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Abstract

The biosynthetic production of branched-chain higher alcohols (BCHAs) such as isopentanol (IpOH) in yeast presents a sustainable solution to produce carbon-neutral transportation fuels. Previous attempts to optimize the production of IpOH in yeast have focused on manipulating the expression of several genes directly involved in BCHA synthesis, but other endogenous proteins that may indirectly regulate flux through these pathways remain to be identified. The activity of such proteins may affect IpOH synthesis by directing cofactor production and turnover, mitochondrial transport of metabolites, ribosomal activity, or by directly competing for metabolic intermediates. In this study, we use libraries of camelid nanobodies and a GFP-based biosensor to perform a screen in S. cerevisiae for proteins whose activities affect IpOH production. We find that previous work on this project likely failed due to a lack of diversity in the nanobody plasmid library used. We perform a new screen using this IpOH biosensor and two different, more diverse nanobody libraries to identify sorted yeast collections that exhibit levels of fluorescence shifted above and below that of a baseline biosensor control strain. This study represents a substantial step toward demonstrating the viability of using camelid nanobodies in conjunction with an IpOH biosensor for a high-throughput screen to examine the metabolism of microbes used in synthetic biology. Further success may reveal new information about metabolic regulation in yeast and can potentially lead to the development of a strain that produces IpOH at increased titers.