Method for Increasing Expression Level of Recombinant Proteins in Bacillus Subtilis Through Co-expressing Bacillus-derived Enhancer Factor

Patent metadata

Field	Value
Publication number	US-2025034539-A1
Application number	US-202418919792-A
Country	US
Kind code	A1
Filing date	Oct 18, 2024
Priority date	May 23, 2023
Publication date	Jan 30, 2025
Grant date	—

Abstract

Disclosed is a method for increasing expression level of recombinant proteins in Bacillus subtilis through co-expressing Bacillus -derived enhancer factor, belonging to the technical field of enzyme engineering. According to the present disclosure, genes encoding recombinant proteins are respectively co-expressed with Bacillus -derived PonA, PonA truncated forms, OppA or SppA, so as to improve the expression level of the recombinant proteins. The results show that the method provided by the present disclosure can increase the expression level of ultra-high temperature amylase, medium temperature amylase or sucrose isomerase at the shake flask level to 3.96 times, 1.49 times and 2.26 times. The expression level of ultra-high temperature amylase can be increased by 26% in high-density fermentation of a 3L bioreactor. The B. subtilis provides a new developable target as an recombinant protein expression host, and provides technical support for the optimization and modification of the enhancer factors.

First claim

What is claimed is:

1 . Recombinant Bacillus subtilis , expressing recombinant proteins, and proteins shown in (a) or (b):
(a) class A penicillin-binding proteins PonA derived from Bacillus; (b) truncated forms of class A penicillin-binding proteins DcPonA derived from Bacillus; wherein the recombinant proteins comprise ultra-high temperature amylase, medium temperature amylase or sucrose isomerase; wherein the proteins PonA having amino acid sequences as shown in any one of SEQ ID NOs:1 to 5; and wherein the truncated forms of the proteins PonA having amino acid sequences as set forth in any one of SEQ ID NOs:6 to 10.

2 . The recombinant B. subtilis according to claim 1 , wherein pUB110 is used as an expression vector to express ultra-high temperature amylase, medium temperature amylase or sucrose isomerase.

3 . The recombinant B. subtilis according to claim 1 , wherein pAD123 is used as an expression vector to express the class A penicillin-binding proteins PonA or the truncated forms of the class A penicillin-binding proteins PonA.

4 . The recombinant B. subtilis according to claim 1 , wherein the recombinant proteins are expressed through a constitutive promoter P amyQ′ .

5 . The recombinant B. subtilis according to claim 1 , wherein the class A penicillin-binding protein PonA, the truncated form of the class A penicillin-binding protein PonA, an ABC pathway transporter OppA or a signal peptide peptidase SppA is expressed through a promoter P glv or a promoter P HpaII .

6 . The recombinant B. subtilis according to claim 1 , wherein a host comprises B. subtilis SCK6.

7 . A method for increasing expression level of recombinant proteins in B. subtilis , comprising overexpressing class A penicillin-binding proteins PonA derived from Bacillus or truncated forms of class A penicillin-binding proteins PonA while expressing recombinant proteins; and the recombinant proteins comprise ultra-high temperature amylase, medium temperature amylase or sucrose isomerase.

8 . The method according to claim 7 , wherein pUB110 is used as an expression vector to express ultra-high temperature amylase, medium temperature amylase or sucrose isomerase.

9 . The method according to claim 8 , wherein pAD123 is used as an expression vector to express the class A penicillin-binding proteins PonA, the truncated forms of the class A penicillin-binding proteins PonA, an ABC pathway transporter OppA or a signal peptide peptidase SppA.

10 . A method for preparing enzyme proteins, wherein the recombinant B. subtilis according to claim 1 is fermented in a medium to prepare ultra-high temperature amylase, medium temperature amylase or sucrose isomerase.

11 . The method according to claim 10 , wherein the method comprises culturing the recombinant strain in a fermentation medium for a period of time to collect ultra-high temperature amylase, medium temperature amylase or sucrose isomerase.

12 . The method according to claim 10 , wherein feeding is further performed during fermentation.

Assignees

• Univ Jiangnan

Inventors

• Zhang Kang
• Wu Jing
• Zhu Xuyang
• Luo Hui

Classifications

• C12R2001/125

  Bacillus subtilis {; Hay bacillus; Grass bacillus} · CPC title

• C07K14/32 Primary

  from Bacillus (G) · CPC title

• C12Y302/01001

  Alpha-amylase (3.2.1.1) · CPC title

• C12N9/2417 Primary

  from microbiological source · CPC title

• C12N9/90

  Isomerases (5.) · CPC title