Construction and development of an auto-regulatory gene expression system in Bacillus subtilis View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2015-12

AUTHORS

Chengran Guan, Wenjing Cui, Jintao Cheng, Li Zhou, Junling Guo, Xu Hu, Guoping Xiao, Zhemin Zhou

ABSTRACT

BACKGROUND: Bacillus subtilis is an all-important Gram-positive bacterium of valuable biotechnological utility that has been widely used to over-produce industrially and pharmaceutically relevant proteins. There are a variety of expression systems in terms of types of transcriptional patterns, among which the auto-inducible and growth-phase-dependent promoters are gaining increasing favor due to their inducer-independent feature, allowing for the potential to industrially scale-up. To expand the applicability of the auto-inducible expression system, a novel auto-regulatory expression system coupled with cell density was constructed and developed in B. subtilis using the quorum-sensing related promoter srfA (PsrfA). RESULTS: The promoter of the srf operon was used to construct an expression plasmid with the green fluorescent protein (GFP) downstream of PsrfA. The expression displayed a cell-density-dependent pattern in that GFP had a fairly low expression level at the early exponential stage and was highly expressed at the late exponential as well as the stationary stages. Moreover, the recombinant system had a similar expression pattern in wild-type B. subtilis 168, WB600, and WB800, as well as in B. subtilis 168 derivative strain 1681, with the complete deletion of PsrfA, indicating the excellent compatibility of this system. Noticeably, the expression strength of PsrfA was enhanced by optimizing the -10 and -35 core sequence by substituting both sequences with consensus sequences. Importantly, the expression pattern was successfully developed in an auto-regulatory cell-density coupling system by the simple addition of glucose in which GFP could not be strongly expressed until glucose was depleted, resulting in a greater amount of the GFP product and increased cell density. The expression system was eventually tested by the successful over-production of aminopeptidase to a desired level. CONCLUSION: The auto-regulatory cell density coupling system that is mediated by PsrfA is a novel expression system that has an expression pattern that is split between cell-growth and over-expression, leading to an increase in cell density and elevating the overall expression levels of heterologously expressed proteins. The broad applicability of this system and inducer-free expression property in B. subtilis facilitate the industrial scale-up and medical applications for the over-production of a variety of desired proteins. More... »

PAGES

150

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12934-015-0341-2

DOI

http://dx.doi.org/10.1186/s12934-015-0341-2

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1036655403

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/26392346


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Download the RDF metadata as:  json-ld nt turtle xml License info

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Turtle is a human-readable linked data format.

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RDF/XML is a standard XML format for linked data.

curl -H 'Accept: application/rdf+xml' 'https://scigraph.springernature.com/pub.10.1186/s12934-015-0341-2'


 

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