Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum

Microbial Cell Factories

Research

Wolfgang Wiechert¹  Roland Freudl¹  Sarah Jurischka¹  Johannes Hemmerich¹  Peter Rohe²  Marco Oldiges³  Britta Kleine⁴ 

[1] Institute of Bio- and Geosciences-Biotechnology (IBG-1), Forschungszentrum Jülich, Jülich, Germany;Bioeconomy Science Center (BioSC), Jülich, Germany;Institute of Bio- and Geosciences-Biotechnology (IBG-1), Forschungszentrum Jülich, Jülich, Germany;Boehringer Ingelheim Pharma GmbH and Co. KG, Biberach, Germany;Institute of Bio- and Geosciences-Biotechnology (IBG-1), Forschungszentrum Jülich, Jülich, Germany;Institute of Biotechnology, RWTH Aachen University, Aachen, Germany;Bioeconomy Science Center (BioSC), Jülich, Germany;Institute of Bio- and Geosciences-Biotechnology (IBG-1), Forschungszentrum Jülich, Jülich, Germany;Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany;

关键词: Corynebacterium glutamicum;    Industrial enzyme production;    Protein secretion;    Sec signal peptide library;    Microbioreactor;    Mini-Pilot-Plant;   

DOI  :  10.1186/s12934-016-0604-6

 received in 2016-08-21, accepted in 2016-11-24,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundTechnical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. Protein secretion can be achieved via general secretion (Sec) pathway. Specific sequences, signal peptides (SPs), are necessary to direct the target protein into the translocation machinery. For example, >150 Sec-specific SPs have been identified for Bacillus subtilis alone. As the best SP for a target protein of choice cannot be predicted a priori, screening of homologous SPs has been shown to be a powerful tool for different expression organisms. While SP libraries between closely related species were successfully applied to optimize recombinant protein secretion, this was not investigated for distantly related species. Therefore, in this study a Sec SP library from low-GC firmicutes B. subtilis is investigated to optimize protein secretion in high-GC actinobacterium Corynebacterium glutamicum using cutinase from Fusarium solani pisi as model protein.ResultsA homologous SP library (~150 SP) for recombinant cutinase secretion in B. subtilis was successfully transferred to C. glutamicum as alternative secretion host. Cutinase secretion in C. glutamicum was quantified using an automated micro scale cultivation system for online growth monitoring, cell separation and cutinase activity determination. Secretion phenotyping results were correlated to those from a previous study, in which the same SP library was used to optimize secretion of the same cutinase but using B. subtilis as host. Strikingly, behavior of specific SP-cutinase combinations was changed dramatically between B. subtilis and C. glutamicum. Some SPs showed comparable cutinase secretion performances in both hosts, whereas other SPs caused diametrical extracellular cutinase activities.ConclusionThe optimal production strain for a specific target protein of choice still cannot be designed in silico. Not only the best SP for a target protein has to be evaluated each time from scratch, the expression host also affects which SP is best. Thus, (heterologous) SP library screening using high-throughput methods is considered to be crucial to construct an optimal production strain for a target protein.

【 授权许可】

CC BY   
© The Author(s) 2016