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CN-121991957-A - Bacillus subtilis DNA fragment with promoter function and application thereof

CN121991957ACN 121991957 ACN121991957 ACN 121991957ACN-121991957-A

Abstract

The invention belongs to the technical fields of genetic engineering and molecular biology, and relates to a DNA fragment, in particular to a DNA fragment with a promoter function of bacillus subtilis and application thereof in production of sucrose phosphorylase. The single-promoter screening method realizes high-efficiency secretion expression of BbrSP-VF and BadSP-QL in bacillus subtilis, the effect is obviously better than the expression effect of single promoter P43, and the extracellular enzyme activity is respectively improved by 247% and 236%. The DNA fragment with the promoter provided by the invention can realize secretory expression of the protein gene without adding an inducer, and the DNA fragment with the promoter is applied to expression of sucrose phosphorylase, provides an effective means for secretory expression of the protein by bacillus subtilis, and has a good development prospect.

Inventors

  • CHEN XIAOLONG
  • ZHUO MING
  • GAN TIAN
  • ZHANG FAN
  • ZHU LINJIANG
  • FAN YONGXIAN

Assignees

  • 浙江工业大学

Dates

Publication Date
20260508
Application Date
20260211

Claims (10)

  1. 1. A DNA fragment of bacillus subtilis having a promoter function, characterized in that the DNA fragment comprises a core functional region of a nucleotide sequence as shown in SEQ ID No.1, or a variant sequence having at least 90% sequence identity with the nucleotide sequence as shown in SEQ ID No.1 and retaining the promoter function.
  2. 2. The use of a DNA fragment of bacillus subtilis with promoter function according to claim 1 for protein expression.
  3. 3. The use according to claim 2, wherein the protein is a heterologous protein.
  4. 4. The use according to claim 3, wherein the heterologous protein is sucrose phosphorylase.
  5. 5. The use according to claim 4, wherein the sucrose phosphorylase is selected from BbrSP-VF or BadSP-QL.
  6. 6. A vector comprising a DNA segment of bacillus subtilis according to claim 1 having promoter function operably linked downstream of a nucleotide sequence encoding a heterologous protein.
  7. 7. A vector according to claim 6, wherein the nucleotide sequence encoding a heterologous protein encodes a sucrose phosphorylase.
  8. 8. A vector according to claim 7, wherein the sucrose phosphorylase is BbrSP-VF or BadSP-QL.
  9. 9. A recombinant engineered cell obtained by transformation or transfer of the vector of any one of claims 6-8 into a host cell.
  10. 10. A recombinant engineered cell according to claim 9, wherein the host cell is bacillus subtilis.

Description

Bacillus subtilis DNA fragment with promoter function and application thereof Technical Field The invention belongs to the technical fields of genetic engineering and molecular biology, and relates to a DNA fragment, in particular to a DNA fragment with a promoter function of bacillus subtilis and application thereof in production of sucrose phosphorylase. Background Bacillus subtilis (Bacillus subtilis) is a soil-borne gram-positive bacterium of the genus Bacillus. Compared with escherichia coli, the bacillus subtilis has the characteristic advantages of being used as a more attractive exogenous gene expression host, such as no endotoxin and no pathogenicity, being considered as a GRAS safe strain, having excellent secretion capacity, greatly simplifying the subsequent separation and purification steps of products, having low nutrition requirements, clear genetic background, being convenient for genetic modification and metabonomics analysis, having a large amount of information about transcription and translation mechanisms and large-scale fermentation, having no obvious codon usage preference and the like. Because of their unique advantages, bacillus subtilis has become an ideal host strain for the current production of various industrial enzymes, such as amylase, protease and lipase. The promoter is a DNA sequence located upstream of the 5' end of the gene, which activates RNA polymerase to bind precisely to the template DNA and has transcription initiation specificity. Transcription initiation is a critical stage of gene expression. Whereas regulation of transcription level is often affected by the strength, specificity and ability of the promoter sequence to respond to external signals. Different promoters have different activities, some promoters promote efficient gene transcription, and some may inhibit transcription. Therefore, screening for suitable promoters is critical for efficient expression of the protein of interest. By screening and optimizing the promoter for the target gene, the expression level of the target protein can be remarkably improved, and the effect of related research or industrial application can be further improved. Sucrose phosphorylase is a group 18 subunit of glycosyl hydrolase family GH13, and has EC number 2.4.1.7 and belonging to transferase. It possesses excellent substrate hybridization and can transfer glucosyl groups to different acceptors using only inexpensive sucrose as substrate. The glycosylation modification can improve the water solubility, stability, bioavailability and other properties of the compound, and the obtained product can be widely applied to the fields of food, medicine, personal care and the like. Sucrose phosphorylase (BbrSP-VF) derived from bifidobacterium breve can catalyze and synthesize the high-value glycoside product AA-2G with high efficiency (refer to patent CN 114703158A). In addition, sucrose phosphorylase (BadSP-QL) derived from bifidobacterium adolescentis (refer to patent CN 118813571A) can glycosylate and modify resveratrol to synthesize glycoside product 3-ResG, so that the water solubility and the biological activity of the resveratrol are effectively improved. Disclosure of Invention The invention aims to provide a DNA fragment with a promoter function of bacillus subtilis and application thereof in the production of two sucrose phosphorylases. In order to achieve the aim of the invention, the invention is realized by the following technical scheme: a DNA fragment of bacillus subtilis having a promoter function, said DNA fragment comprising a core functional region of a nucleotide sequence as set forth in SEQ ID No.1, or a variant sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID No.1 and retaining the promoter function. The invention successfully separates a DNA fragment which can efficiently express sucrose phosphorylase from bacillus subtilis genome, namely a sequence defined by SEQ ID NO.1 in a sequence table. The sequence is derived from a specific region in the bacillus subtilis genome, and has complete promoter function (named as P spoVG) through experiments. When the P spoVG promoter is applied to drive expression of a reporter gene or an exogenous protein having important industrial application value (such as sucrose phosphorylase), P spoVG exhibits overwhelming advantages compared to the widely used industrial standard of the P43 promoter. Under the condition of not depending on any exogenous inducer, the expression quantity of the target protein controlled by P spoVG is greatly improved, and the target protein is directly converted into the multiple increase of the activity of the related extracellular enzyme, so that the production efficiency and the economic benefit are greatly improved. In addition, as the P spoVG promoter keeps high activity continuously in the bacterial growth process, no additional inducer is needed in the fermentation process, the process flow is simp