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CN-121975710-A - Construction method and application of bacillus amyloliquefaciens chassis strain

CN121975710ACN 121975710 ACN121975710 ACN 121975710ACN-121975710-A

Abstract

The invention provides a construction method of a bacillus amyloliquefaciens chassis strain and application thereof, which takes bacillus amyloliquefaciens HZ12 as a starting strain, screens and identifies two key genes of a non PTS system from a glucose transport path, namely a glucose osmotic enzyme gene Bao-glcP1 from bacillus amyloliquefaciens and a glucokinase gene Cgb-glK from corynebacterium glutamicum, and after the genes are overexpressed, the L-tyrosine yield of engineering bacteria reaches 395.82 mg/L and 385.37 mg/L respectively, which is 36% and 33% higher than that of the starting strain. And then, by strengthening a non-PTS system and weakening the PTS system, reconstructing glucose uptake and phosphorylation pathways of the strain, decoupling the glucose phosphorylation process from PEP, obtaining a chassis strain with high L-tyrosine yield, and improving the L-tyrosine yield to 407.16 mg/L, wherein the yield is improved by 43% compared with that of the original strain. The key speed-limiting enzyme genes aroA and tyrA for L-tyrosine synthesis are further over-expressed in the chassis strain, so that the yield of the L-tyrosine is increased to 1400.53 mg/L, and compared with the original strain, the yield is increased by 3.82 times.

Inventors

  • ZHAO ZIYUE
  • Ji Anying
  • SONG CAN
  • ZHAI ZHENGYUAN
  • SHI YAHONG

Assignees

  • 中原食品实验室

Dates

Publication Date
20260505
Application Date
20260206

Claims (9)

  1. 1. A bacillus amyloliquefaciens chassis strain HZ12 comprises Bao-glcP1 comprises Cgb-glK DeltatsH, and is characterized in that the bacillus amyloliquefaciens chassis strain HZ12 comprises Bao-glcP1 comprises Cgb-glK DeltatsH, the bacillus amyloliquefaciens is taken as a starting strain, glucose osmotic enzyme genes glcP1 and glucose kinase genes glK are overexpressed, and a coding gene ptsH of phosphoryl carrier protein HPr is deleted, so that the bacillus amyloliquefaciens chassis strain HZ12 comprises Bao-glcP1 comprises Cgb-glK DeltatsH.
  2. 2. The bacillus amyloliquefaciens chassis strain HZ 12:Bao-glcP 1:Cgb-glK DeltatsH, which is disclosed in claim 1, wherein the glucose penetrating enzyme gene glcP1 is derived from bacillus amyloliquefaciens, and the nucleotide sequence of the glucose penetrating enzyme gene glcP1 is shown as SEQ ID No. 2.
  3. 3. The bacillus amyloliquefaciens chassis strain HZ 12:Bao-glcP 1:Cgb-glK ΔptsH according to claim 1, wherein the glucokinase gene glK is derived from corynebacterium glutamicum, and the nucleotide sequence of the glucokinase gene is shown as SEQ ID NO. 7.
  4. 4. The construction method of the bacillus amyloliquefaciens chassis strain HZ 12:Bao-glcP 1:Cgb-glK delta ptsH according to any one of claims 1-3, which is characterized by comprising the following steps: S1, using a genome of bacillus amyloliquefaciens as a template, amplifying a homology arm A, a homology arm B and a gene fragment P43+UTR1+Bao-glcP1+ TamyL, connecting the homology arm A, the homology arm B and the P43+UTR1+Bao-glcP1+ TamyL fragment by SOE-PCR to obtain a fusion fragment, using restriction enzyme XbaI and BamHI to double-cleave the fusion fragment and a temperature-sensitive knockout plasmid T2 (2) -ori, connecting the fusion fragment after enzyme cleavage and the T2 (2) -ori by using T4 ligase, and transforming a connection product into escherichia coli competent cells to obtain an integrated expression vector T2 (2) -XKDG of the gene Bao-glcP1; S2, electrically converting an integrated expression vector T2 (2) -XKDG of Bao-glcP1 into competent cells of bacillus amyloliquefaciens, identifying to obtain a positive transformant, and screening and identifying single-exchange and double-exchange strains of the positive transformant to obtain an integrated strain HZ12 of Bao-glcP1; S3, using a genome of bacillus amyloliquefaciens as a template, amplifying a homology arm A, a homology arm B and a gene fragment P43+Cgb-glK + TamyL, connecting the homology arm A and the homology arm B with the gene fragment P43+Cgb-glK + TamyL by SOE-PCR to obtain a fusion fragment, using restriction enzymes XbaI and BamHI to double-digest the fusion fragment and a temperature-sensitive knockout plasmid T2 (2) -ori, connecting the digested fusion fragment and the digested fusion fragment T2 (2) -ori by using T4 ligase, and transforming a connection product into competent cells of escherichia coli to obtain an integrated expression vector T2 (2) -glK of a gene Cgb-glK; s4, electrically converting an integrated expression vector T2 (2) -XKDE:: cgb-glK into an integrated strain HZ12:: bao-glcP1 constructed in S2 to obtain the integrated strain HZ 12::: bao-glcP1:: cgb-glK; S5, using a genome of bacillus amyloliquefaciens as a template, amplifying a homologous arm A and a homologous arm B, connecting the homologous arm A and the homologous arm B through SOE-PCR to obtain a fusion fragment, using restriction endonucleases XbaI and BamHI to double-digest the fusion fragment and a temperature-sensitive knockout plasmid T2 (2) -ori, connecting the digested fusion fragment and the digested T2 (2) -ori with T4 ligase, and transforming competent cells of the escherichia coli by using a connection product to obtain a knockout vector T2 (2) delta ptsH of a gene ptsH; S6, electrotransformation of the knockout vector T2 (2) delta ptsH into the integrated strain HZ12:: bao-glcP1:: cgb-glK constructed in S4 to obtain the chassis strain HZ 12::: bao-glcP 1::: cgb-glK delta ptsH.
  5. 5. A bacillus amyloliquefaciens genetic engineering bacterium HZ13/tyrA-aroA is characterized in that the bacillus amyloliquefaciens genetic engineering bacterium HZ13/tyrA-aroA is constructed on the basis of Bao-glcP 1:Cgb-glK DeltatsH and the DAHP synthase gene aroA and the prephenate dehydrogenase gene tyrA of any one of claims 1-3.
  6. 6. The bacillus amyloliquefaciens chassis strain HZ12 of any one of claims 1-3, bao-glcP1, cgb-glK ΔptsH or the application of the bacillus amyloliquefaciens genetically engineered bacterium HZ13/tyrA-aroA in the production of L-tyrosine.
  7. 7. The use according to claim 6, wherein the specific method for producing L-tyrosine comprises the steps of: S1, activating the bacillus amyloliquefaciens chassis strain HZ12 of any one of claims 1-3, wherein Bao-glcP1, cgb-glK delta ptsH or bacillus amyloliquefaciens genetic engineering bacterium HZ13/tyrA-aroA of claim 5 on an LB plate, and then picking single colony to inoculate in an LB liquid culture medium, and culturing for 8-12 h at 35-38 ℃ and 180-220 rpm to obtain seed liquid; s2, inoculating the seed solution into a fermentation medium with an inoculum size of 3-5%, and fermenting at 35-38 ℃ and 143-170 rpm for 36-40 h to produce the L-tyrosine.
  8. 8. The application of the fermentation medium according to claim 7, wherein the composition of the fermentation medium comprises 4-6 g/L glucose 20~25 g/L,(NH 4 ) 2 SO 4 2.5~4.5 g/L,K 2 HPO 4 6~7 g/L,KH 2 PO 4 1~2 g/L,MgSO 4 ·7H 2 O 1~2g/L, sodium citrate, 4-6 g/L peptone, 5-7 g/L yeast extract and 1-mL/L trace elements.
  9. 9. The use according to claim 8, wherein the trace element comprises sodium citrate 9~12 g/L,FeSO 4 ·7H 2 O 3~5 g/L,CaCl 2 3~5 g/L,MnSO 4 ·5H 2 O 0.5~2 g/L,CoCl 2 ·6H 2 O 0.2~0.6 g/L,ZnSO 4 ·7H 2 O 0.1~0.4 g/L,AlCl 3 ·6H 2 O 0.05~0.2 g/L,CuCl 2 ·H 2 O 0.05~0.2 g/L,H 3 BO 4 0.001~0.006 g/L.

Description

Construction method and application of bacillus amyloliquefaciens chassis strain Technical Field The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a construction method and application of a bacillus amyloliquefaciens chassis strain. Background L-tyrosine is taken as an important aromatic amino acid, is not only one of key components of protein synthesis, but also an important precursor of various high-added-value bioactive compounds (such as levodopa, resveratrol and various alkaloids), and has wide market prospect in the fields of medicines, nutritional health-care products and fine chemical engineering. With the rapid development of synthetic biology and metabolic engineering technology, green biological production of L-tyrosine by using a microbial cell factory has become a mainstream direction for replacing the conventional chemical extraction method. Among many microbial hosts, bacillus amyloliquefaciens is considered as an excellent platform for the production of amino acids and derivatives thereof by virtue of its excellent protein secretion ability, clear genetic background, and well-established strains. However, the natural metabolic network of the strain is highly synthesized in L-tyrosine, and the utilization efficiency of carbon sources, especially the uptake and primary metabolic pathways of glucose, often become key bottlenecks for limiting the accumulation of target products. In bacillus amyloliquefaciens, uptake and phosphorylation of glucose is primarily dependent on the phosphotransferase system (PTS). PTS is a highly efficient but metabolically costly system for phosphorylating glucose into glucose-6-phosphate by directly utilizing phosphoenolpyruvate (PEP) as a phosphate donor while it is transported into cells. Although this process achieves simultaneous transport and activation of sugars, it also leads to irreversible consumption of PEP. Notably, PEP is a key metabolic precursor to the initiation of the aromatic amino acid synthesis pathway. In the shikimate pathway, condensation of PEP with erythrose 4-phosphate under the catalysis of DAHP synthase is the rate limiting step of the whole pathway. In E.coli, intracellular 50% of PEP flows to the PTS system for glucose transport. Thus, there is a direct competition for PEP between PTS-mediated glucose uptake and L-tyrosine synthesis, and this inherent metabolic conflict is believed to be a central factor that restricts further enhancement of L-tyrosine production, as schematically shown in fig. 1. Based on this, substitution of PTS with phosphoenolpyruvate independent uptake mechanism and phosphorylation function may help to promote L-tyrosine synthesis in Bacillus amyloliquefaciens. Disclosure of Invention Aiming at the technical problems, the invention aims to provide a construction method of a bacillus amyloliquefaciens chassis strain and application thereof, which reconstruct a glucose transport system of bacillus amyloliquefaciens and improve the yield of L-tyrosine. Through excavating and identifying key gene targets involved in glucose uptake and phosphorylation in bacillus amyloliquefaciens, a novel efficient glucose utilization way matched with L-tyrosine synthesis is constructed in bacillus amyloliquefaciens through screening and introducing high-performance heterologous glucose transport proteins and glucokinase. And the expression level of the key genes is further improved through the optimization of UTR. Finally, by weakening the endogenous PTS system and strengthening the non-PTS pathway, the phosphorylation process of glucose is decoupled from PEP, and ATP is relied on for energy supply, thus providing a new strategy and material basis for realizing efficient microbial manufacture of the amino acid and the derivative thereof. The bacillus amyloliquefaciens chassis strain HZ12 comprises Bao-glcP1 comprises Cgb-glK DeltatsH, bacillus amyloliquefaciens is taken as a starting strain, and a coding gene ptsH of phosphoryl carrier protein HPr is deleted through over-expression of glucose osmotic enzyme genes glcP1 and glucose kinase genes glK, so that the bacillus amyloliquefaciens chassis strain HZ12 comprises Bao-glcP1 comprises Cgb-glK DeltatsH. Preferably, the glucose penetrating enzyme gene glcP1 is derived from bacillus amyloliquefaciens, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2. Preferably, the glucokinase gene glK is derived from corynebacterium glutamicum, and the nucleotide sequence of the glucokinase gene is shown as SEQ ID NO. 7. The construction method of the bacillus amyloliquefaciens chassis strain HZ12 comprises the following steps of Bao-glcP1, cgb-glK delta ptsH: S1, using a genome of bacillus amyloliquefaciens as a template, amplifying a homology arm A, a homology arm B and a gene fragment P43+UTR1+Bao-glcP1+ TamyL, connecting the homology arm A, the homology arm B and the P43+UTR1+Bao-glcP1+ TamyL fragment by SOE-PCR to obtain a f