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CN-121991869-A - Bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase and construction method and application thereof

CN121991869ACN 121991869 ACN121991869 ACN 121991869ACN-121991869-A

Abstract

The invention discloses a bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase (4 GT), and a construction method and application thereof. The engineering strain is constructed by taking bacillus subtilis DB403 as a host and introducing recombinant plasmid pHTN13/P spovG -P spovG -SP sacB -4 GT. The invention combines the strong constitutive serial promoter P spovG -P spovG with the high-efficiency signal peptide SP sacB and combines with the 4GT gene, thereby remarkably improving the secretory expression efficiency of the 4GT in bacillus subtilis. Under the optimized fermentation condition, the enzyme activity of the shake flask fermentation extracellular 4GT of the engineering strain can reach 88.53U/mL, and compared with the original strain, the engineering strain is improved by 96.21%. The engineering strain constructed by the invention can be used for producing the 4GT enzyme preparation with low cost and high efficiency, and has important application value in the fields of starch modification, functional food development and the like.

Inventors

  • JIANG DEMING
  • WEN YUANYUAN
  • SUN YANGNING
  • GAO HONGLIANG
  • HUANG JING
  • CHANG ZHONGYI
  • LI XIAOFANG
  • WANG YINGYU
  • XIANG RUFA

Assignees

  • 华东师范大学
  • 上海高更食品科技股份有限公司

Dates

Publication Date
20260508
Application Date
20260120

Claims (10)

  1. 1. A recombinant bacillus subtilis engineering strain is characterized by taking bacillus subtilis DB403 as a host and comprising a recombinant expression cassette, wherein the recombinant expression cassette comprises a promoter, a signal peptide, a coding gene of 4-alpha-glycosyltransferase or maltotriose transferase or 1, 4-alpha-glucan branching enzyme, and the strain can secrete and express the active 4-alpha-glycosyltransferase or maltotriose transferase or 1, 4-alpha-glucan branching enzyme; Wherein, the The promoter includes one or more of P grac 、P 43 、P 556 、P spovG 、P yvyD 、P haG 、P lytR 、P sigW 、P xylA 、P yqfD 、P mmgA 、P gerBC 、P bsamy 、P phrC 、P gsiB 、P nprE 、P bltD ; The signal peptide includes one or more of SP samyQ 、SP sacB 、SP lytF 、SP yybN 、SP ybbE 、SP ydbK 、SP yddT 、SP phrC 、SP cccA 、SP yxiT 、SP yvbX 、P spovG .
  2. 2. The recombinant bacillus subtilis engineering strain according to claim 1, wherein the strain takes bacillus subtilis DB403 as a host and comprises a recombinant expression cassette, wherein the recombinant expression cassette comprises a P spovG promoter, a coding sequence of a signal peptide SP sacB and a coding gene of 4-alpha-glycosyltransferase which are connected in series, and the strain can secrete and express the active 4-alpha-glycosyltransferase.
  3. 3. The recombinant bacillus subtilis engineering strain according to claim 1 wherein the recombinant expression cassette is located in one or more of plasmids pHTN13, pHT01, pHT43, pWB980, pBE.
  4. 4. A method, comprising one or more of the following: (1) A method of constructing a recombinant Bacillus subtilis engineering strain according to any one of claims 1 to 3, comprising constructing the recombinant Bacillus subtilis engineering strain by introducing and expressing the recombinant expression cassette according to any one of claims 1 to 3 into a Bacillus subtilis DB403 as a host; (2) A method for producing a 4-alpha-glycosyltransferase comprising fermenting the recombinant Bacillus subtilis engineering strain according to any one of claims 1 to 3, and obtaining the 4-alpha-glycosyltransferase from the fermentation broth.
  5. 5. The method according to claim 4, wherein in (2), the fermentation medium used for the fermentation comprises a carbon source and a nitrogen source, wherein the carbon source comprises soluble starch, the nitrogen source comprises tryptone, and/or the inoculum size of the fermentation is 1.0% -3.0%.
  6. 6. The method according to claim 4, wherein in (2), the composition of the fermentation medium comprises 1.0% -10.0% of the soluble starch, 0.5% -3.0% of tryptone, 20-28g/L of yeast extract, 1.8-2.6 g/L of K 2 HPO 4 14-19g/L,KH 2 PO 4 , a solvent comprising water and the like, and the pH is 6.8-7.2.
  7. 7. A fermentation medium is characterized by comprising 1.0% -10.0% of soluble starch, 0.5% -3.0% of tryptone, 20-28 g/L of yeast extract, 1.8-2.6 g/L of K 2 HPO 4 14-19 g/L,KH 2 PO 4 and a solvent comprising water and the like, wherein the pH is 6.8-7.2.
  8. 8. A substance, characterized in that the substance comprises one or more of the following: (1) A primer, the nucleotide sequence of which comprises one or more of the nucleotide sequences shown as SEQ ID NOS.4-65; (2) A primer pair, the nucleotide sequence of which comprises one or more of the nucleotide sequences shown as SEQ ID NOs 4-65; (3) A nucleotide sequence for a 4-alpha-glycosyltransferase, said nucleotide sequence being shown in SEQ ID No. 1; (4) A protein related to 4-alpha-glycosyltransferase, the amino acid sequence of the protein is shown as SEQ ID NO. 2; (5) A plasmid comprising one or more of pHTN13/P grac -SP samyQ -4GT、pHTN13/P grac -SP sacB -4GT、pHTN13/P grac -SP lytF -4GT、pHTN13/P grac -SP yybN -4GT、pHTN13/P grac -SP ybbE -4GT、pHTN13/P grac -SP ydbK -4GT、pHTN13/P grac -SP yddT -4GT、pHTN13/P grac -SP phrC -4GT、pHTN13/P grac -SP cccA -4GT、pHTN13/P grac -SP yxiT -4GT、pHTN13/P grac -SP yvbX -4GT、pHTN13/P 43 -SP samyQ -4GT、pHTN13/P 556 -SP samyQ -4GT、pHTN13/P spovG -SP samyQ -4GT、pHTN13/P yvyD -SP samyQ -4GT、pHTN13/P haG -SP samyQ -4GT、pHTN13/P lytR -SP samyQ -4GT、pHTN13/P sigW -SP samyQ -4GT、pHTN13/P xylA -SP samyQ -4GT、pHTN13/P yqfD -SP samyQ -4GT、pHTN13/P mmgA -SP samyQ -4GT、pHTN13/P gerBC -SP samyQ -4GT、pHTN13/P bsamy -SP samyQ -4GT、pHTN13/P phrC -SP samyQ -4GT、pHTN13/P gsiB -SP samyQ -4GT、pHTN13/P nprE -SP samyQ -4GT、pHTN13/P bltD -SP samyQ -4GT、pHTN13/P spovG -P haG -SP samyQ -4GT、pHTN13/P spovG -P yvyD -SP samyQ -4GT、pHTN13/P spovG -P 43 -SP samyQ -4GT、pHTN13/P spovG -P spovG -SP samyQ -4GT、pHTN13/P spovG -P spovG -SP sacB -4GT.
  9. 9. Use of a recombinant bacillus subtilis engineering strain according to any one of claims 1-3, or a method according to any one of claims 4-6, or a medium according to claim 7, or a substance according to claim 8 in the fermentative production of 4-alpha-glycosyltransferase, a bacillus subtilis genetic engineering strain for the production of high yield 4-alpha-glycosyltransferase 4GT, high efficiency fermentative production, large scale/economical production of 4GT enzyme preparation, high efficiency secretory expression systems, starch modification and functional food development fields, food medicine fields.
  10. 10. The recombinant Bacillus subtilis engineering strain, method, medium, substance according to any one of claims 1 to 9, wherein the gene encoding 4-alpha-glycosyltransferase is derived from Thermus thermophilus HB8 Thermus thermophilus HB, ATCC27634, and/or, The nucleotide sequence of the coding gene of the 4-alpha-glycosyltransferase is shown as SEQ ID NO. 1; the amino acid sequence of the SP samyQ signal peptide is the same as the amino acid sequence from 1 st to 31 st in the alpha-amylase precursor protein shown in NCBI accession number AAA22192.1, the amino acid sequence of the SP sacB signal peptide is the same as the amino acid sequence from 1 st to 29 st in the SacB protein shown in NCBI accession number CCD10844.1, the SP yybN signal peptide is the same as the amino acid sequence from 1 st to 30 nd in the DUF2712 domain protein shown in NCBI accession number WP_6728, the SP cccA signal peptide is the same as the amino acid sequence from 1 st to 27 st in the cytochrome c-550 protein shown in NCBI accession number MCSY 9210509.1, the SP lytF signal peptide is the same as the amino acid sequence shown in NCBI accession number AFG28268.1, the SP ybbE signal peptide is the same as the amino acid sequence shown in NCBI accession number AFG28210.1, the SP ydbK signal peptide is the amino acid sequence same as the amino acid sequence from 1 st to 30 st in the NCBI accession number WP 009968442.1, the SP cccA signal peptide is the same as the amino acid sequence from 1 st to 27 th in the cytochrome c-550 protein shown in NCBI accession number MCS, the amino acid sequence is the amino acid sequence from 1 st to 27 in the NCBI accession number NCG; The nucleotide sequence of the P promoter is identical to the nucleotide sequence from the first position to the second position in the sequence shown in NCBI accession number CP; the nucleotide sequence of the P promoter is identical with the nucleotide sequence of the complementary strand from the first position to the second position in the sequence shown in NCBI accession number CP; the nucleotide sequence of the P promoter is identical with the nucleotide sequence of the complementary strand from the first position to the second position in the sequence shown in NCBI accession number CP; the nucleotide sequence of the promoter is identical to the nucleotide sequence of the first position in the sequence shown in NCBI accession number CP, the nucleotide sequence of the promoter is identical to the nucleotide sequence of the complementary strand of the first position in the sequence shown in NCBI accession number CP, the nucleotide sequence of the promoter is identical to the nucleotide sequence of the complementary strand of the first position in the sequence shown in NCBI accession number CP, the nucleotide sequence of the promoter is identical to the nucleotide sequence from 3486142 to 3486441 in the sequence shown in NCBI accession number CP133412.1, the nucleotide sequence of the promoter P bsamy is identical to the nucleotide sequence of the complementary strand from 3852127 to 3852326 in the sequence shown in NCBI accession number CP054050.1, the nucleotide sequence of the promoter P phrC is identical to the nucleotide sequence from 413520 to 413819 in the sequence shown in NCBI accession number CP133412.1, the nucleotide sequence of the promoter P gsiB is identical to the nucleotide sequence from 478116 to 478421 in the sequence shown in NCBI accession number CP133412.1, the nucleotide sequence of the promoter P nprE is identical to the nucleotide sequence of the complementary strand from 1518759 to 1519160 in the sequence shown in NCBI accession number CP035411.1, and the nucleotide sequence of the promoter P bltD is identical to the nucleotide sequence from 2514262 to 2514562 in the sequence shown in NCBI accession number CP 133412.1.

Description

Bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase and construction method and application thereof Technical Field The invention belongs to the technical field of genetic engineering and microbial fermentation, relates to a bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase, a construction method and application thereof, and in particular relates to a construction method and application of a bacillus subtilis (Bacillus subtilis) genetic engineering strain for realizing efficient secretory expression of 4-alpha-glycosyltransferase (4 GT) through optimization of an expression element. Background The 4-alpha-glycosyltransferase (4-alpha-glycosyltransferase, 4GT, EC 2.4.1.25) can catalyze the hydrolysis and transfer of alpha-1, 4 glycosidic bonds in starch molecules, so that the starch main chain is shortened, the side chain is prolonged, the retrogradation resistance, the solubility and the digestion property of the starch are obviously improved, and the application prospect in the fields of foods, cosmetics and medicines is wide. 4GT derived from Thermus thermophilus HB8 (Thermus thermophilus HB, ATCC 27634) has been of great interest for its good thermal stability. However, the natural thermophilic strain has harsh culture conditions, low enzyme activity and high fermentation cost, and severely restricts industrialization thereof. Although the escherichia coli expression system has realized the heterologous expression of 4GT, the problems of low enzyme activity, difficult purification caused by intracellular expression, risk of endotoxin pollution and the like exist, and the application of the escherichia coli expression system in the field of food and medicine is limited. Bacillus subtilis (Bacillus subtilis) is an ideal heterologous protein expression host as a recognized food safety class (GRAS) microorganism with excellent protein secretion capacity. However, the enzyme activity of the recombinant expression 4GT in bacillus subtilis is generally low, and the lack of systematic collaborative optimization research on multi-level regulatory elements such as promoters, signal peptides and the like leads to the failure of the secretion expression efficiency of the 4GT to meet the industrial application requirement. Therefore, the development of the genetic engineering strain capable of efficiently secreting and expressing 4GT in bacillus subtilis has important significance for breaking through the industrial bottleneck and promoting the application of 4GT in the related fields. Disclosure of Invention In order to solve the technical problems of low secretion efficiency and low enzyme activity of bacillus subtilis expressed 4-alpha-glycosyltransferase (4 GT) in the existing research, the invention provides a bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase and a construction method and application thereof for the first time based on the technical means in the technical fields of the existing genetic engineering and microbial fermentation, and particularly provides a bacillus subtilis genetic engineering strain for high yield of 4-alpha-glycosyltransferase which is constructed through multi-scale and step-by-step expression element optimization, a construction method thereof and application thereof in high-efficiency fermentation production. The invention has the core innovation that the unique combination of the tandem double promoter P spovG-PspovG and the signal peptide SP sacB is provided and verified for the first time by constructing and screening a specific promoter library and a signal peptide library from a huge genetic element library of bacillus subtilis, has obvious synergistic effect on 4GT secretory expression, and further develops a soluble starch-tryptone fermentation medium which is precisely matched with the unique combination. The integration scheme finally enables the extracellular enzyme activity of the shake flask fermentation of 4GT to reach 88.53U/mL, and compared with the original optimized starting strain, the extracellular enzyme activity of the shake flask fermentation of 4GT is improved by 96.21%. The invention provides a recombinant bacillus subtilis (Bacillus subtilis) engineering strain, which takes bacillus subtilis DB403 as a host and comprises a recombinant expression cassette, wherein the recombinant expression cassette comprises a promoter, a signal peptide, a coding gene of 4-alpha-glycosyltransferase (4-alpha-glycosyltransferase, 4GT, EC 2.4.1.25) or maltotriose transferase or 1, 4-alpha-glucan branching enzyme and the like, and the recombinant bacillus subtilis engineering strain can secrete and express the active 4-alpha-glycosyltransferase or maltotriose transferase or 1, 4-alpha-glucan branching enzyme. In a specific embodiment, the promoter comprises one or more of Pgrac、P43、P556、PspovG、PyvyD、PhaG、PlytR、PsigW、PxylA、PyqfD、PmmgA、PgerBC、Pbsamy、PphrC、P