Kluyveromyces lactis as an attractive expression platform for production of PSI-like antimicrobial peptides

CURTO, P.; LUFRANO, D.; PINTO, C.; CUSTÓDIO, V.; GOMES, A.C.; VAIRO CAVALLI, S.; FARO, C.; SIMÕES, I.

Simposio; 26th International Conference on Yeast Genetics and Molecular Biology; 2013

Goethe University Frankfurt

Typical plant aspartic protease zymogens are characterized by containing a recognizable saposin-like domain (plant specific insert - PSI). As a common feature to saposin-like proteins, the membrane interaction ability of several PSI domains was confirmed and a role as defensive weapons against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents for the development of new biocontrol tools or alternative therapeutic applications is yet to be fully explored, as these domains have been heterologously produced in E. coli with very low production yields. Here, we report the development of a Kluyveromyces lactis-based expression platform for the production of the PSI domain of cirsin in high yields. Upon optimization, we successfully generated K. lactis transformants expressing and secreting correctly processed PSI. This domain was shown to be glycosylated and we further demonstrated that this non-conventional yeast can be an effective platform for production of both wt cirsin PSI and its non-glycosylated form. A purification protocol was established with protein yields of 4.33 mg/L for wt and 4.67 mg/L for non-glycosylated PSI; the highest reported for a PSI domain expressed without solubility enhancement tags. Moreover, bioactivity assays towards phytopathogenic fungi confirmed that cirsin PSI is produced in a biologically active form in K. lactis and provide the first evidences for its anti-fungal activity. This is the first report on the production of a PSI domain in an eukaryotic expression system which, therefore, emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.