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

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, w...

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Autores principales: Tran, Dinh Thi Minh, Phan, Trang Thi Phuong, Doan, Thanh Thi Ngoc, Tran, Thuoc Linh, Schumann, Wolfgang, Nguyen, Hoang Duc
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599426/
https://www.ncbi.nlm.nih.gov/pubmed/33163371
http://dx.doi.org/10.1016/j.btre.2020.e00540
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author Tran, Dinh Thi Minh
Phan, Trang Thi Phuong
Doan, Thanh Thi Ngoc
Tran, Thuoc Linh
Schumann, Wolfgang
Nguyen, Hoang Duc
author_facet Tran, Dinh Thi Minh
Phan, Trang Thi Phuong
Doan, Thanh Thi Ngoc
Tran, Thuoc Linh
Schumann, Wolfgang
Nguyen, Hoang Duc
author_sort Tran, Dinh Thi Minh
collection PubMed
description 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.
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spelling pubmed-75994262020-11-05 Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis Tran, Dinh Thi Minh Phan, Trang Thi Phuong Doan, Thanh Thi Ngoc Tran, Thuoc Linh Schumann, Wolfgang Nguyen, Hoang Duc Biotechnol Rep (Amst) Review 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. Elsevier 2020-10-09 /pmc/articles/PMC7599426/ /pubmed/33163371 http://dx.doi.org/10.1016/j.btre.2020.e00540 Text en © 2020 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Review
Tran, Dinh Thi Minh
Phan, Trang Thi Phuong
Doan, Thanh Thi Ngoc
Tran, Thuoc Linh
Schumann, Wolfgang
Nguyen, Hoang Duc
Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
title Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
title_full Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
title_fullStr Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
title_full_unstemmed Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
title_short Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis
title_sort integrative expression vectors with pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599426/
https://www.ncbi.nlm.nih.gov/pubmed/33163371
http://dx.doi.org/10.1016/j.btre.2020.e00540
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