Genetic platform for improved expression and secretion of enzymes and antimicrobial peptides

Wei, Long; Schwaneberg, Ulrich (Thesis advisor); Elling, Lothar (Thesis advisor)

Aachen : RWTH Aachen University (2020, 2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020

Abstract

Enzymes are biological catalysts that are produced by living organisms. Their properties make them indispensable in different fields of industry and research. Obtaining enzymes in sufficient quantities is important for their application, as well as for their scientific analysis. Heterologous enzyme production is a common method to achieve high enzyme titers and employing secretion can improve enzyme yield as well as reduce downstream processing. The goal of this thesis was to establish a platform for enzyme production that can be used for simple and straightforward optimization of enzyme expression and secretion. The main objective was the generation of a signal peptide library fusion platform that can be used to screen for suitable signal peptides. To this end, a cloning strategy was established for simple and fast library generation. Subsequently, a signal peptide library was built with signal peptides from B. subtilis. This library served as starting point for subcloning into different vectors and for generating fusion libraries of different target genes. Two different vectors were used for subcloning: a C. glutamicum vector to enable enzyme production in the gram positive bacterium and a vector containing the fluorescent reporter protein Pp1FbFP to enable monitoring the target protein secretion during the expression process. A cutinase, a phytase and a diguanylate cyclase were cloned into the library to test the library’s applicability. For cutinase, the signal peptide screening resulted in a threefold increase in activity, which was achieved by the signal peptide YobB. The best signal peptide for the phytase AppA showed 3.5-fold improvement compared to the starting variant. For the diguanylate cyclase tDGCsh, secretion could not be observed. Fusion with signal peptides does not only enable enzyme secretion, it can also be used for the secretion of peptides. Secretory production of antimicrobial peptides in C. glutamicum was tested for the four antimicrobial peptides AFP1, LCI, Psoriasin and VarvF. With the signal peptide NprE, LCI and Psoriasin were secreted at high levels. Titers of up to 130 mg/L have been reached for LCI while Psoriasin concentration was estimated to be 54 mg/L. Directed evolution was performed to improve different signal peptides towards more efficient secretion. For this purpose, the signal peptides PhrF and YbdN were chosen. Two rounds of directed evolution for PhrF resulted in a 2.4-fold increase in activity. YbdN was evolved in one round of mutagenesis, resulting in a 1.36-fold improvement. Coexpression of enzymes with additional unfused signal peptides resulted in significantly improved enzyme secretion. The effect of coexpressed signal peptides on secretion was strongly depended on the expression conditions and the used signal peptide. To further improve enzyme production, vector backbone evolution with the epMEGAWHOP method was performed for the compact E. coli vector pALXtreme-1a and the E. coli-C. glutamicum shuttle vector pEKEx2. The activity of the model enzyme cutinase was increased up to 2.5-fold after two evolution rounds for pEKEx2 and up to ninefold after three rounds of directed evolution for pALXtreme-1a.

Institutions

• Department of Biology [160000]
• Chair of Biotechnology [162610]