CN-121991870-A - Bacillus subtilis genetic engineering strain for high yield of starch branching enzyme and construction method and application thereof

CN121991870ACN 121991870 ACN121991870 ACN 121991870ACN-121991870-A

Abstract

The invention discloses a bacillus subtilis genetic engineering strain for high-yield starch branching enzyme (1, 4-alpha-glucan branching enzyme, GBE), a construction method and application thereof. The strain takes bacillus subtilis DB403 (B.subtilis DB 403) as an original host, obtains an optimized host strain by knocking out upp genes, alpha-amylase genes amyA, autolyzed key genes sigD and the like, obtains an optimized recombinant expression plasmid by systematically screening bacillus subtilis high-copy vectors and strong promoters, and introduces the expression plasmid into the host strain to construct the engineering strain. The GBE intracellular enzyme activity of the strain reaches 18104.32U/mL, which is improved by 64.58% compared with the original system, and the strain has wide application prospect in the field of starch modification and functional food development.

Inventors

JIANG DEMING
Zhou Juexin
GAO HONGLIANG
HUANG JING
NIU YANNING
CHANG ZHONGYI

Assignees

华东师范大学

Dates

Publication Date: 20260508
Application Date: 20260122

Claims (10)

1. A bacillus subtilis genetically engineered strain, characterized in that the strain comprises a host strain and a recombinant expression plasmid; the host strain is prepared by taking bacillus subtilis DB403 as an original host and knocking out one or more of upp genes, alpha-amylase genes amyA and autolysis related genes; Wherein the recombinant expression plasmid comprises a vector and a promoter.
2. A genetically engineered strain of Bacillus subtilis according to claim 1, wherein the vector comprises one or both of pWB980-ori, pHTN07, and/or, The promoter includes one or more of P43, pspovg, pbc, pgrac.
3. The genetically engineered strain of bacillus subtilis of claim 1, wherein the genetically engineered strain of bacillus subtilis comprises pWB980-ori/P43-GBE.
4. A method, comprising one or more of the following: (1) A method for preparing the bacillus subtilis genetic engineering strain according to claim 1, which comprises the steps of respectively constructing the host strain according to claim 1 and the recombinant expression plasmid according to claim 1, and then introducing the recombinant expression plasmid into the host strain to construct the bacillus subtilis genetic engineering strain; (2) A method for producing a starch branching enzyme, which comprises fermenting the recombinant Bacillus subtilis engineering strain according to any one of claims 1 to 3, collecting thalli, and dissolving to obtain the starch branching enzyme.
5. The method according to claim 4, wherein in (2), The fermentation time is 12-156h, and/or, The centrifugation conditions for collecting the thalli comprise centrifugation at 8000-12000r/min for 10-20min and/or, The solubilized reagent includes Arg.
6. A primer, characterized in that the nucleotide sequence of the primer comprises one or more of the nucleotide sequences shown in SEQ ID NOS.17-122.
7. A primer pair, wherein the nucleotide sequence of the primer pair comprises one or more of the nucleotide sequences shown in SEQ ID NOs 17-122.
8. A plasmid, characterized in that, the plasmid includes one or more of pWB980-ori/P43-GBE、pHTN07/Pspovg-GBE、pHTN07/P43-GBE、pWB980-ori/Pspovg-GBE、pHTN07/Pgrac-GBE、pWB980-ori/Pgrac-GBE、pWB980-ori/Pbc-GBE、pHTN07/Pbc-GBE.
9. The use of the bacillus subtilis genetically engineered strain according to any one of claims 1-3, or the method according to claim 4 or 5, or the primer pair according to claim 6, or the plasmid according to claim 7, in high-yield starch branching enzymes, bacillus subtilis genetically engineered strains for preparing high-yield starch branching enzymes, low-cost/stable/efficient production of GBE enzyme preparations, starch modification and functional food development, large-scale application of GBE, genetically engineered strains with easy purification of development products/small background interference, and matched processes thereof.
10. The genetically engineered strain of Bacillus subtilis, the method, the primer pair, the plasmid, the use according to claim 1 to 9, wherein the vector comprises one or more of pWB980-ori, pHTN07, and/or, The promoter comprises one or more of P43, pspovg, pbc, pgrac, and/or, The nucleotide sequence of the GBE coding gene is shown as SEQ ID NO. 1, the nucleotide sequence of the P43 promoter is shown as SEQ ID NO. 2, the nucleotide sequence of the Pspovg promoter is shown as SEQ ID NO. 3, the nucleotide sequence of the Pgrac promoter is shown as SEQ ID NO. 4, the nucleotide sequence of the Pbc promoter is shown as SEQ ID NO. 5, the nucleotide sequence of the upp is shown as SEQ ID NO. 6, the nucleotide sequence of the amyA is shown as SEQ ID NO. 7, the nucleotide sequence of the lytC is shown as SEQ ID NO. 8, the nucleotide sequence of the lytD is shown as SEQ ID NO. 9, the nucleotide sequence of the lytE is shown as SEQ ID NO. 10, the nucleotide sequence of the lytF is shown as SEQ ID NO. 110, the nucleotide sequence of the lytG is shown as SEQ ID NO. 12, the nucleotide sequence of the lytH is shown as SEQ ID NO. 13, the nucleotide sequence of the cwlC is shown as SEQ ID NO. 6, the nucleotide sequence of the cwlC is shown as SEQ ID NO. 14 or the nucleotide sequence of the 4225/5345 is shown as SEQ ID NO. 14, The bacillus subtilis genetic engineering strain is an engineering strain for high-expression of starch branching enzyme GBE and/or, The amyA knockout excludes the effect of non-target amylase enzymes in the starch branching enzyme GBE modified starch process, and/or, The sigD knockout delays the autolysis process of the host bacteria, increases the cell biomass and increases the GBE expression level of the starch branching enzyme, and/or, The knockout of the amyA gene and the sigD gene is realized by using the upp gene as a counter-screening marker through a homologous recombination technology and/or, The Arg solution can create an alkaline environment to promote the autonomous dissolution of inclusion bodies, and is favorable for converting starch branching enzyme into a soluble form with a correct folding conformation.

Description

Bacillus subtilis genetic engineering strain for high yield of starch branching enzyme 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 starch branching enzyme (1, 4-alpha-glucan branching enzyme, GBE) and a construction method and application thereof, and in particular relates to a bacillus subtilis genetic engineering strain which is prepared by systematically screening expression vectors and knocking out host specific genes to cooperatively improve the GBE expression level. Background Starch branching enzymes, which may also be referred to as 1,4- α -glucan branching enzymes (1, 4- α -glucan branching enzyme, GBE, ec 2.4.1.18), are key enzymes for catalyzing starch molecules to produce α -1,6 glycosidic bond branches, and for modifying the physicochemical properties (e.g., aging resistance, freeze-thaw stability) of starch. GBE derived from hyperthermophiles (e.g., aquifex aeolicus VF) has potential for industrial use due to its good thermal stability. Bacillus subtilis (Bacillus subtilis) is used as a safe expression host, but faces two major bottlenecks when the heterologous GBE is expressed efficiently, namely 1) alpha-amylase secreted by the host (mainly encoded by amyA genes) can degrade reaction substrate starch, seriously interfere with enzyme activity measurement and application effects of the GBE, and 2) a cell autolysis system regulated by lytC, lytD, sigD isogenes is activated in the middle and later stages of fermentation, so that biomass of thalli is reduced and target protein is degraded, and yield is limited. In the prior art, although there are attempts to express GBE in bacillus subtilis, the problems of strong background interference, short fermentation period, unstable yield and the like are common. Comprehensive strategies have not been reported to enhance cell stability and protein accumulation by systematically knocking out amyA genes to eliminate background interference, in combination with screening for knocking out key autolytic related genes, thereby greatly improving GBE yield. Therefore, the development of the genetic engineering strain and the matched process thereof, which can stably and efficiently express the GBE in the bacillus subtilis, have easy purification of products and small background interference, has great significance for promoting the large-scale application of the GBE. Disclosure of Invention The invention provides a bacillus subtilis genetic engineering strain for high-yield starch branching enzyme, a construction method and application thereof, and aims to solve the technical problems of strong host background interference, short fermentation period, low yield and instability in the prior art of bacillus subtilis expression GBE. The strain construction strategy has also been demonstrated to increase the expression levels of other glycosidases universally. The invention provides and verifies a strategy combining multidimensional optimization of a host chassis and systematic screening of an expression element in bacillus subtilis for the first time so as to realize efficient expression of glycosidic transferases such as starch branching enzyme (GBE). The innovation of the concept is that the method is characterized in that the method is used for defining that the background interference (amyA knockout) of host alpha-amylase is a necessary premise for accurately evaluating and improving the activity of target glycosidase in a starch substrate system, recognizing that the simple improvement of the expression intensity can aggravate the metabolic load of cells and induce advanced autolysis, so that the stability of the host cells is required to be synchronously enhanced, and the experiment shows that among a plurality of autolysis related genes, the effect of knocking out transcription regulatory factors sigD on prolonging the fermentation period, improving the biomass and the accumulation of target proteins is most obvious, thereby providing a new theoretical basis for target selection, and the method is used for carrying out matched screening on a multi-gene knocking-out host and a multi-vector/promoter combination for the first time, so that an optimized 'host-vector' expression system is obtained, and obvious synergistic effect is generated. In the process of implementing the conception, the invention mainly overcomes the following technical difficulties of realizing continuous and accurate knockout of a plurality of genes (upp, amyA, sigD and the like) in bacillus subtilis, ensuring the efficiency of gene operation and the stability of genome, rapidly and accurately identifying key genes with the greatest influence on the yield of target protein from a plurality of autolyzed related genes, and screening out the combination which is most matched with an optimized host chassis a