Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

Microbial Cell Factories

Research

Tina N. Ploss¹  Patrick Lorenz¹  Anja Lingner²  Sjouke Piersma³  Jan Maarten van Dijl³  Ewoud Reilman³  Carmine G. Monteferrante⁴  Emma L. Denham⁵  Jari Vehmaanperä⁶ 

[1] AB Enzymes GmbH, Feldbergstrasse 78, 64293, Darmstadt, Germany;AB Enzymes GmbH, Feldbergstrasse 78, 64293, Darmstadt, Germany;c-LEcta GmbH, Perlickstraße 5, 04103, Leipzig, Germany;Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RD, Groningen, The Netherlands;Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RD, Groningen, The Netherlands;Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands;Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RD, Groningen, The Netherlands;Division of Biomedical Sciences, Warwick Medical School, University of Warwick, CV4 7AL, Coventry, UK;Roal Oy, Tykkimäentie 15b, 05200, Rajamäki, Finland;

关键词: Bacillus subtilis;    Amylase;    Heterogeneity;    Secretion stress;    Transcription;    Translation;   

DOI  :  10.1186/s12934-016-0455-1

 received in 2016-02-05, accepted in 2016-03-22,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundBacillus 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.ResultsHere 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.ConclusionExpression 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.

【 授权许可】

CC BY   
© Ploss et al. 2016