Pntb mutant, genetically engineered bacteria containing the mutant and construction method and application thereof

Abstract

The present application relates to the field of genetic engineering, and specifically provides a PntB mutant for improving L-tryptophan yield, a genetically engineered bacterium containing the mutant, a construction method and application thereof, the present application aims at the problem of NADPH deficiency in the production process of aromatic amino acids, especially L-tryptophan, obtains a pyridine nucleotide transhydrogenase beta subunit mutant PntB(A167T) through site-directed mutagenesis, and constructs a genetically engineered bacterium containing the mutant, the genetically engineered bacterium is applied in L-tryptophan fermentation, promotes the conversion of NADH to generate NADPH, the concentration of intracellular NADPH is increased by 4.8 times, the ability of the genetically engineered strain BDP2 to produce L-tryptophan is increased by 38.8% relative to the initial strain BDX1, the yield of L-tryptophan reaches 1.02g / L, and finally the efficient synthesis of L-tryptophan is realized.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically providing a PntB mutant that increases L-tryptophan production, a genetically engineered bacterium containing the mutant, its construction method, and its applications. Background Technology

[0002] L-Tryptophan is an essential amino acid crucial for human health. It is not only a building block of protein synthesis but also a precursor to serotonin (5-hydroxytryptamine) and melatonin, both of which play important roles in regulating mood, sleep, and appetite. L-Tryptophan is widely found in protein-rich foods such as dairy products, meat, fish, and nuts. While sufficient L-Tryptophan can usually be obtained from the diet, in certain situations, such as mood swings or sleep disorders, people may consider using L-Tryptophan supplements. However, high doses of supplements can cause side effects, so it is advisable to consult a professional before use. Historically, a health incident in Japan in 1989 due to L-Tryptophan supplement contamination prompted increased global regulation of dietary supplements. Overall, L-Tryptophan plays an indispensable role in maintaining normal physiological functions and promoting health.

[0003] As an important reducing agent, 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 facilitate the reaction. The synthetic pathway of aromatic amino acids involves multiple steps, many of which involve enzymatic reactions that require NADPH. For example, the formation of shikimic acid, a precursor in aromatic amino acid synthesis, requires 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. This is usually achieved by adjusting the substrate in the culture medium to increase NADPH production: for example, adding certain sugars or amino acids can promote reducing power generation. Additionally, increasing NADPH production can enhance reducing power: this can be achieved by overexpressing certain 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. This β subunit contains an N-terminal domain with 6-8 transmembrane regions and a cytoplasmic C-terminal domain that binds NADP(H). Early studies showed that the PntB(G314E) mutation leads to the loss of both energy-dependent and energy-independent pyridine nucleotide transhydrogenase activities. In *E. coli*, PntB can convert NADH to NADPH, thereby increasing intracellular NADPH supply. Summary of the Invention

[0005] The purpose of this invention is to modify the gene involved in reducing power conversion in the L-tryptophan synthesis of Escherichia coli through genetic engineering methods, thereby obtaining a strain that expands L-tryptophan production.

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

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

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

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

[0010] 3) Shake-flask fermentation was performed to verify the L-tryptophan producing 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. Attached Figure Description

[0012] Figure 1 PH5a-aroG fbr -trpE fbr DCBA plasmid map

[0013] Figure 2 Results of shake-flask fermentation of L-tryptophan producing strains BDX1 and BDP2

[0014] Figure 3 Results of NADPH concentration determination in fermentation broths of L-tryptophan producing strains BDX1 and BDP2 Detailed Implementation

[0015] The following examples are provided to help to better understand the present invention, but are not intended to limit the invention.

[0016] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

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

[0018] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0019] The amino acid sequence of the 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 *E. coli* MG1655 as a template, the aroG fragment with the adapter was amplified using primers aroG-F and aroG-R. Using PH5a plasmid as a template, the PH5a-M fragment with the adapter was amplified using primers PH5a-MF and PH5a-MR. Using the aroG and PH5a-M fragments as templates, the aroG-F and PH5a-MR primers were used to amplify the aroG-M-Gibson fragment for Gibson assembly. Using MG1655 as a template, the trpEDCBA-Gibson fragment with the adapter was amplified using primers trpEDCBA-F and trpEDCBA-R. The plasmid backbone with the adapter was amplified using PH5a-ver-F and PH5a-ver-R, and then assembled with the aroG-M-Gibson and trpEDCBA-Gibson fragments to obtain the PH5a-aroG-trpEDCBA plasmid using Gibson assembly. Using PH5a-aroG-trpEDCBA plasmid as a template, and aroG fbr -F and trpE fbr -R primers amplify the PH5a-1-Gibson fragment with the adapter, using aroG fbr -R and trpE fbr -F primers amplify the PH5a-2-Gibson fragment with the adapter. The PH5a-1-Gibson fragment and the PH5a-2-Gibson fragment are then assembled with Gibson to obtain PH5a-aroGibson. fbr -trpE fbr DCBA plasmid, plasmid map as shown Figure 1 As shown.

[0023] Construction of the Cas9-PPSA plasmid: Using wild-type Escherichia coli MG1655 as a template, the ppsA-UP fragment with the adapter was amplified using primers ppsA-up-F and ppsA-up-R, and the ppsA-Down fragment with the adapter was amplified using primers ppsA-down-F and ppsA-down-R. The ppsA-UP and ppsA-Down fragments were then assembled into the ppsA-UD fragment. Using the Cas9 plasmid as a template, the plasmid backbone ppsA-ver1 fragment was amplified using primers ppsA-N20-F and ppsA-ver-R, and the plasmid backbone tnaA-ver2 fragment was amplified using primers ppsA-ver-F and ppsA-N20-R. The plasmid backbones ppsA-ver1 and ppsA-ver2, along with the fragment ppsA-UD, were combined using Gibson assembly (a method invented by Gibson et al. to achieve intermolecular ligation of multiple DNA fragments in a single reaction) to obtain the cas9-ppsA plasmid.

[0024] Construction of the Cas9-trpR plasmid: Using wild-type E. coli MG1655 as a template, the trpR-UP fragment with the adapter was amplified using primers trpR-up-F and trpR-up-R, and the trpR-Down fragment with the adapter was amplified using primers trpR-down-F and trpR-down-R. The trpR-UP and trpR-Down fragments were then assembled into the trpR-UD fragment. Using the Cas9 plasmid as a template, the plasmid backbone trpR-ver1 fragment was amplified using primers trpR-N20-F and trpR-ver-R, and the plasmid backbone trpR-ver2 fragment was amplified using primers trpR-ver-F and trpR-N20-R. The plasmid backbones trpR-ver1 and trpR-ver2, along with the fragment trpR-UD, were combined using Gibson assembly (the Gibson assembly method, invented by Gibson et al., involves the intermolecular ligation of multiple DNA fragments in a single reaction) to obtain the cas9-trpR plasmid.

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

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

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

[0028]

[0029]

[0030] Plasmid PH5a-aroG fbr -trpEDCBA fbr The tryptophan-producing strain BDX1 was constructed by transforming strain KW and overexpressing the ppsA gene by introducing the tac promoter into its genome, and then knocking out the regulatory factor trpR and the L-tryptophan degradation gene tnaA. The strains and plasmids used in this section are as follows:

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

[0032]

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

[0034] The inventors screened highly active PntB and successfully screened out PntB mutants with enhanced catalytic activity.

[0035] Construction of the Cas9-pntB plasmid: Using MG1655 as a template, the pntB-up fragment with adapter was amplified using primers pntB-up-F and pntB-up-R, and the pntB-down fragment with adapter was amplified using primers pntB-down-F and pntB-down-R. Using the pntB-up and pntB-down fragments as templates, the pntB-UD fragment for Gibson assembly was amplified using primers pntB-up-F and pntB-down-R. Using the Cas9 plasmid as a template, the plasmid backbone pntB-ver1 with adapter was amplified using primers pntB-ver-F and pntB-N20-R, and the plasmid backbone pntB-ver2 with adapter was amplified using primers pntB-N20-F and pntB-ver-R. The plasmid backbones pntB-ver1 and pntB-ver2, along with the pntB-UD fragment above, were assembled using Gibson 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-producing 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 strain BDX1, and induction was performed by adding arabinose to the culture medium to finally construct strain BDP2 containing the mutant PntB (A167T). The strains and plasmids used in this section are as follows:

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

[0041]

[0042] Example 3: Fermentation of L-tryptophan-producing strains

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

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

[0045] (2) Seed culture: Use an inoculation loop to pick a single colony from a fresh activated slant and place it in seed culture medium (50 mL LB medium in a 500 mL Erlenmeyer flask, sealed with sealing film). Incubate at 37℃ and 220 r / min for 6-8 h until OD. 600 Approximately 2-3.

[0046] (3) Shake-flask batch fermentation: The seed culture was inoculated at a rate of 10% into a tetracycline-resistant fermentation basal medium (500 mL Erlenmeyer flask, 50 mL inoculation volume, sealed with sealing film), and L-tryptophan was fermented in batches at 37℃ and 220 r / min for 36-42 h with shaking. Table 5 shows the shake-flask fermentation medium.

[0047] Table 5. L-Tryptophan Fermentation Medium Formulation

[0048] Culture medium components 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: Detection of fermentation strains by high performance liquid chromatography (HPLC)

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

[0051] The HPLC conditions were as follows: ZORBAX Eclipse AAA (amino acid analysis) 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. A RID and VWD detector were connected in series. The detection cell temperature was controlled at 40℃. The injection volume was 10 μL. The analysis time was 26 min. 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 fermentation culture of strains KW, BDX1, and BDP2 for 38-42 hours, the supernatant of the fermentation broth was analyzed by HPLC. The results are as follows: Figure 2 As shown in the figure. The fermentation results show that, based on strain BDX1, the introduction of the PntB mutant PntB (A167T) increased the tryptophan production capacity of strain BDP2 by 38.8% compared to strain BDX1, achieving an L-tryptophan yield of 1.02 g / L. Based on this, the NADPH levels during the fermentation process of strains BDX1 and BDP2 were measured, and the results are shown in the figure. Figure 3 As shown, the intracellular NADPH concentration increased by 4.8 times after the introduction of the PntB(A167T) mutant into strain BDP2, directly proving that the mutant PntB(A167T) can more effectively catalyze the conversion of NADH to NADPH.

Claims

1. A method for constructing a genetically engineered bacterium containing a PntB mutant, characterized by: The genome in the starting strain Escherichia coli strain KW is introduced into a tac promoter overexpression ppsA gene, a regulatory factor is knocked out trpR and an L-tryptophan decomposition gene tnaA , to obtain a strain BDX1, the 167th amino acid residue A167 in the pyridine nucleotide transhydrogenase beta subunit PntB is mutated into T through site-directed mutation, and is integrated into the strain BDX1 to obtain a strain BDP2, which is the genetically engineered bacteria.

2. The method for constructing the genetically engineered bacteria according to claim 1, characterized in that: Specifically, comprising the following steps: 1) In the starting strain, a recombinant plasmid PH5a-aroG aroG fbr and trpEDCBA fbr -trpEDCBA fbr -trpEDCBA fbr , overexpressing ppsA gene, knocking out trpR gene, knocking out gene tnaA , and obtaining strain BDX1; 2) In the BDX1 strain obtained in step 1), the 167th amino acid residue A167 of the pyridine nucleotide transhydrogenase β subunit PntB is mutated into T by site-directed mutation to obtain the mutant PntB(A167T), thereby obtaining the target strain BDP2.

3. The genetically engineered bacteria according to claim 1 are applied in fermentation for producing L-tryptophan.

Images