Ploss, Tina N., Reilman, Ewoud, Monteferrante, Carmine G., Denham, Emma, Piersma, Sjouke, Lingner, Anja, Vehmaanperä, Jari, Lorenz, Patrick and van Dijl, Jan Maarten (2016) Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions. Microbial Cell Factories, 15 (1). doi:10.1186/s12934-016-0455-1 ISSN 1475-2859.

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Abstract

Background
Bacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M.

Results
Here we show that activity of the htrB promoter as induced by overproduction of AmyM was “noisy”, which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions.

Conclusion
Expression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

Item Type:	Journal Article
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology
Divisions:	Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences > Microbiology & Infection Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School
Library of Congress Subject Headings (LCSH):	Amylases, Bacillus subtilis, Proteolytic enzymes
Journal or Publication Title:	Microbial Cell Factories
Publisher:	BioMed Central Ltd.
ISSN:	1475-2859
Official Date:	29 March 2016
Dates:	Date Event 29 March 2016 Published 22 March 2016 Accepted 5 February 2016 Submitted
Volume:	15
Number:	1
DOI:	10.1186/s12934-016-0455-1
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access (Creative Commons)

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