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

metabolic engineering

fatty acid derivatives

protein engineering

Saccharomyces cerevisiae

tolerance

transporter

KA-salen, Kemihuset
Opponent: Brian Pfleger, University of Wisconsin-Madison, USA

Författare

Yating Hu

Chalmers, Biologi och bioteknik, Systembiologi

Heterologous transporter expression for improved fatty alcohol secretion in yeast

Metabolic Engineering,; Vol. 45(2018)p. 51-58

Artikel i vetenskaplig tidskrift

Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast.

ACS Synthetic Biology,; Vol. 2(2018)p. 584-590

Artikel i vetenskaplig tidskrift

Zhiwei Zhu*, Yating Hu*, Paulo Teixeira, Rui Pereira, Yun Chen, Verena Siewers and Jens Nielsen, Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids

Yating Hu*, Zhiwei Zhu*, Margit Winkler, Verena Siewers and Jens Nielsen, Engineering a carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols

The growing demand of sustainable development for modern society alongside concerns about climate change caused by greenhouse gas emissions from fossil fuels have become the driven force for the development of microbial production of fuels and oleochemicals. And the advances in biotechnology contribute to the metabolic engineering of microorganisms to meet the requirement of industrial-scale production. In this respect, the budding yeast, Saccharomyces cerevisiae, has been extensively studied as the popular chassis due to the ease of genetic modification and the robustness and tolerance towards harsh conditions. Among the numerous metabolic pathways found in nature, fatty acid synthesis pathway is an attractive route with the potential possibility to expend its product portfolio to liquid transportation fuels and high value oleochemicals.

In this thesis, the biosynthesis of fatty acid-derivatives, including fatty alcohols, alkanes and medium-chain fatty acids, was investigated in S. cerevisiae. With the purpose of meeting commercial requirement, traditional pathway engineering was conducted to enhance the precursor supply for these chemicals. Besides, the key enzyme engineering, transporter engineering, together with optimizing the cultivation conditions were all complemented to allow for the high-level production of FA derivatives in yeast. In conclusion, this study provided novel insight into establishment of a versatile yeast platform for FA derivatives production through multidimensional engineering approaches, which will further inspire the production of other chemicals in S. cerevisiae.

Model-Based Construction And Optimisation Of Versatile Chassis Yeast Strains For Production Of Valuable Lipid And Aromatic Compounds (CHASSY)

Europeiska kommissionen (Horisont 2020), 2016-12-01 -- 2020-11-30.

Bioteknisk produktion av värdeskapande ingredienser

Stiftelsen för Strategisk forskning (SSF), 2015-01-01 -- 2020-12-31.

Stiftelsen för Strategisk forskning (SSF), 2015-01-01 -- 2020-12-31.

Ämneskategorier

Annan maskinteknik

Biokemi och molekylärbiologi

Biokatalys och enzymteknik

Drivkrafter

Hållbar utveckling

Infrastruktur

Chalmers infrastruktur för masspektrometri

Styrkeområden

Livsvetenskaper och teknik (2010-2018)

ISBN

978-91-7905-174-7

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4641

Utgivare

Chalmers tekniska högskola

KA-salen, Kemihuset

Opponent: Brian Pfleger, University of Wisconsin-Madison, USA

Mer information

Senast uppdaterat

2019-09-12