(228c) Partnering Anaerobic Fungi and Engineered Kluyveromyces Marxianus Enables Direct and Efficient Production of Fragrances and Fuels from Agricultural Residues

Recalcitrant plant biomass is an abundant and economical although underutilized source of trapped carbon to produce value-added chemicals. Current pretreatment methods to release this carbon are energy intensive, expensive, and frequently rely on non-green chemicals. Moreover, these processes can generate microbial inhibitors that reduce downstream production efficiency. To address this challenge, we propose the use of anaerobic fungi as a sustainable approach to release trapped carbon from plant biomass at ambient conditions. However, some carbon is ‘lost’ to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two-stage bioprocessing system pairing the anaerobic fungus Piromyces indianae with the yeast Kluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. P. indianae efficiently hydrolyzed untreated substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol. These products are then fed to K. marxianus to produce up to 2.4 g/L ethyl acetate. Knocking out an alcohol acyl transferase and upregulating aromatic amino acid biosynthesis redirects production to advanced biofuels and fragrances 2-phenylethanol and isoamyl alcohol. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media. More importantly, this system outperforms traditional 2-stage bioprocesses that commonly only rely on sugar handoff due to the additional capture of produced ethanol and organic acids by K. marxianus; our analyses suggest that the vast majority of available carbon in fungal hydrolysates is not in the form of simple sugars. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility.