Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast
Graphical abstract
Introduction
Medium chain aliphatic hydrocarbons (C7–C13) are the predominant components in petroleum-based gasoline or jet fuels (Edwards, 2003, Sheppard et al., 2016), and also serve as solvents and chemicals (Choi and Lee, 2013, Kourist, 2015). A variety of enzymes committed to the formation of hydrocarbons via the deoxygenation of fatty acids (Kourist, 2015, Rude et al., 2011, Rui et al., 2015, Rui et al., 2014) or fatty aldehydes (Aarts et al., 1995, Marsh and Waugh, 2013, Qiu et al., 2012, Schirmer et al., 2010) have been discovered in nature (Fig. 1). In addition, the production of straight chain alkanes or 1-alkenes via biocatalysis (Amaya et al., 2016, Dennig et al., 2015, Foo et al., 2017, Liu et al., 2014, Yan et al., 2015, Zachos et al., 2015, Zhang et al., 2013) or heterologous biosynthesis (Akhtar et al., 2013, Bernard et al., 2012, Buijs et al., 2015, Cao et al., 2016, Chen et al., 2015, Choi and Lee, 2013, Coursolle et al., 2015, Foo et al., 2017, Harger et al., 2013, Howard et al., 2013, Kallio et al., 2014, Liu et al., 2014, Schirmer et al., 2010, Sheppard et al., 2016, Song et al., 2016, Xu et al., 2016, Yan et al., 2016, Zhou et al., 2016a, Zhou et al., 2016b) has been implemented. Yeast, an important industrial workhorse (Becker and Wittmann, 2015, Nielsen, 2015), is considered to be very suitable for the production of these fuel molecules (Hong and Nielsen, 2012). However, in vivo synthesis of medium chain aliphatic hydrocarbons in yeast is not realized yet. This is mostly because of the scarcity of medium chain fatty acid (MCFA) precursors in native yeast cells. Recently, we have modified fungal type I fatty acid synthases (FASs) by inserting a heterologous thioesterase to release MCFAs and introducing mutations into the ketoacyl synthase domain (G1250S and M1251W in Fas2) to restrict the elongation of fatty acids (Gajewski et al., 2017a, Zhu et al., 2017). These modifications in FASs have allowed yeast to produce MCFAs. Here we further enabled the in vivo synthesis of medium chain alkanes and 1-alkenes in yeast by exploring the activities of hydrocarbon-forming enzymes towards medium chain substrates, and blocking the formation of by-products.
Section snippets
Plasmids, strains and culture conditions
Strains and plasmids used in this study were listed in Table S1 and Table S2, respectively. E. coli DH5α was used for plasmid amplification. If not specified, E. coli cells were cultivated in Luria-Bertani (LB) medium at 37 °C and 200 rpm. 80 mg/L of ampicillin was supplemented for plasmid selection. Transformation of E. coli was according to a previously described protocol (Inoue et al., 1990). All S. cerevisiae strains used in this study were derived from CEN.PK113-11C (MATa SUC2 MAL2-8c his3Δ1
Medium chain fatty acid production
Although MCFAs are valuable precursors for the synthesis of oleochemicals and biofuels, very few of them are generated by native FASs. Recently, we and others have engineered fungal type I FASs for MCFA production (Gajewski et al., 2017b, Xu et al., 2016, Zhu et al., 2017). By embedding a heterologous short chain acyl-ACP/CoA thioesterase into the reaction compartments of FAS, as well as introducing mutations into the ketoacyl synthase domain, the engineered ScFAS28 produced substantial MCFAs
Conclusion
In this study, we engineered yeast to enable the biosynthesis of gasoline or jet fuels range hydrocarbons by introducing the modified FASs from S. cerevisiae to supply medium chain fatty acid precursors, and expression of downstream alkane or 1-alkene forming pathways to produce medium chain hydrocarbons. For the two-step synthesis of alkanes, the poor catalytic activities of ADOs and the drain of aldehyde intermediates into alcohols posed a serious obstacle to further improvement of alkane
Competing financial interests
The authors declare no competing financial interest.
Acknowledgment
This work was financially supported by the Novo Nordisk Foundation, the Knut and Alice Wallenberg Foundation, Vetenskapsrådet and Total New Energy. We thank the Chalmers Mass Spectrometry Infrastructure for assistance with GC/MS analysis
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- 1
Present address: Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian, China.
- 2
Present address: Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, United States.
- 3
Present address: Biopetrolia AB, Systems and Synthetic Biology, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.
- 4
Present address: Evolva Biotech, Lersø Parkallé 40–42, DK-2100 Copenhagen, Denmark.