Integrative expression vectors with P grac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Dinh Thi Minh Tran; Trang Thi Phuong Phan; Thanh Thi Ngoc Doan; Thuoc Linh Tran; Wolfgang Schumann; Hoang Duc Nguyen

doi:10.1016/j.btre.2020.e00540

Integrative expression vectors with P grac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Biotechnol Rep (Amst). 2020 Oct 9:28:e00540. doi: 10.1016/j.btre.2020.e00540. eCollection 2020 Dec.

Authors

Dinh Thi Minh Tran^{1

2

3

4}, Trang Thi Phuong Phan^{1

5

4}, Thanh Thi Ngoc Doan^{1

6}, Thuoc Linh Tran^{1

4}, Wolfgang Schumann^{1

7}, Hoang Duc Nguyen^{1

3

4}

Affiliations

¹ Center for Bioscience and Biotechnology, University of Science, 227 Nguyen Van Cu Dist. 5, Ho Chi Minh City, Viet Nam.
² Department of Biology, Ho Chi Minh City University of Education, 280 An Duong Vuong, Dist. 5, Ho Chi Minh City, Viet Nam.
³ Department of Microbiology, University of Science, 227 Nguyen Van Cu Dist. 5, Ho Chi Minh City, Viet Nam.
⁴ Vietnam National University, Ho Chi Minh City, Viet Nam.
⁵ Laboratory of Molecular Biotechnology, University of Science, 227 Nguyen Van Cu Dist. 5, Ho Chi Minh City, Viet Nam.
⁶ Agriculture and Food Technology Faculty, Tien Giang University, 119, Ap Bac, My Tho City, Tien Giang, Viet Nam.
⁷ Institute of Genetics, University of Bayreuth, D-95440, Bayreuth, Germany.

Abstract

Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the β-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.

Keywords: BgaB, β-galactosidase; IPTG, isopropylthiogalactoside; Inducer-free; Integrative expression vector; LB, Luria broth; MCS, multiple cloning site; MUG, methylumbelliferyl β-d-galactopyranoside; Pgrac01 promoter; Pgrac100 promoter; Pgrac212 promoter; pHT vector.