LL05 was obtained from Chinese patent: ZL202110059483.2.

[0024] (2) Enzymes and kits: DpnI enzyme was purchased from Thermo Fisher Scientific. PrimeSTAR Max DNA Polymerase was purchased from TaKaRa. Plasmid extraction kit and gel extraction kit were purchased from Omega. SSCS E. coli competent cell preparation solution was purchased from Generay.

[0025] (3) Reagents: Yeast powder and peptone were purchased from OXOID; NaCl and glucose were purchased from Sangon Biotech (Shanghai) Co., Ltd.; glycerol was purchased from Tianjin Standard Chemical Reagent Co., Ltd.; all other reagents were domestically produced (all of which can be purchased from ordinary biochemical reagent companies).

[0026] 2. Culture medium

[0027] LB medium for Escherichia coli (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl);

[0028] Escherichia coli seed culture medium (glycerol 10 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L);

[0029] Escherichia coli PY30 medium (glycerol 15 g / L, yeast extract 5 g / L, peptone 10 g / L, NaCl 5 g / L, glucose 1 g / L, MgSO4·7H2O 200 mg / L, FeSO4·7H2O 10 mg / L, MnSO4·5H2O 10 mg / L, pH 6.8).

[0030] Example 1: Construction of wild-type Escherichia coli erythrose-4-phosphate dehydrogenase expression plasmid

[0031] Following optimization based on the *E. coli* codon, the *E. coli* erythrosine-4-phosphate dehydrogenase gene (encoding the amino acid sequence SEQ ID NO. 2) was obtained using whole-genome synthesis technology (Beijing Qingke Biotechnology Co., Ltd.). A tac promoter and an RBS sequence (TTGACAATTAATCATCGGCTCGTATAATGTGTGGAAGGTTAAAAAACTATTTAAAAAGTAGGTCTGTTT) were added to the N-terminus of the protein. This sequence was then constructed into the pACYCduet-1 vector by a gene synthesis company, replacing the original T7 promoter and RBS sequence. The constructed plasmid was named pACYCduet-1-pTac-Epd, and its map is shown below. Figure 1 As shown.

[0032] Nucleotide sequence of the Escherichia coli erythrose-4-phosphate dehydrogenase gene (SEQ ID NO.1):

[0033]

[0034] Amino acid sequence of Escherichia coli erythrose-4-phosphate dehydrogenase (SEQ ID NO.2):

[0035] MTVRVAINGFGRIGRNVVRALYESGRRAEITVVAINELADAAGMAHLLKYDTSHGRFAWEVRQERDQLFVGDDAIRVLHERSLQSLPWRELGVDVVLDCTGVYGSREHGEAHIAAGAKKVLFSHPGSNDLDATVVYGVNQDQLRAEHRIVSNASCTTNCIIPVIKLLDDA YGIESGTVTTIHSAMHDQQVIDAYHPDLRRTRAASQSIIPVDTKLAAGITRFFPQFNDRFEAIAVRVPTINVTAIDLSVTVKKPVKANEVNLLLQKAAQGAFHGIVDYTELPLVSVDFNHDPHSAIVDGTQTRVSGAHLIKTLVWCDNEWGFANRMLDTTLAMATVAFR.

[0036] Example 2: Site-directed gene mutation

[0037] This invention analyzes the crystal structure and catalytic mechanism of wild-type *Escherichia coli* erythrose-4-phosphate dehydrogenase (PDB: 2X5J), simulates the substrate-enzyme docking mechanism, screens for designable residues, and then rationally designs the amino acid sequences of mutants. Experimental verification identified 46 effective mutants with single-point modification: II13S; G14A; N16F; V17I; T31I; T31W; T31Q; L38T; A41L; Q63Y; Q67M; L78F; H79Q; E80Q; E80L; L83I; L86I; V95I; C99V; G109A; P125Q; P125L; P1 25M; V134I; Q142Y; L143I; A153G; T156N; T177L; T179I; A184S; A184V; K214R; K214H; A 216V; G218A; G218W; Q225M; A234S; T239V; T243V; A244L; L247I; S248T; L281F; N318T.

[0038] By optimizing single-point effective mutants through two-point and multi-point combinations, 28 effective mutants with better synergistic effects were identified, namely G14A / A234S; I13S / E80Q; P125Q / A153G; N16F / N318T; G14A / A234S / T31W; G14A / A234S / T31Q; G14A / A234S / T31I; G14A / A234S / L86I; G14A / A234S / P125M; G14A / A234S / T243V; I13S / E80Q / T31W; I13S / E80Q / T31I; I13S / E80Q / L86I; and I13S / E80Q. / P125M;P125Q / A153G / L86I;P125Q / A153G / P125M; G14A / A234S / T31I / V17I; G14A / A234S / T31I / Q67M; G14A / A234S / T31I / L143I; G1 4A / A234S / T31I / T177L; G14A / A234S / T31I / A244L; G14A / A234S / T31I / S248T; I13S / E80Q / P125M / V17I; I13S / E80Q / P125M / Q67M; I13S / E80Q / P125M / L143I; I13S / E80Q / P125M / T177L; I13S / E80Q / P125M / A244L.

[0039] Using the recombinant plasmid pACYCduet-1-pTac-Epd as a PCR amplification template, each erythrose-4-phosphate dehydrogenase mutant was obtained by introducing one or more point mutations through one or more rounds of PCR reactions (primers are shown in Table 2).

[0040] Taking the construction of pACYECduet-1-pTac-Epd-I13S plasmid as an example, the PCR system (Table 1) is as follows:

[0041] Table 1 PCR reaction system

[0042] Components Volume (µl) Prime STAR Mix 20 Template DNA (10 ng / μl) 1 10µM forward primer 2 10µM reverse primer 2 <![CDATA[dd H2O]]> 15 Total volume 40

[0043] PCR amplification conditions: 98℃ for 30 s; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min / 2 kb, 30 cycles; 72℃ for 4 min. The PCR template was pACYCduet-1-pTac-Epd-WT, and the primers were I13S-F and I13S-R. After recovery, the PCR product was digested with DpnI at 37℃ for 4 hours to remove the template DNA. The product was then recovered and dissolved in 30 μL of sterile dd H2O and transformed into DH5α. Single colonies were picked for sequencing, and plasmids with correct sequencing results were extracted for later use. The remaining mutant plasmids were constructed using the same method.

[0044] Table 2 Primers used for mutations in Example 2

[0045] mutation site Primer name Primer sequence (5'-3') I13S I13S-F ATCAACGGCTTCGGTCGTAGCGGCCGTAACG I13S-R CTACGACCGAAGCCGTTGATCGCTACACGAAC G14A G14A-F CAACGGCTTCGGTCGTATCGCCCGTAACGTT G14A-R GCGATACGACCGAAGCCGTTGATCGCTACAC N16F N16F-F GCTTCGGTCGTATCGGCCGTTTCGTTGTTCGTG N16F-R AAACGGCCGATACGACCGAAGCCGTTGATCGCT V17I V17I-F TCGGTCGTATCGGCCGTAACATTGTTCGTGCTC V17I-R TGTTACGGCCGATACGACCGAAGCCGTTGATC T31I T31I-F CTGGTCGTCGGTGCTGAAATCATCGTTGTTGCTAT T31I-R ATGATTTCAGCACGACGACCAGATTCGTACAG T31W T31W-F CTGGTCGTCGGTGCTGAAATCTGGGTTGTTGCTA T31W-R CCAGATTTCAGCACGACGACCAGATTCGTACAG T31Q T31Q-F CTGGTCGTCGGTGCTGAAATCCAGGTTGTTGCTA T31Q-R CTGGATTTCAGCACGACGACCAGATTCGTACAG L38T L38T-F CCGTTGTTGCTATCAACGAAACGGCTGACGCT L38T-R GTTTCGTTGATAGCAACAACGGTGATTTCAGC A41L A41L-F CTATCAACGAACTGGCTGACCTTGCTGGTATGG A41L-R AGGTCAGCCAGTTCGTTGATAGCAACAACGGT Q63Y Q63Y-F GTTTCGCTTGGGAAGTTCGTTTTGAACGTGACC Q63Y-R AAAACGAACTTCCCAAGCGAAACGACCGTGAGAG Q67M Q67M-F AAGTTCGTCAGGAACGTGACATGCTGTTCGTTG Q67M-R ATGTCACGTTCCTGACGAACTTCCCAAGCGAAAC L78F L78F-F GTGACGACGCTATCCGTGTTTTTCACGAACGTT L78F-R AAAAACACGGATAGCGTCGTCACCAACGAACAG H79Q H79Q-F GACGCTATCCGTGTTCTGCAGGAACGTTCTC H79Q-R GGTGCAGAACACGGATAGCGTCGTCACCAACG E80Q E80Q-F ACGCTATCCGTGTTCTGCACCAACGTTCTCT E80Q-R GGTGCAGAACACGGATAGCGTCGTCACCAAC E80L E80L-F ACGCTATCCGTGTTCTGCACTTACGTTCTCTG E80L-R AAGTGCAGAACACGGATAGCGTCGTCACCAAC L83I L83I-F GTGTTCTGCACGAACGTTCTATTCAGTCTCTGC L83I-R AATAGAACGTTCGTGCAGAACACGGATAGCGTC L86I L86I-F ACGAACGTTCTCTGCAGTCTATTCCGTGGCGTG L86I-R AATAGACTGCAGAGAACGTTCGTGCAGAACACG V95I V95I-F GGCGTGAACTGGGTGTTGACATTGTTCTGGACT V95I-R TGTCAACACCCAGTTCACGCCACGGCAGAGAC C99V C99V-F GTGTTGACGTTGTTCTGGACGTCACCGGTGTTT C99V-R ACGTCCAGAACAACGTCAACACCCAGTTCACG G109A G109A-F TTTACGGTTCTCGTGAACACGCTGAAGCTCACAT G109A-R GCGTGTTCACGAGAACCGTAAACACCGGTGCAG P125Q P125Q-F AAAAAGTTCTGTTCTCTCACCAGGGTTCTAACG P125Q-R TGGTGAGAGAACAGAACTTTTTTAGCACCAGCAG P125L P125L-F AAAAAGTTCTGTTCTCTCACCTTGGTTCTAACG P125L-R AAGGTGAGAGAACAGAACTTTTTTAGCACCAGCAG P125M P125M-F AAAAAGTTCTGTTCTCTCACATGGGTTCTAAC P125M-R TAGTGAGAGAACAGAACTTTTTTAGCACCAGC V134I V134I-F CTAACGACCTGGACGCTACCATTGTTTACGGTGT V134I-R TGGTAGCGTCCAGGTCGTTAGAACCCGGGTG Q142Y Q142Y-F TTTACGGTGTTAACCAGGACTATCTGCGTGCTG Q142Y-R ATAGTCCTGGTTAACACCGTAAACAACGGTAGC L143I L143I-F ACGGTGTTAACCAGGACCAGATTCGTGCTGAACAC L143I-R AATCTGGTCCTGGTTAACACCGTAAACAACGGT A153G A153G-F AACACCGTATCGTTTCTAACGGTTCTTGCACCAC A153G-R CCGTTAGAAACGATACGGTGTTCAGCACGCAG T156N T156N-F TCGTTTCTAACGCTTCTTGCAACACCAACTGCAT T156N-R TTGCAAGAAGCGTTAGAAACGATACGGTGTTC T177L T177L-F CTTACGGTATCGAATCTGGTCTCGTTACCACCAT T177L-R AGACCAGATTCGATACCGTAAGCGTCGTCCAG T179I T179I-F GTATCGAATCTGGTACCGTTATCACCATCCACT T179I-R ATAACGGTACCAGATTCGATACCGTAAGCGTCG A184S A184S-F CCGTTACCACCATCCACTCTTCTATGCACGACC A184S-R AAGAGTGGATGGTGGTAACGGTACCAGATTC A184V A184V-F CCGTTACCACCATCCACTCTGTTATGCACGACC A184V-R ACAGAGTGGATGGTGGTAACGGTACCAGATTC K214R K214R-F CTATCATCCCGGTTGACACCAGACTGGCTGCTGGT K214R-R CTGGTGTCAACCGGGATGATAGACTGAGAAGCAG K214H K214H-F CTATCATCCCGGTTGACACCCATCTGGCTGCTG K214H-R ATGGGTGTCAACCGGGATGATAGACTGAGAAGC A216V A216V-F TCCCGGTTGACACCAAACTGGTTGCTGGTATAAC A216V-R ACCAGTTTGGTGTCAACCGGGATGATAGACTGAG G218A G218A-F TTGACACCAAACTGGCTGCTGCTATAACAAGGT G218A-R GCAGCAGCCAGTTTGGTGTCAACCGGGATGAT G218W G218W-F TTGACACCAAACTGGCTGCTTGGATAACAAGGT G218W-R CCAAGCAGCCAGTTTGGTGTCAACCGGGATGAT Q225M Q225M-F GTATAACAAGGTTTTTTCCGATGTTCAACGACC Q225M-R ATCGGAAAAAACCTTGTTATACCAGCAGCCAG A234S A234S-F ACGACCGGTTCGAAGCTATCAGTGTTCGTGTT A234S-R CTGATAGCTTCGAACCGGTCGTTGAACTGCGG T239V T239V-F CTATCGCTGTTCGTGTTCCGGTCATCAACGTTAC T239V-R ACCGGAACACGAACAGCGATAGCTTCGAACCGGT T243V T243V-F GTGTTCCGACCATCAACGTTGTCGCTATCGACC T243V-R ACAACGTTGATGGTCGGAACACGAACAGCGAT A244L A244L-F TTCCGACCATCAACGTTACCCTTATCGACCTGT A244L-R AGCGGTAACGTTGATGGTCGGAACACGAACAGC L247I L247I-F TCAACGTTACCGCTATCGACATCTCTGTTACCGT L247I-R GATGTCGATAGCGGTAACGTTGATGGTCGGAAC S248T S248T-F ACGTTACCGCTATCGACCTGACTGTTACCGTT S248T-R TCAGGTCGATAGCGGTAACGTTGATGGTCGGAAC L281F L281F-F TATCGTTGACTACACCGAATTTCCGCTGGTTT L281F-R AAATTCGGTGTAGTCAACGATACCGTGGAACGC N318T N318T-F AAACCCTGGTTTGGTGCGACACCGAATGGGGTT 　 N318T-R GTGTCGCACCAAACCAGGGTTTTGATCAGGTG

[0046] Example 3: Construction of wild-type and mutant recombinant strains of Escherichia coli erythrose-4-phosphate dehydrogenase

[0047] (1) Preparation of Escherichia coli LL05 competent cells

[0048] Escherichia coli LL05 (this strain is from authorized patent: ZL202110059483.2) was streaked onto LB agar plates and incubated at 37°C for 12 h. Single colonies were then inoculated into 5 mL of liquid LB medium. The initial OD of the bacterial culture in the shake flask after inoculation was determined according to the following criteria. 600 =0.1 Inoculate the bacterial culture from the test tube into a 250 mL shake flask containing 30 mL of liquid LB, and incubate at 37°C until OD. 600 =0.4~0.6, incubate on ice for 30 min, centrifuge at 8000 rpm for 10 min at 4 ℃. Remove the supernatant, resuspend the remaining bacteria in 3 mL SSCS solution, and place on ice for 30 min. The competent cells are now ready. Aliquot and place on ice for later use.

[0049] (2) Transformation

[0050] The wild-type and mutant plasmids from Examples 1 and 2 were transformed into the prepared LL05 competent cells, respectively. After a 30-min ice bath, they were heat-shocked for 45 s, ice-shocked for 3 min, and then recovered for 1 h in a shaker at 37°C and 200 rpm before being plated on LB agar plates. Single colonies from the plates were picked and inoculated into LB medium containing 5 mL of 34 µg / mL chloramphenicol. The culture was incubated at 37°C for 16 h, and the culture was stored in 20% glycerol at -80°C. This yielded the wild-type and mutant Escherichia coli recombinant strains of erythrose-4-phosphate dehydrogenase.

[0051] Example 4: Plate fermentation of recombinant erythrose-4-phosphate dehydrogenase bacteria

[0052] The recombinant bacteria of wild-type and mutant erythrose-4-phosphate dehydrogenase, frozen at -80℃ in Example 3, were streaked onto LB agar plates containing 34 µg / mL chloramphenicol and incubated overnight at 37℃. Single colonies were picked and inoculated into seed culture medium containing 5 mL of 34 µg / mL chloramphenicol. After incubation at 37℃ for 16 h, the inoculum was inoculated into 24-well plates containing 2 mL of 34 µg / mL chloramphenicol at an OD=0.1 inoculation rate and incubated at 37℃, 800 rpm, and 80% humidity for 70 h. After fermentation, fermentation broth with similar OD values ​​was centrifuged at 8000 rpm for 3 min to obtain the fermentation supernatant, which was used to determine the vitamin B6 yield.

[0053] Example 5: High-performance liquid chromatography (HPLC) determination of vitamin B6-pyridoxine (PN) yield

[0054] The Escherichia coli erythrose-4-phosphate dehydrogenase is a dehydrogenase based on E4P, which oxidizes the terminal aldehyde of E4P into an acid, and then, through the DXP-dependent pathway, finally generates the product form of vitamin B6, pyridoxine (PN), in Escherichia coli LL05.

[0055] The main purpose of this embodiment is to determine the difference in extracellular PN production after fermentation of wild-type and mutant recombinant strains of erythrose-4-phosphate dehydrogenase in Example 4. If the modified mutant has higher catalytic efficiency, the corresponding recombinant strain can produce more PN after fermentation. Calculating the relative PN production of the mutant and wild-type strains after fermentation reflects the modification effect of the *E. coli* erythrose-4-phosphate dehydrogenase mutant.

[0056] The formula for calculating the relative yield of PN is PN (PN yield of Epd mutant recombinant bacteria) / PN0 (PN yield of wild-type Epd recombinant bacteria) * 100%.

[0057] The detection instrument was a Thermo Fisher Ultimate 3000 high-performance liquid chromatograph, and the detection column used was an acid-resistant COSMOSIL 5C18-AR-II Packed Column (Cosmosil, 4.6 mm ID × 250 mm, 5 µm).

[0058] The detection method was as follows: Mobile phase A: 33 mM H3PO4, pH adjusted to 2.5 with 10 mM KOH, then 0.8 mM sodium 1-octanesulfonate was added; Mobile phase B: 80% acetonitrile; Flow rate: 0.8 ml / min; Injection volume: 20 μl; HPLC conditions: 0-5 min gradient from 100% A to 99% A / 1% B, 5-10 min gradient to 81% A / 19% B, 10-20 min gradient to 72% A / 28% B, 20-25 min gradient to 37% A / 63% B, 25-30 min gradient to 100% A, column equilibration for 5 min; Column temperature: 35 ℃; Detector: FLD detector, excitation wavelength 293 nm, emission wavelength 395 nm. The peak elution time for PN standard detection by HPLC was 21.6 min.

[0059] Depend on Figure 2 As shown, based on the rational design and modification strategy, the erythrose-4-phosphate dehydrogenase mutant recombinant strain constructed in this invention, after fermentation, showed a 4%-116% increase in vitamin B6 (PN) production compared to the wild-type Epd recombinant strain; after the superposition of some single-point mutations, the mutant strain showed a 35%-212% increase in vitamin B6 (PN) production compared to the wild-type recombinant strain. Figure 3 The relative yields of vitamin B6 (PN) after fermentation by all erythrose-4-phosphate dehydrogenase mutant recombinant strains are shown in Table 3. Table 3 shows the relative biomass (OD) of the Epd wild-type and mutant recombinant strains using LL05 Escherichia coli as a chassis after fermentation. 600 There was no significant difference. Therefore, the modified erythrose-4-phosphate dehydrogenase mutant is more conducive to the microbial fermentation production of vitamin B6, and the mutant has great application value.

[0060] Table 3. Relative yield and biomass of vitamin B6 (PN) after fermentation by wild-type and mutant recombinant strains of Epd

[0061] Mutation site Engineered strain PN relative yield (%) Biomass (OD600) pACYCduet-1-pTac-Epd (wild type) LL05-pACYCduet-1-pTac-Epd-WT 100.00 9.97 Epd(I13S) LL05-Epd(I13S) 131.45 9.60 Epd(G14A) LL05-Epd(G14A) 132.18 9.98 Epd(N16F) LL05-Epd(N16F) 120.43 9.39 Epd(V17I) LL05-Epd(V17I) 156.70 9.47 Epd(T31I) LL05-Epd(T31I) 214.94 9.56 Epd(T31W) LL05-Epd(T31W) 155.60 9.41 Epd(T31Q) LL05-Epd(T31Q) 167.77 9.62 Epd(L38T) LL05-Epd(L38T) 205.12 10.38 Epd(A41L) LL05-Epd(A41L) 209.51 9.18 Epd(Q63Y) LL05-Epd(Q63Y) 195.83 9.08 Epd(Q67M) LL05-Epd(Q67M) 159.31 10.24 Epd(L78F) LL05-Epd(L78F) 106.58 10.80 Epd(H79Q) LL05-Epd(H79Q) 120.38 10.12 Epd(E80Q) LL 202.13 10.24 ​ ​ 129.98 9.98 ​ ​ 209.73 9.93 ​ ​ 112.30 8.85 ​ ​ 180.82 10.20 ​ ​ 204.65 9.44 ​ ​ 169.21 9.75 ​ ​ 104.46 10.41 ​ ​ 118.93 10.06 ​ ​ 165.82 10.53 ​ ​ 150.20 8.55 ​ ​ 105.42 10.14 ​ ​ 177.76 10.88 ​ ​ 115.50 10.65 ​ ​ 109.30 10.62 ​ ​ 185.40 10.10 ​ ​ 132.11 7.80 ​ ​ 206.91 10.15 ​ ​ 121.77 9.86 ​ ​ 117.18 9.61 ​ ​ 127.30 7.84 ​ ​ 109.76 9.83 ​ ​ 122.19 9.90 ​ ​ 215.29 10.04 ​ ​ 137.20 10.08 ​ ​ 106.27 9.48 ​ LL05-Epd(T238V) 178.27 9.98 Epd(T243V) LL05-Epd(T243V) 150.80 7.32 Epd(A244L) LL05-Epd(A244L) 215.40 7.86 Epd(L247I) LL05-Epd(L247I) 147.69 8.07 Epd(S248T) LL05-Epd(S248T) 136.35 8.37 Epd(L281F) LL05-Epd(L281F) 128.00 9.31 Epd(N318T) LL05-Epd(N318T) 185.45 7.41 Epd(G14A / A234S) LL05-Epd(G14A / A234S) 184.69 7.84 Epd(I13S / E80Q) LL05-Epd(I13S / E80Q) 196.72 8.69 Epd(P125Q / A153G) LL05-Epd(P125Q / A153G) 157.50 9.82 Epd(N16F / N318T) LL05-Epd(N16F / N318T) 180.55 8.28 Epd(G14A / A234S / T31W) LL05-Epd(G14A / A234S / T31W) 204.39 10.8 Epd(G14A / A234S / T31Q) LL05-Epd(G14A / A234S / T31Q) 223.12 9.11 Epd(G14A / A234S / T31I) LL05-Epd(G14A / A234S / T31I) 203.11 10.88 Epd(G14A / A234S / L86I) LL05-Epd(G14A / A234S / L86I) 211.10 9.80 Epd(G14A / A234S / P125M) LL05-Epd(G14A / A234S / P125M) 246.26 9.77 Epd(G14A / A234S / T243V) LL05-Epd(G14A / A234S / T243V) 185.55 10.64 Epd(I13S / E80Q / T31W) LL05-Epd(I13S / E80Q / T31W) 192.78 9.86 Epd(I13S / E80Q / T31I) LL05-Epd(I13S / E80Q / T31I) 198.62 9.71 Epd(I13S / E80Q / L86I) LL05-Epd(I13S / E80Q / L86I) 215.49 8.62 Epd(I13S / E80Q / P125M) LL05-Epd(I13S / E80Q / P125M) 231.16 8.53 Epd(P125Q / A153G / L86I) LL05-Epd(P125Q / A153G / L86I) 176.60 9.13 Epd(P125Q / A153G / P125M) LL05-Epd(P125Q / A153G / P125M) 188.90 7.56 Epd(G14A / A234S / T31I / V17I) LL05-Epd(G14A / A234S / T31I / V17I) 193.19 9.65 Epd(G14A / A234S / T31I / Q67M) LL05-Epd(G14A / A234S / T31I / Q67M) 243.72 10.98 Epd(G14A / A234S / T31I / L143I) LL05-Epd(G14A / A234S / T31I / L143I) 135.61 9.26 Epd(G14A / A234S / T31I / T177L) LL05-Epd(G14A / A234S / T31I / T177L) 312.00 9.53 Epd(G14A / A234S / T31I / A244L) LL05-Epd(G14A / A234S / T31I / A244L) 138.11 9.67 Epd(G14A / A234S / T31I / S248T) LL05-Epd(G14A / A234S / T31I / S248T) 126.03 9.02 Epd(I13S / E80Q / P125M / V17I) LL05-Epd(I13S / E80Q / P125M / V17I) 176.78 10.10 Epd(I13S / E80Q / P125M / Q67M) LL05-Epd(I13S / E80Q / P125M / Q67M) 186.80 9.19 Epd(I13S / E80Q / P125M / L143I) LL05-Epd(I13S / E80Q / P125M / L143I) 236.48 8.20 Epd(I13S / E80Q / P125M / T177L) LL05-Epd(I13S / E80Q / P125M / T177L) 210.13 8.78 Epd(I13S / E80Q / P125M / A244L) LL05-Epd(I13S / E80Q / P125M / A244L) 199.69 7.94

Claims

1. A mutant of erythrose-4-phosphate dehydrogenase, which, based on the amino acid sequence of erythrose-4-phosphate dehydrogenase as shown in SEQ ID NO.2, contains only one of the following mutations: T31W, T31Q, L38T, A41L, E80Q, E80L, C99V, G218A, G218W, A244L, G14A / A234S / T31W, G14A / A234S / T31Q, G14A / A234S / T31I, G14A / A234S / L86I, G14A / A234S / P125M, I13S / E80Q / L86I, I13S / E80Q / P125M, G14A / A234S / T31I / Q67M, ​​G14A / A234S / T31I / T177L, I13S / E80Q / P125M / L143I or I13S / E80Q / P125M / T177L.

2. The encoding gene of the erythrose-4-phosphate dehydrogenase mutant as described in claim 1.

3. The recombinant expression vector for the encoding gene of the erythrose-4-phosphate dehydrogenase mutant as described in claim 2.

4. Recombinant bacteria containing the coding gene of the erythrose-4-phosphate dehydrogenase mutant as described in claim 2 or the recombinant expression vector as described in claim 3.

5. The recombinant bacteria as described in claim 4, characterized in that, It's E. coli.

6. The use of the erythrose-4-phosphate dehydrogenase mutant as described in claim 1, its encoding gene, or the recombinant bacteria as described in claim 4 in the preparation of vitamin B6.

7. The application as described in claim 6, characterized in that, This includes the steps of producing vitamin B6 by culturing the recombinant bacteria as described in claim 4 or 5, and collecting the vitamin B6.

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