Multidimensional engineering for the production of fatty acid derivatives in Saccharomyces cerevisiae

Doctoral thesis, 2019

Saccharomyces cerevisiae, also known as budding yeast, has been important for human society since ancient time due to its use during bread making and beer brewing, but it has also made important contribution to scientific studies as model eukaryote. The ease of genetic modification and the robustness and tolerance towards harsh conditions have established yeast as one of the most popular chassis in industrial-scale production of various compounds. The synthesis of oleochemicals derived from fatty acids (FAs), such as fatty alcohols and alka(e)nes, has been extensively studied in S. cerevisiae, which is due to their key roles as substitutes for fossil fuels as well as their wide applications in other manufacturing processes. Aiming to meet the commercial requirements, efforts in different engineering approaches were made to optimize the TRY (titer, rate and yield) metrics in yeast.

The major aim of this thesis was to enable a versatile yeast platform for the production of FA derivatives through diverse engineering strategies. We tested several membrane transporters for the potential to mediate fatty alcohol export in S. cerevisiae. A novel function of the mammalian transporter FATP1 was identified as it was able to benefit fatty alcohol efflux in a high fatty alcohol production strain. According to the results, human FATP1 led to an improvement of extracellular fatty alcohols (2.6-fold increase) and cell fitness compared with the control strain. FATP1 was then introduced into an engineered S. cerevisiae strain carrying a heterologous 1-alkene biosynthetic pathway for improved 1-alkene secretion and production. Combined with an optimization of fatty acid metabolism and the electron transport system, a final titer of 35.3 mg/L of 1-alkenes was achieved with more than 80% being secreted.

Medium-chain fatty acids (MCFAs) are non-inherent fatty acids in yeast whose microbial synthesis is considered to be challenging. Through expressing either an engineered native fatty acid synthase (FAS) or an engineered bacterial type I FAS, the synthesis of MCFAs has been successfully implemented in yeast. In our work, directed evolution of the native transporter Tpo1 and adaptive laboratory evolution were performed to increase the tolerance against MCFAs. Together with further augmentation of the metabolic flux towards MCFAs and optimization of the cultivation process this resulted in >1 g/L MCFA production. Based on the MCFA production platform, we attempted to synthesize medium-chain fatty alcohols (MCFOHs, C6-C12) in yeast. Different protein engineering strategies were designed to engineer the carboxylic acid reductase from Mycobacterium marinum (MmCAR), a key enzyme involved in fatty acid conversion. We successfully changed the substrate specificity towards MCFAs and improved the enzyme catalytic activity via directed evolution, using both rational and semi-rational approaches. With further deleting the TPO1 transporter gene and combining different MmCAR mutations, a final production of 250 mg/L MCFOHs was achieved, a 3-fold increase compared with the control strain. 

In conclusion, we provided new insight into the establishment of yeast platforms for the production of FA derivatives through multidimensional engineering strategies.