Function, Control and Engineering of Microbial Methylotrophy

Methanol is an attractive low-cost substrate for biotechnology that does not need agricultural land for its production and can be produced sustainably from the greenhouse gas CO2. Methylotrophy, i.e. the ability of microorganisms to use methanol as carbon and energy source, has evolved in several groups of bacteria, and in a branch of budding yeasts. In yeasts, the methanol assimilation pathway is encapsulated in peroxisomes which may protect the cytosol from toxic intermediates while in bacteria the processes are cytosolic and the reactions are thoroughly balanced to prevent the accumulation of toxic compounds. Research questions We plan to elucidate the role of compartmentation on the functioning of the methanol metabolism in the naturally methylotrophic yeast Komagataella phaffii, and engineered Escherichia coli, by answering the following research questions: • Why and to which degree is compartmentation essential for methylotrophy in yeast? • Can we establish synthetic methylotrophy in bacteria using an “artificial” methylotrophic organelle? Approach We will re-target the entire assimilation pathway to the cytosol of K. phaffii, and exchange the first enzyme from an O2-dependent oxidase to an NAD+ dependent dehydrogenase to understand if any, or all pathway reactions depend on the peroxisomal localization. Artificial organelles based on bacterial microcompartments will be built to harbor the pathway, and introduced into E. coli to create synthetic E. coli strains with the yeast methanol utilization pathway. Functionality of the pathway variants will be assessed by in vitro and in vivo 13C based metabolomics, and the metabolic network and its interplay will be further balanced by adaptive laboratory evolution.