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Significance The yeastSaccharomyces cerevisiae has been utilized extensively as a cell factory to produce numerous products. The de novo synthesis of medium-chain fatty alcohols (MCFOHs) through conversion of free fatty acids by carboxylic acid reductase (CAR) has been studied in yeast. However, the broad substrate specificity of CAR is one of the biggest challenges to improve MCFOH production. Here, different protein engineering strategies were employed to modify the CAR enzyme toward a more specific catalytic activity on shorter chain fatty acids. For this, we created a high-throughput screening assay relying on the detoxification of the toxic substrate to select for efficient enzyme variants. This study represents an important step forward in the selective synthesis of MCFOHs in yeast.

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast