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.
Highlights
Technical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery
Dynamic harvest procedure reproduces signal peptide screening results from cutinase secreting B. subtilis strains with lower statistical error In this study, an integrated system of microbioreactor and liquid handling robot [22] was applied for evaluation of SP performance with respect to the secretory production of cutinase using C. glutamicum
To allow a direct comparison of the results for SP impact on cutinase secretion with B. subtilis obtained by Brockmeier et al [16], four B. subtilis expression strains with different signal peptides (YwmC, AmyE, NprE and YpjP) were re-assessed using the MPP cultivation and harvest setup
Summary
Technical bulk enzymes represent a huge market, and the extracellular production of such enzymes is favorable due to lowered cost for product recovery. A major part of industrial biotechnology is the production of technical bulk enzymes, with an estimated market of 1 billion US-$ in 2010 [1] To supply this market with sufficient quantities and in an economically feasible manner, platform technologies are developed continuously. Corynebacterium glutamicum as host organism for protein production For the production of technical bulk enzymes, different Gram-positive expression hosts are available. These mostly monoderm bacteria are robust in terms of cultivation conditions, low nutritional demand, and are able to secrete proteins into the extracellular medium [3, 4]. This system was recently improved for secretory antibody Fab fragment production by deletion of cspB and pbp1a [11]
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