(157a) An Autoinducible Fungus to Increase Production of Biofuels and Bioproducts

While bacteria have many autoinducible systems, eukaryotes, including the industrial host Saccharomyces cerevisiae, have few comparable systems. The most common systems use costly antibiotics (e.g. tetracycline) or special growth medias that require time-consuming preculture (e.g. galactose and sucrose). Consequentially, inducible systems are rarely utilized in commercial applications, despite the obvious benefits of decoupling the growth mode from the production mode. Decoupling is particularly valuable to the production of compounds that are toxic to the production organism, or require allocation of resources such as co-factors and ATP. Although many valuable biochemicals have toxic biosynthetic intermediates in S. cerevisiae, such as in the mevalonate pathway, a lack of autoinducible systems result in fermentations that rely on constitutive promoters. The goal of this work was to rewire the yeast mating signaling pathway for inducing expression of a gene (or genes) of interest. Our autoinduction system is based the engineering of three distinct modules that will set the threshold for autoinduction, prevent premature activation, and activate metabolite production. The “timing knob” module determines the cell density where activation of the target genes begins. We have developed a range of autoinducible strains integrating the MFɑ1 and STE2 genes under different promoters. Using fluorescent reporters, we observe a clear shift in the production of the GOI with increasing levels of alpha factor (ie, the inducer) at similar levels to those observed with the strongest constitutive promoters. We have cloned a variety of endogenous promoters to drive expression of alpha factor, and optimization of our expression system has the potential to improve yield, titer, process efficiency and process economics for companies producing a variety of materials, chemicals and fuels in yeast cell factories.