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

© 2020 The Authors..

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.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:28

Enthalten in:

Biotechnology reports (Amsterdam, Netherlands) - 28(2020) vom: 03. Dez., Seite e00540

Sprache:

Englisch

Beteiligte Personen:

Tran, Dinh Thi Minh [VerfasserIn]
Phan, Trang Thi Phuong [VerfasserIn]
Doan, Thanh Thi Ngoc [VerfasserIn]
Tran, Thuoc Linh [VerfasserIn]
Schumann, Wolfgang [VerfasserIn]
Nguyen, Hoang Duc [VerfasserIn]

Links:

Volltext

Themen:

BgaB, β-galactosidase
IPTG, isopropylthiogalactoside
Inducer-free
Integrative expression vector
Journal Article
LB, Luria broth
MCS, multiple cloning site
MUG, methylumbelliferyl β-d-galactopyranoside
PHT vector
Pgrac01 promoter
Pgrac100 promoter
Pgrac212 promoter

Anmerkungen:

Date Revised 12.11.2020

published: Electronic-eCollection

Citation Status PubMed-not-MEDLINE

doi:

10.1016/j.btre.2020.e00540

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM317311476