Synthetic growth-coupling of heterologous enzyme activities in microbial cells

Evolution arises from an impactful and fascinating law of biology: survival of the fittest. Interestingly, this is broadly speaking also the destiny of most engineering projects, including those on developing novel solutions for the synthesis of valuable bioactive compounds using biocatalysts, such as enzymes and microorganisms. The solutions that are most cost and resource efficient will make it to the market and contribute to realizing a more sustainable society in the future. More than ever are we equipped with techniques to construct and modify enzymes and biosynthetic pathways towards compounds of our interest, yet we lack methods to design and identify the best variants among those. The work within this thesis centers around a powerful and handy genetic tool that can enable researchers to screen and select the best enzyme variants out: coupling of the activity of enzymes to the growth of microorganisms. Generally, growth is a simple phenotype to screen for, and since microorganisms strive towards better growth, growth-coupling (GC) enables both directed evolution of enzymes and application of evolution experiments to screen and/or evolve the most active enzymes and pathways. Simply, GC couples the objective of both engineers and microorganisms: better enzyme activity for better growth. More active enzymes can enable better production of the bioactive compounds of our interest. The focus of this work has been to growth-couple the activity of methyltransferases (MTase GC). Methyltrans-ferases are enzymes that are ubiquitous in natural product pathways and promising enzymatic biocatalysts for in vitro synthesis of bioactive compounds of societal interest. In this work we present a MTase GC design and focus on its implementation and improvement in the eukaryotic production chassis, the yeast Saccharomyces cerevisiae. We follow-up on the initial proof-of-concept work by improving the resolution of the GC, demonstrate coupling of multiple methyltransferases to growth and present how to screen different enzyme variants within a library of catechol O-methyltransferases. The MTase GC design leverages a conserved feature of the metabolism of living organisms: the global methyl group donor, S-adenosylmethionine. In an attempt to investigate this ancient part of the yeast metabolism, we evolved MTase GC design strains and discovered consistent mutations in genes encoding a central transporter and a regulatory cofactor protein of the (sulfur) metabolism, indicative of adaptation being mediated by a multitude of specific mechanisms.The work presented throughout the thesis provides the scientific community with a new tool for engineering better production of methylated compounds using the yeast S. cerevisiae and contributes novel insights into its buffered metabolic and regulatory networks.