Optimising production of mannosylerythritol lipids by Pseudozyma graminicola through omic and molecular approaches

Abstract

Mannosylerythritol lipids (MEL) are biosurfactants produced primarily by basidiomycete fungi. MELs have potential applications in a wide variety of fields, ranging from medicine to industrial emulsifiers, making them an important target for investigation. MEL production is species-specific, with different proportions of the four variants MEL A-D, being produced and secreted. Of the MEL producers characterised to date all have a five-gene biosynthetic MEL cluster, directly involved on the synthesis of the molecule. However, the mechanisms involved in the regulation of these genes remains unclear. The work described in this thesis contributes to the understanding of MEL production by Pseudozyma graminicola, which primarily produces MEL-C. First, we sequenced and annotated the P. graminicola genome, from which we identified the MEL cluster. Based on comparative genomic analysis, we report P. graminicola as a potential biotrophic plant pathogen. Then, we developed a 1HMR based semi-quantitative method to confirm and monitor MEL production. Alongside this we identified the optimal conditions for the production of MELs using two fermentation systems. Transcription of the MEL cluster genes was monitored during MEL producing and non-producing fermentation conditions using RNAseq and RT PCR. We developed a transformation protocol, which was applied to deletion of emt1 to validate the gene cluster function. We also used the same approach to investigate the roles of three transcription factors, areA, gti1 and pac2 with potential regulation roles in MEL production. Based on this analisis, deletion of gti1 and pac2 appear to result in reduced and enhanced MEL production, repectivly. Further detailed analysis of these mutant is now required to confirm and potentially exploit these findings. The developments described in this thesis contribute to better understanding the biology of P. graminicola associated to MEL production, providing the knowledge necessary for future development of high-yield strains.