PntB mutant, genetically engineered bacterium containing PntB mutant, and construction method and application of PntB mutant

Abstract

The invention relates to the field of gene engineering, and particularly provides a PntB mutant for improving the yield of L-tryptophan, a genetically engineered bacterium containing the mutant as well as a construction method and application of the genetically engineered bacterium, aiming at the problem that NADPH (nicotinamide adenine dinucleotide phosphate) is insufficient in the production process of aromatic amino acid, especially L-tryptophan, pyridine nucleotide transhydrogenase beta subunit mutant PntB (A167T) is obtained through site-specific mutagenesis, and the PntB mutant PntB (A167T) is used for improving the yield of L-tryptophan. According to the present invention, the mutant is constructed, the genetically engineered bacterium containing the mutant is constructed, the genetically engineered bacterium is applied to L-tryptophan fermentation so as to promote the conversion of NADH to generate NADPH, the intracellular NADPH concentration is increased by 4.8 times, the L-tryptophan production ability of the genetically engineered strain BDP2 is increased by 38.8% compared with the initial strain BDX1, the L-tryptophan yield reaches 1.02 g / L, and the efficient synthesis of the L-tryptophan is finally achieved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and specifically provides a PntB mutant for improving L-tryptophan production, a genetic engineering bacterium containing the mutant, and a construction method and application thereof. Background Art

[0002] L-Tryptophan is an essential amino acid that is vital to human health. It is not only a basic unit for protein synthesis, but also a precursor for the synthesis of serotonin (5-hydroxytryptamine) and melatonin, both of which play an important role in regulating mood, sleep and appetite. L-Tryptophan is widely found in protein-rich foods such as dairy products, meat, fish and nuts. Although it is usually possible to get enough L-tryptophan from the diet, in some cases, such as mood swings or sleep disorders, people may consider using L-tryptophan supplements. However, high-dose supplements may cause side effects, so professionals should be consulted before use. Historically, a health incident caused by contamination of L-tryptophan supplements occurred in Japan in 1989, which prompted global tightening of regulation of dietary supplements. Overall, L-tryptophan plays an indispensable role in maintaining normal physiological functions of the human body and promoting health.

[0003] As an important reductant, NADPH participates in multiple metabolic reactions, especially those synthetic pathways that require reducing power. In the synthesis of aromatic amino acids, NADPH provides the necessary reducing power to promote the reaction. The synthetic pathway of aromatic amino acids includes multiple steps, many of which involve enzymatic reactions that require NADPH. For example, the production of shikimic acid, a precursor of aromatic amino acid synthesis, requires the participation of NADPH. Increasing the supply of NADPH can increase the rate of these reactions, thereby increasing the amount of aromatic amino acids produced as the final product. Usually, the substrate in the culture medium is adjusted to increase the generation of NADPH: for example, adding certain sugars or amino acids can promote the generation of reducing power; in addition, the reducing power is enhanced by increasing the generation of NADPH: this can be achieved by overexpressing some reduction-related enzymes (glucose-6-phosphate dehydrogenase G6PD, soluble pyridine nucleotide transhydrogenase SthA, and pyridine nucleotide transhydrogenase PntAB).

[0004] PntB encodes the β-subunit of a membrane-bound proton pump, pyridine nucleotide transhydrogenase, which contains an N-terminal domain with 6-8 transmembrane regions and a cytoplasmic C-terminal domain that binds NADP(H). Early studies have shown that the PntB(G314E) mutation results in the loss of both energy-dependent and energy-independent pyridine nucleotide transhydrogenase activities. In Escherichia coli, PntB can convert NADH to NADPH, thereby increasing the intracellular NADPH supply level. Summary of the invention

[0005] The purpose of the present invention is to transform the gene involved in reducing power conversion in L-tryptophan synthesis of Escherichia coli by genetic engineering method, so as to obtain a strain for expanding L-tryptophan production.

[0006] The method for constructing the L-tryptophan genetically engineered bacteria comprises the following steps:

[0007] 1) Escherichia coli strain KW (Chen, Y et al. Journal of Industrial Microbiol &

[0008] Biotechnology, 2018, 45(5): 357-367.) to construct a gene containing aroG fbr and trpEDCBA fbr Recombinant plasmid PH5a-aroG fbr -trpEDCBA fbr , overexpressing the ppsA gene, knocking out the trpR gene, and knocking out the tnaA gene on the genome to construct strain BDX1;

[0009] 2) In the BDZ1 strain, the amino acid residue A167 at position 167 of the β-subunit of pyridine nucleotide transhydrogenase PntB was mutated to T by site-directed mutagenesis to obtain a mutant PntB (A167T), thereby constructing the strain BDP2, and the amino acid sequence of PntB (A167T) is shown in SEQ ID NO: 1;

[0010] 3) Shake flask fermentation verification of L-tryptophan production strains KW, BDX1 and BDP2;

[0011] The introduction of the PntB (A167T) mutant can balance the supply of NADH and NADPH in the production process of aromatic amino acids, thereby promoting the effective accumulation of aromatic amino acids and laying the foundation for the industrial production of L-tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :PH5a-aroG fbr -trpE fbr DCBA Plasmid Map

[0013] Figure 2 : Shake flask fermentation results of L-tryptophan producing strains BDX1 and BDP2

[0014] Figure 3 :Results of NADPH concentration determination in fermentation broth of L-tryptophan producing strains BDX1 and BDP2 DETAILED DESCRIPTION

[0015] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0016] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0017] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0018] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0019] The amino acid sequence of mutant PntB (A167T) is shown in SEQ ID NO: 1. SEQ ID NO: 1 is MSGGLVTAAYIVAAILFIFSLAGLSKHETSRQGNNFGIAGMAIALIATIFGPDTGNVGWILLAMVIGGAIGIRLAKKVEMTEMPELVAILHSFVGLAAVLVGFNSYLHHDAGMAPILVNIHLTEVFLGIFIGAVTFTGSVVAFGKLCGKISSKPLMLPNRHKMNLATLVVSFLLLIVFVRTDSVGLQVLALLIMTAIALVFGWHLVASIGGADMPVVVSMLNSYSGWAA AAAGFMLSNDLLIVTGALVGSSGAILSYIMCKAMNRSFISVIAGGFGTDGSSTGDDQEVGEHREITAEETAELLKNSHSVIITPGYGMAVAQAQYPVAEITEKLRARGINVRFGIHP VAGRLPGHMNVLLAEAKVPYDIVLEMDEINDDFADTDTVLVIGANDTVNPAAQDDPKSPIAGMPVLEVWKAQNVIVFKRSMNTGYAGVQNPLFFKENTHMLFGDAKASVDAILKAL.

[0020] The KW strain is described in the following literature: Chen, Y. et al. Rational design and analysis of an Escherichia coli strain for high-efficiency tryptophan production. Journal of Industrial Microbiol & Biotechnology, 45(5), 357-367(2018).

[0021] Example 1: Construction of L-tryptophan producing strain BDX1

[0022] PH5a-aroG fbr -trpE fbr DCBA plasmid construction: Using wild-type Escherichia coli MG1655 as a template, primers aroG-F and aroG-R were used to amplify the aroG fragment with a linker. Using PH5a plasmid as a template, PH5a-MF and PH5a-MR were used to amplify the PH5a-M fragment with a linker. Using aroG fragment and PH5a-M fragment as templates, aroG-F and PH5a-MR primers were used to amplify the aroG-M-Gibson fragment for Gibson assembly. Using MG1655 as a template, primers trpEDCBA-F and trpEDCBA-R were used to amplify the trpEDCBA-Gibson fragment with a linker. Using PH5a-ver-F and PH5a-ver-R, the plasmid backbone with a linker was amplified, and the PH5a-aroG-trpEDCBA plasmid was obtained by Gibson assembly with the above aroG-M-Gibson fragment and trpEDCBA-Gibson fragment. Using PH5a-aroG-trpEDCBA plasmid as template, aroG fbr -F and trpE fbr -R primers were used to amplify the PH5a-1-Gibson fragment with adapters, and aroG fbr -R and trpE fbr The PH5a-2-Gibson fragment with a linker was amplified by -F primer, and the PH5a-1-Gibson fragment and the PH5a-2-Gibson fragment were assembled by Gibson assembly to obtain the PH5a-aroG fbr -trpE fbr DCBA plasmid, plasmid map as shown Figure 1 shown.

[0023] Construction of cas9-ppsA plasmid: Using wild-type E. coli MG1655 as template, primers ppsA-up-F and ppsA-up-R were used to amplify the ppsA-UP fragment with a linker, and primers ppsA-down-F and ppsA-down-R were used to amplify the ppsA-Down fragment with a linker. The ppsA-UP fragment and ppsA-Down fragment were assembled into a ppsA-UD fragment. Using cas9 plasmid as template, primers ppsA-N20-F and ppsA-ver-R were used to amplify the plasmid backbone ppsA-ver1 fragment, and primers ppsA-ver-F and ppsA-N20-R were used to amplify the plasmid backbone tnaA-ver2 fragment. The plasmid backbones ppsA-ver1, ppsA-ver2 and the above fragment ppsA-UD were assembled by Gibson assembly (the Gibson assembly method was invented by Gibson et al. to achieve intermolecular connection of multiple DNA fragments in one reaction) to obtain the cas9-ppsA plasmid.

[0024] Construction of cas9-trpR plasmid: Using wild-type E. coli MG1655 as a template, primers trpR-up-F and trpR-up-R were used to amplify the trpR-UP fragment with a linker, and primers trpR-down-F and trpR-down-R were used to amplify the trpR-Down fragment with a linker. The trpR-UP fragment and trpR-Down fragment were assembled into a trpR-UD fragment. Using cas9 plasmid as a template, primers trpR-N20-F and trpR-ver-R were used to amplify the plasmid backbone trpR-ver1 fragment, and primers trpR-ver-F and trpR-N20-R were used to amplify the plasmid backbone trpR-ver2 fragment. The plasmid backbones trpR-ver1, trpR-ver2 and the above fragment trpR-UD were assembled by Gibson assembly (the Gibson assembly method was invented by Gibson et al. to achieve intermolecular connection of multiple DNA fragments in one reaction) to obtain the cas9-trpR plasmid.

[0025] Cas9-tnaA plasmid construction: Using MG1655 as a template, primers tnaA-up-F and tnaA-up-R were used to amplify the tanA-UP fragment with a linker, and primers tnaA-down-F and tnaA-down-R were used to amplify the tnaA-Down fragment with a linker. The tnaA-UP fragment and the tnaA-Down fragment were assembled into the tnaA-UD fragment. Using the cas9 plasmid as a template, the plasmid backbone tnaA-ver1 fragment was amplified with primers tnaA-N20-F and tnaA-ver-R, and the plasmid backbone tnaA-ver2 fragment was amplified with primers tnaA-ver-F and tnaA-N20-R. The plasmid backbone tnaA-ver1, tnaA-ver2 and the above fragment tnaA-UD were combined by Gibson assembly (the Gibson assembly method was invented by Gibson et al. to achieve intermolecular connection of multiple DNA fragments in one reaction) to obtain the cas9-tnaA plasmid.

[0026] The primers used in this section are as follows:

[0027] Table 1 Primers used to construct L-tryptophan producing strain BDX1

[0028]

[0029]

[0030] Plasmid PH5a-aroG fbr -trpEDCBA fbr The strain KW was transformed, and the ppsA gene was overexpressed by introducing the tac promoter into its genome, and the regulatory factor trpR and the L-tryptophan decomposition gene tnaA were knocked out to construct the tryptophan production strain BDX1. The strains and plasmids used in this section are as follows:

[0031] Table 2 Strains and plasmids used to construct L-tryptophan production strain BDX1

[0032]

[0033] Example 2: Construction of L-tryptophan producing strain BDP2

[0034] The inventors screened for highly active PntB and successfully screened out PntB mutants with improved catalytic ability.

[0035] Cas9-pntB plasmid construction: Using MG1655 as a template, primers pntB-up-F and pntB-up-R were used to amplify the pntB-up fragment with a linker, and primers pntB-down-F and pntB-down-R were used to amplify the pntB-down fragment with a linker. Using the pntB-up fragment and the pntB-down fragment as templates, primers pntB-up-F and pntB-down-R were used to amplify the pntB-UD fragment for Gibson assembly. Using the cas9 plasmid as a template, primers pntB-ver-F and pntB-N20-R were used to amplify the plasmid backbone pntB-ver1 with a linker, and primers pntB-N20-F and pntB-ver-R were used to amplify the plasmid backbone pntB-ver2 with a linker. The plasmid backbones pntB-ver1 and pntB-ver2 were assembled with the above pntB-UD fragments by Gibson assembly to obtain the plasmid cas9-pntB.

[0036] The primers used in this section are as follows:

[0037] Table 3 Primers used to construct L-tryptophan production strain BDP2

[0038] Primer name Nucleotide sequence (5'-3') Primer pntB-up-F (SEQ ID NO: 38) GAAGAATCCATGGGCCTGTGGGTGGGGCAATTGGTATCCG Primer pntB-up-R (SEQ ID NO: 39) AGGAAGGAAACGACCAGAGTCGCCAGGTTCATTTTGTGAC Primer pntB-down-F (SEQ ID NO: 40) ACAAAATGAACCTGGCGACTCTGGTCGTTTCCTTCCTG Primer pntB-down-R (SEQ ID NO: 41) AGAATCCAAGCTTCCATTCAAGGAACGGTTCATCGCCTTA Primer pntB-ver-F (SEQ ID NO: 42) TAAGGCGATGAACCGTTCCTTGAATGGAAGCTTGGATTCTC Primer pntB-ver-R (SEQ ID NO: 43) GGATAACCAATTGCCCCACCACAGGCCCATGGATTCTTCG Primer pntB-N20-F (SEQ ID NO: 44) GATCTTAGCCCGTCACAAAATGAACCTGGGTTTTAGAGCTAGAAATAGC Primer pntB-N20-R (SEQ ID NO: 45) CTCTAAAACCCAGGTTCATTTTGTGACGGGCTAAGATCTGACTCCATAAC

[0039] The plasmid cas9-pntB was introduced into the strain BDX1, and arabinose was added to the culture medium for induction, and finally the strain BDP2 containing the mutant PntB (A167T) was constructed. The strains and plasmids used in this section are as follows:

[0040] Table 4 Strains and plasmids used to construct L-tryptophan production strain BDP2

[0041]

[0042] Example 3, Fermentation of L-tryptophan producing strain

[0043] The shake flask fermentation process of Escherichia coli L-tryptophan production strains KW, BDX1 and BDP2 is as follows:

[0044] (1) Slant activation culture: Take out the preserved strain from the -80°C freezer, streak it onto a solid medium containing tetracycline resistance, and culture it at 37°C for 12-18 hours.

[0045] (2) Seed culture: Use an inoculation loop to pick a single colony from the fresh activated slant and place it in a seed basic culture medium (500 mL of LB culture medium in a 500 mL Erlenmeyer flask, sealed with a sealing film), and culture at 37°C and 220 rpm for 6-8 h until the OD 600 About 2-3.

[0046] (3) Shake flask batch fermentation: The seed culture solution was inoculated with 10% of the inoculum into a tetracycline-resistant basic fermentation medium (500 mL Erlenmeyer flask, 50 mL of liquid, sealed with a sealing film), and L-tryptophan batch fermentation was performed at 37°C and 220 r / min for 36-42 h. Shake flask fermentation medium Table 5:

[0047] Table 5 L-tryptophan fermentation medium formula

[0048] Medium composition content glucose 20g / L <![CDATA[(NH4)2SO4]]> 10g / L <![CDATA[KH2PO4]]> 5g / L yeast 2g / L mops 0.4M <![CDATA[MgSO4]]> 5g / L <![CDATA[FeSO47H2O]]> 15mg / L Sodium citrate 0.5g / L VB1 100mg / L <![CDATA[CuSO4·5H2O]]> 4mg / L <![CDATA[ZnSO4·7H2O]]> 4mg / L <![CDATA[MnSO4H2O]]> 15mg / L

[0049] Example 4, high performance liquid chromatography (HPLC) detection of fermentation strains

[0050] The fermentation broth was centrifuged at 5500 rpm / min for 15-20 min in a refrigerated centrifuge, and the supernatant was collected and filtered through a 0.22 μm filter membrane before HPLC detection.

[0051] HPLC conditions were as follows: the chromatographic column was a ZORBAX Eclipse AAA (amino acid analysis) chromatographic column, mobile phase A: 40 mM Na2HPO4, pH 7.8, mobile phase B: methanol: acetonitrile: water = 45:45:10, v / v / v. The elution gradient was 0-1 min, 100% A; 9.8 min: 43% A + 57% B; 10 min: 100% B; 12 min: 100% B; 12.5 min: 100% A. The flow rate was 2.0 mL / min, RID and VWD detectors were connected in series, the detection cell temperature was controlled at 40°C, the injection volume was 10 μL, the analysis time was 26 min, and the UV detection wavelength was 338 nm.

[0052] Example 5. Fermentation results and analysis of L-tryptophan producing strains KW, BDX1 and BDP2

[0053] After 38-42h of fermentation, the supernatant of the fermentation broth of strains KW, BDX1 and BDP2 was tested by HPLC. Figure 2 As shown in the fermentation results, it can be seen that the introduction of the PntB mutant PntB (A167T) on the basis of strain BDX1 increased the ability of strain BDP2 to produce tryptophan by 38.8% compared with strain BDX1, and the L-tryptophan production reached 1.02 g / L. On this basis, the NADPH content of strains BDX1 and BDP2 during fermentation was measured, and the results are shown in Figure 3 As shown, after the introduction of the PntB(A167T) mutant into strain BDP2, the concentration of intracellular NADPH increased by 4.8 times, directly proving that the mutant PntB(A167T) can more efficiently catalyze NADH to generate NADPH.

Claims

1. A PntB mutant, characterized in that: The amino acid residue A167 at position 167 of the pyridine nucleotide transhydrogenase β subunit PntB is site-directedly mutated to T, and its amino acid sequence is shown in SEQ ID NO:

1.

2. A genetically engineered bacterium containing the PntB mutant of claim 1, characterized in that: The amino acid residue A167 at position 167 of the pyridine nucleotide transhydrogenase β subunit PntB is site-directedly mutated to T, and its amino acid sequence is shown in SEQ ID NO:

1.

3. The method for constructing the genetically engineered bacteria according to claim 2, characterized in that: The tac promoter is introduced into the genome of the starting strain to overexpress the ppsA gene, and the regulatory factor trpR and the L-tryptophan decomposition gene tnaA are knocked out to obtain the strain BDX1. The 167th amino acid residue A167 of the pyridine nucleotide transhydrogenase β subunit PntB is mutated to T through site-directed mutagenesis and integrated into the strain BDX1 to obtain the strain BDP2, which is the genetically engineered bacteria.

4. The method for constructing the genetically engineered bacteria according to claim 3, characterized in that: Specifically, the following steps are included: 1) In the starting strain, construct a strain containing the gene aroG fbr and trpEDCBA fbr Recombinant plasmid PH5a-aroG fbr -trpEDCBA fbr , the ppsA gene was overexpressed on the genome, the trpR gene was knocked out, and the tnaA gene was knocked out to construct the strain BDX1; 2) In the BDX1 strain obtained in step 1), the amino acid residue A167 at position 167 of the pyridine nucleotide transhydrogenase β subunit PntB was mutated to T by site-directed mutagenesis to obtain a mutant PntB (A167T), thereby constructing the target strain BDP2.

5. The method for constructing a genetically engineered bacterium according to claim 3 or 4, characterized in that: The starting strain is Escherichia coli strain KW.

6. Use of the genetically engineered bacteria according to claim 2 in producing L-tryptophan by fermentation.

Images