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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0604-6) contains supplementary material, which is available to authorized users.

Combinatorial impact of Sec signal peptides from Bacillus subtilis and bioprocess conditions on heterologous cutinase secretion by Corynebacterium glutamicum

“…Similar secretion performance was achieved when using the same SPs in closely related Bacillus species, namely B. subtilis and B. licheniformis (Degering et al, 2010). However, when comparing SPs for secretion of the same protein in more distantly related host organisms like B. subtilis and C. glutamicum, an erratic correlation of the secretion performance is found (Hemmerich, Rohe, et al, 2016).…”

“…Application examples of cutinase comprise laundry and dishwashing detergents, food industry and textile processing (Chen, Su, Chen, & Wu, 2013). SPs from B. subtilis were shown to enable heterologous cutinase secretion in C. glutamicum (Rohe, Venkanna, Kleine, Freudl, & Oldiges, 2012), yet results from SP library screening in B. subtilis, to identify SPs causing high cutinase secretion efficiency in this organism, cannot be transferred to C. glutamicum (Hemmerich, Rohe, et al, 2016). Because it is not known that which SP is best suited to secrete cutinase with C. glutamicum for various bioprocess conditions a priori, five Sec SPs from B. subtilis were chosen, associated with a high (Epr), average (YwmC), and low (YpjP) cutinase secretion performance in B. subtilis (Brockmeier et al, 2006).…”

“…Similar secretion performance was achieved when using the same SPs in closely related Bacillus species, namely B. subtilis and B. licheniformis (Degering et al, ). However, when comparing SPs for secretion of the same protein in more distantly related host organisms like B. subtilis and C. glutamicum , an erratic correlation of the secretion performance is found (Hemmerich, Rohe, et al, ). The a priori prediction of the best performing SP for a target protein of interest failed so far and the very specific, but still unpredictable, best combination of SP and target protein has to be evaluated from scratch (Brockmeier et al, ).…”

Microbioreactor Systems for Accelerated Bioprocess Development

“…The integration of MBR systems into robotic environments greatly enhances their capabilities towards an improved quantitative microbial phenotyping and bioprocess development . In particular, liquid handling robotics allow for automated manipulation of growing cultures, e.g., by adding inducers, taking samples, adding nutrients and adjusting pH.…”

Comprehensive exploration of the translocation, stability and substrate recognition requirements in VIM-2 lactamase

“…The less than optimal residue level fitness of wtVIM-2 may be because we employ E. coli as a host while natural VIM variants are often found in Pseudomonas 62 , and/or the signal peptide is not selected to produce maximum expression in natural environments. It has been shown that different signal peptides produce variable expression levels and translocation rates for a given protein, both of which affect the final resistance, especially in different host organisms 23,57,58,[63][64][65] . Furthermore, the signal peptide is frequently mutated in naturally occurring VIM-type variants (see section on 'Natural VIM variation' below), suggesting changes in the signal peptide sequences may have played significant roles in dissemination of MBL genes to different hosts and adaptation to higher antibiotic concentrations.…”