Project Title: Bioprospecting for novel fungal and plant heme-dependent monooxygenases, expressed in yeasts
Supervisor: Prof. Toni Glieder, Graz University of Technology, Austria
Objectives: Based on the development and use of new cloning vectors for Pichia pastoris and fission yeast, innovative cytochrome P450 enzymes from eukaryotes should be discovered. Their expression will be realized by functional coexpression of heme and reductase domains from cDNA libraries in yeasts using bidirectional promoters or monocistronic expression with 2A sequences; As an attractive alternative, catalyzing similar reactions as P450 enzymes expression libraries will also be screened for new heme peroxygenase enzymes from eukaryotes. Another aim is to support other ESRs in the same workpackage in screening for improved activities for the hydroxylation of heterocyclic compounds and to apply the new and engineered enzymes in prep scale biotransformations.
Methodology: The work will involve various molecular biology and biochemical techniques and special training and applications for gene expression and library generation in Pichia pastoris. The focus will be on enzymes from eukaryotes to obtain complementary enzymes to known and well established bacterial P450s. Enzyme discovery and screening will be performed in close collaboration with partners in Aachen and Pavia where secondments are planed. LC-, GC-MS analytics will be applied for product analytics and screening. In silico and functional screening of ccale up experiments in the final phase of the thesis work is planed in collaboration with industrial partners in Scotland and the Netherlands.
Expected Results: (i) Generally applicable new vectors for eukaryotic mononctional oxygenase discovery by functional library generation and screening; (ii) Innovative plant and or fungal heme oxygenases with complementary substrate/product profiles; (iii) Standard protocols for whole cell biotransformation of heterocyclic compounds.