Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast

Highlights

• Medium chain fatty acids were obtained by engineering fungal fatty acid synthases.

• Medium chain alkanes were produced by expressing an alkane biosynthesis pathway.

• Among 13 candidates, the ADO from Thermosynechococcus elongatus showed superior activity.

• Targeting of the alkane-forming pathway into peroxisomes improved alkane production.

• Medium chain 1-alkenes were synthesized by expressing UndA decarboxylase.

Abstract

Microbial synthesis of medium chain aliphatic hydrocarbons, attractive drop-in molecules to gasoline and jet fuels, is a promising way to reduce our reliance on petroleum-based fuels. In this study, we enabled the synthesis of straight chain hydrocarbons (C7–C13) by yeast Saccharomyces cerevisiae through engineering fatty acid synthases to control the chain length of fatty acids and introducing heterologous pathways for alkane or 1-alkene synthesis. We carried out enzyme engineering/screening of the fatty aldehyde deformylating oxygenase (ADO), and compartmentalization of the alkane biosynthesis pathway into peroxisomes to improve alkane production. The two-step synthesis of alkanes was found to be inefficient due to the formation of alcohols derived from aldehyde intermediates. Alternatively, the drain of aldehyde intermediates could be circumvented by introducing a one-step decarboxylation of fatty acids to 1-alkenes, which could be synthesized at a level of 3 mg/L, 25-fold higher than that of alkanes produced via aldehydes.

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.