Engineering Yeast Metabolism for Production of Sesquiterpenes
Doktorsavhandling, 2016

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.

Saccharomyces cerevisiae






metabolic engineering

Opponent: Brian Pfleger, University of Wisconsin-Madison, United States of America


Stefan Tippmann

Chalmers, Biologi och bioteknik, Systembiologi

From flavors and pharmaceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae

Biotechnology journal,; Vol. 8(2013)p. 1435-1444

Artikel i vetenskaplig tidskrift

Production of farnesene and santalene by Saccharomyces cerevisiae using fed-batch cultivations with RQ-controlled feed

Biotechnology and Bioengineering,; Vol. 113(2016)p. 72-81

Artikel i vetenskaplig tidskrift

Affibody scaffolds improve production of sesquiterpenes in Saccharomyces cerevisiae.

Industrial production of petroleum derived chemicals is well established and extremely efficient. However, in connection with diminishing oil resources and an increasing environmental burden, there is a strong demand for more sustainable processes in the chemical industry. Within the past 25 years, the science of Metabolic Engineering has emerged as a key technology to enable production of chemicals by microbial fermentation from renewable carbon sources. In this respect, the yeast Saccharomyces cerevisiae plays an important role, since it is tolerant towards industrial conditions (e.g. low pH, high sugar concentrations) and its metabolism can be engineered efficiently to expand its product portfolio. Several metabolic engineering efforts have focused on production of terpenes, which represent a large and diverse group of natural products with numerous applications as fragrances, pharmaceuticals, surfactants, lubricants and emollients.

In this study, production of farnesene was investigated in S. cerevisiae, which can be used as a diesel fuel in its hydrogenated form farnesane. Cellular metabolism needs to be rewired to produce farnesene efficiently in S. cerevisiae. For this purpose, different aspects affecting product formation have been addressed, such as the construction of enzyme clusters as a potential tool to allow for improved substrate channeling from one enzyme to another. Besides, changes in the gene expression profile and metabolite abundances upon engineering metabolism were investigated to improve the understanding of farnesene synthesis in S. cerevisiae. In conclusion, this study established novel metabolic engineering approaches and served the identification of new ones, which will support future research regarding the production of these compounds in S. cerevisiae.



Biokatalys och enzymteknik


Livsvetenskaper och teknik



Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4140


Chalmers tekniska högskola


Opponent: Brian Pfleger, University of Wisconsin-Madison, United States of America