Intramembrane protease RasP boosts protein production in Bacillus

Microbial Cell Factories

Research

Jan Maarten van Dijl¹  Jolanda Neef¹  Vivianne J. Goosens²  Cristina Bongiorni³  Brian Schmidt³ 

[1] Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, P.O. Box 30001, Hanzeplein 1, 9700 RB, Groningen, The Netherlands;Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, P.O. Box 30001, Hanzeplein 1, 9700 RB, Groningen, The Netherlands;Department of Bioengineering, Centre for Synthetic Biology, Imperial College London, SW7 2AZ, London, UK;DuPont Industrial Biosciences, 925 Page Mill Road, 94304, Palo Alto, CA, USA;

关键词: Bacillus;    RasP;    Site-2 protease;    Secretion;    Amylase;    Protease;   

DOI  :  10.1186/s12934-017-0673-1

 received in 2016-12-30, accepted in 2017-03-31,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe microbial cell factory Bacillus subtilis is a popular industrial platform for high-level production of secreted technical enzymes. Nonetheless, the effective secretion of particular heterologous enzymes remains challenging. Over the past decades various studies have tackled this problem, and major improvements were achieved by optimizing signal peptides or removing proteases involved in product degradation. On the other hand, serious bottlenecks in the protein export process per se remained enigmatic, especially for protein secretion at commercially significant levels by cells grown to high density. The aim of our present study was to assess the relevance of the intramembrane protease RasP for high-level protein production in B. subtilis.ResultsDeletion of the rasP gene resulted in reduced precursor processing and extracellular levels of the overproduced α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis. Further, secretion of the overproduced serine protease BPN’ from Bacillus amyloliquefaciens was severely impaired in the absence of RasP. Importantly, overexpression of rasP resulted in threefold increased production of a serine protease from Bacillus clausii, and 2.5- to 10-fold increased production of an AmyAc α-amylase from Paenibacillus curdlanolyticus, depending on the culture conditions. Of note, growth defects due to overproduction of the two latter enzymes were suppressed by rasP-overexpression.ConclusionHere we show that an intramembrane protease, RasP, sets a limit to high-level production of two secreted heterologous enzymes that are difficult to produce in the B. subtilis cell factory. This finding was unexpected and suggests that proteolytic membrane sanitation is key to effective enzyme production in Bacillus.

【 授权许可】

CC BY   
© The Author(s) 2017