Engineering amino acid metabolism and transport in yeast

Industrialization and technological advances facilitated the drastic growth of the global population, which is now reliant on fossil fuels for electricity, transport, and the production of consumption goods. The bio-based production of chemicals can reduce the chemical industry's contribution to climate change and its impact on the environment by producing compounds from renewable sources using microorganisms. Dietary supplements, in particular, are difficult to synthesize chemically but are much easier to produce using microbial cell factories. The natural antioxidant ergothioneine has potential as a healthy aging dietary supplement to prevent neurodegenerative and cardiovascular disease. Ergothioneine is a high-value compound well suited for production in microbial cell factories, as it is difficult to synthesize chemically. This thesis describes the engineering of the amino acid metabolism and transport of yeast to produce ergothioneine. As the uptake and secretion of metabolites are often overlooked in the field of metabolic engineering, the use of transporters in microbial cell factories was reviewed, with a summary of the current methods for transporter identification and characterization.

Then, a study on the characterization of the energetically efficient Mae2p dicarboxylic acid transporter of Schizosaccharomyces pombe is presented. Subsequently, this thesis offers the reader a comprehensive, multi-disciplinary review of the nutraceutical ergothioneine. Finally, three studies describe the engineering of ergothioneine production in Saccharomyces cerevisiae and Yarrowia lipolytica. First, the ergothioneine production capabilities of S. cerevisiae were tested. Second, the amino acid metabolism in S. cerevisiae was extensively engineered, together with amino acid and ergothioneine transport, for the high-level production of ergothioneine. Third, the ergothioneine biosynthesis pathway was inserted in the host Y. lipolytica to produce ergothioneine through fermentation. Overall, this thesis presents knowledge and applications of transporters in microbial cell factories, shows the development of ergothioneine-producing yeast strains, and provides insight into amino acid metabolism and transport.