Biosensor-Mediated Screening of Nanobody Libraries for Enhanced Biofuel Production in Saccharomyces cerevisiae

Abstract

Biofuels are an attractive, sustainable alternative to traditional fossil-derived liquid fuels used to power the transportation sector. Branched-chain higher alcohols (BCHAs) offer higher energy densities and an easier path to transition renewable fuels than the more commonly used ethanol. The BCHA isopentanol is a particularly attractive gasoline substitute. In this work, we harness the yeast Saccharomyces cerevisiae, equipped with a biosensor whose green fluorescence is linearly correlated to isopentanol production. This system is used to screen nanobody libraries in a high-throughput manner to efficiently identify nanobodies that have significant impacts on isopentanol titers in yeast. Using fluorescence-activated cell sorting (FACS), we successfully sorted yeast strains that had higher or lower GFP fluorescence compared to the positive control. We isolated the nanobodies from these strains for further analysis, finding evidence of sequence redundancy in the plasmids that encode the nanobodies. This is a novel approach to identify proteins tangentially involved in isopentanol production in yeast. Furthermore, proteins being bound by nanobodies will be the targets of future engineering efforts.