SUPERVISOR: Diethard MATTANOVICH

PROJECT ASSIGNED TO: Lisa SANVITO

Single carbon substrates (C1) gain increasing interest in biotechnology, as they are independent from agricultural production and can be obtained from CO2, offering a promising solution to global challenges such as the food crises and climate change. Among them, methanol and formate offer the advantage of being liquid, easy to store and transport, and to mix with aqueous culture media.

The methylotrophic yeast Komagataella phaffii is an established host for recombinant protein productions. It can use methanol as carbon and energy source thanks to the xylulose monophosphate (XuMP) pathway. It was proven that it can also use formate as carbon and energy source via the reductive glycine pathway (rGly), while co-assimilating CO2. In this case, growth is very slow due to metabolic burdens.

In this project, we aim to apply model-driven synthetic pathway designs to enable more energy and carbon efficient C1 utilization pathways that consequently lead to higher product yields.

At first, novel synthetic C1 utilization pathways and heterologous enzymes which could improve K. phaffii growth on methanol/formate will be identified and strains will be engineered according to these findings using CRISPR-Cas9 technology. The effect of knocking out or overexpressing genes involved in these pathways will also be evaluated to gain a better understanding of how these C1 substrates are metabolised in yeast. Secondly, adaptive laboratory evolution experiments will be carried out to enhance growth and tolerance to these C1 substrates. Thirdly, these strains will be characterized by whole genome sequencing while the pathway characterization will rely on 13C tracer studies which will be analysed with mass spectrometry.

This PhD project is part of the Cluster of Excellence Circular Bioengineering (COE), Program 2 “Self-Sustainable Microbial Systems”.