The potential of biotechnology to replace fossil fuel-based processes for the production of value-added molecules will depend largely on its economic competitiveness. To this end, there have been decades of work to engineer the ability of the versatile industrial cellular factory Saccharomyces cerevisiae to grow on 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 so far generally relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. Previous work by our lab has demonstrated that activating the galactose (GAL) regulon (a REG approach), the regulatory metabolic 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 build on this foundation by exploring 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 requiring 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 appears to be both substrate- and media-specific. Ongoing work seeks to identify the genes and gene expression patterns that underlie the observed phenotypic benefits across substrates. Taken together, these results both 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 attractive substrates beyond those described here.

Engineering a global regulatory system potentiates rapid growth of Saccharomyces cerevisiae on numerous non-native substrates

ACS Fall 2021

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