¹³C-metabolic flux analysis of recombinant yeasts for biofuels applications

Abstract

Fossil fuels have powered the transportation industry since the Industrial Revolution. However, rising transportation energy demand and new knowledge about the environmental impact of burning fossil fuels have motivated the development of technologies for sustainable production of renewable, carbon-neutral liquid fuels. To that end, biological systems may be leveraged to fix carbon dioxide and to catalyze the conversion of renewable feed stocks to fuel molecules. Today, the gasoline additive ethanol and biodiesel are produced by yeast fermentation of sugars derived from cornstarch and sucrose and transesterification of vegetable oils, respectively. However ethanol has many drawbacks as a fuel additive, and both biofuels are currently produced from edible feed stocks. For biofuels to contribute significantly to meeting total transportation energy demand, processes for production of fuel molecules from non-food feed stocks must be engineered. Two promising solutions are fermentation of sugars derived from "woody," lignocellulosic biomass and production of fuels from volatile fatty acids (VFAs) such as acetate, which can be produced by fermentation of organics in municipal solid waste and sewage or syngas. The production of biofuels from lignocellulosic material or VFAs will require metabolic engineering of biocatalysts to improve yields, productivities, and final titers. These metabolic engineering efforts can be facilitated by ¹³C-Metabolic Flux Analysis (MFA), a method for elucidating the otherwise unobservable intracellular metabolic fluxes in biological systems. We first developed protocols for extraction and LC-MS/MS analysis of intracellular metabolites, which provides data that may be used for metabolic flux estimation. We then performed an analysis of both the measurement and modeling errors associated with using these data for flux determination. Finally, we applied ¹³C-MFA to two industrially relevant systems: 1) Fermentation of xylose, a sugar present in lignocellulosic biomass, to ethanol in Saccharomyces cerevisiae, and 2) overproduction of fatty acids that may be transesterified to biodiesel from either glucose or acetate in the oleaginous yeast Yarrowia lipolytica. These experiments identified a potential bottleneck in xylose fermentation in S. cerevisiae and the primary source of NADPH for fatty acid biosynthesis in Y. lipolytica, and also suggested potential strategies for improving lipid yields in Y. lipolytica.