Engineering Yeast Metabolism for Production of Sesquiterpenes

Abstract: Sesquiterpenes belong to the large and diverse group of isoprenoids, which are ubiquitous in the plant kingdom and have various applications in the chemical industry as fragrances, pharmaceuticals and as substitutes for petroleum-based gasoline, diesel and jet fuels. In this project, production of sesquiterpenes was studied in Saccharomyces cerevisiae using farnesene as an example with the objective to gain new insights into the synthesis of these compounds and to evaluate different metabolic engineering strategies.

Farnesyl diphosphate (FPP) represents the universal precursor for all sesquiterpenes and different strategies were addressed to increase production of this intermediate and to allow for its efficient conversion to farnesene. As FPP is provided by the mevalonate pathway, we aimed at increasing the flux through the pathway by incorporation of malonyl-CoA using a recently identified acetoacetyl-CoA synthase from Streptomyces sp. strain CL190. While the enzyme had detrimental effects on growth as well as on product formation, it was able to compensate for the loss of the essential, endogenous acetoacetyl-CoA thiolase. Additionally, a homologous enzyme with superior efficiency could be identified. Secondly, as FPP is required as substrate for different pathways and represents a metabolic branch point, a novel tool for enzyme co-localization was developed to divert more flux towards farnesene. The system utilizes scaffolds based on affibodies and could improve product yields by more than twofold. Furthermore, the role of terpene synthases on the production of farnesene was elucidated by comparing farnesene synthases with different plant origins, i.e. Malus domestica, Citrus junos and Artemisia annua. While the selected candidates produced similar amounts of farnesene (up to 170 mg/L), these enzymes appeared to be superior in comparison to other sesquiterpene synthases, i.e. santalene synthase. Lastly, the response to increased product formation was investigated using transcriptome and metabolome analysis, which highlighted changes across various metabolic pathways and identified pantothenic acid as a potential target for future engineering strategies.

In conclusion, the study evaluates different metabolic engineering strategies for production of sesquiterpenes in S. cerevisiae and provides new insights into the cellular response during their production. Additionally, utilization of affibodies as a novel tool in metabolic engineering to increase chemical production in S. cerevisiae was highlighted.