Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

Abstract

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleTJE

“…1). More importantly, by taking advantage of heterologous P450 redox partners, the engineered E. coli 8 and Saccharomyces cerevisiae 25 cells with OleT JE expression were able to produce 97.6 mg/L and 3.7 mg/L total α-alkenes, respectively.…”

Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway

“…Given the high hydrophobicity of hydrocarbons, a role in regulating membrane properties seems more likely. From a biotechnological perspective, the discovery of the microalgal alkane synthase(s) would also expand the repertoire of enzymes available to harness hydrocarbon synthesis in microbes (André et al, 2013;Choi and Lee, 2013;Liu et al, 2014;Chen et al, 2015). The presence of hydrocarbons of various chain length in C. variabilis cells shows that microalgal hydrocarbon-forming enzymes may in fact have a broad substrate specificity.…”

Biobased production of alkanes and alkenes through metabolic engineering of microorganisms

“…Therefore, researchers are trying to find better enzyme candidates from various resources by applying data derived from bioinformatics studies and sequence alignment analysis. In a previous study of OleT, several homologous genes of oleT JE presented different productivity and distribution of chain-lengths, and a codon-optimized version of the best enzyme enabled increased production of alkenes [10]. Structure-based engineering of enzymes can also change the active site and hereby improve the enzyme activity.…”

“…In the case of OleT co-expressed with CamAB and formate dehydrogenase (FDH), the optimal temperature for enzyme activity was different depending on the FA chain-length (highest activity at 4°C for C4–C9 and C18, and room temperature for C10–C16) [22]. As titer is an accumulated metric it is closely correlated with cell growth and the alkane/alkene titer was therefore found to increase with the use of rich media for engineered strains of E. coli expressing Ado and S. cerevisiae expressing oleT [10, 57]. …”

“…PCC6803Alkanes (C15, C17), Alkene (C17)26 mg/L[63]FAR-AD E. coli Alkanes (C13, iso-C13, C15, iso-C15, C16, C17), Alkenes (C13, C15, C16, C17)5 mg/L[28]CAR-AD E. coli Alkanes (C11, C13), Alkenes (C15, C17)2 mg/L[2]DOX-AD S. cerevisiae Alkanes (C14, C16)73. 5 μg/L[24]OleT E. coli Alkenes (C11, C13, C15, C15:2, C17:2)97.6 mg/L[41] S. cerevisiae Alkenes (C11–C19)3.7 mg/L[10]UndA E. coli Alkenes (C9–C13)6 mg/L[53]UndB E. coli Alkenes (C5–C17)55 mg/L[52]OleABCD E. coli Alkenes (27:3, 27:2, 29:2, 29:3)40 μg/L[5]Ols Synechoccus sp. PCC7002Alkenes (C19, C19:2)4.2 mg/L/OD 730 [43] S. globisporus Alkene (15:7)129.3 mg/L[14] E. coli Alkene (C15)140 mg/L[40] …”