Engineering a Global Regulatory System Potentiates Rapid Growth of Saccharomyces Cerevisiae on Numerous Non-Native Substrates

There have been decades of effort to engineer the industrial cellular factory Saccharomyces cerevisiae to utilize cheap and renewable sources of carbon and energy such as lignocellulosic biomass, glycerol, and various C1 compounds. However, the implementation of heterologous catabolic pathways to enable the assimilation of these non-native substrates has generally relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. Our lab recently demonstrated that activating the galactose (GAL) regulon (a REG approach), the regulatory structure responsible for coordinating growth on the native substrate galactose, during growth on the non-native sugar xylose results in higher growth rates and more efficient substrate utilization compared with traditional constitutive expression (a CONS approach). The present study seeks to investigate whether the large-scale gene expression changes observed upon activation of the GAL regulon are broadly beneficial to growth on non-native substrates. We start by constructing and characterizing a self-activating variant of the sensor-protein (GAL3-SA) and find that expression of this mutant is sufficient to fully activate the GAL regulon without an inducing substrate. We find that despite moving from substrate-specific activation (in the native GAL regulon) to non-specific activation, cells carrying GAL3-SA do not seem to incur serious fitness costs or sacrifice the previously observed benefits during growth. Finally, we compare CONS and REG approaches to growth on the renewable and structurally distinct substrates arabinose, cellobiose, and glycerol and observe that while a REG approach is superior in all cases, the magnitude of the benefit is both substrate- and media-specific. Ongoing work seeks to identify gene expression patterns that underlie the observed phenotypic benefits across substrates. These results validate GAL regulon activation as a general approach to engineering more efficient catabolism of non-native substrates and provide a tool (GAL3-SA) for applying it to other substrates.