CRISPR-Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in .

Background: The thermotolerant yeast shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C, C, and C sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis.

Results: Enabled by the design of hybrid RNA polymerase III promoters, this work adapts the CRISPR-Cas9 system from for use in . The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol--acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the ATF disrupted strain revealed that Atf activity contributes to ethyl acetate biosynthesis, but the knockout reduced ethyl acetate titers by only ~15%. Overexpression experiments revealed that Adh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the ADH2 disrupted strain showed that the knockout almost completely eliminated ethanol production and resulted in the accumulation of acetaldehyde.

Conclusions: Newly designed RNA polymerase III promoters for sgRNA expression in enable a CRISPR-Cas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically ADH1-7 and ATF. Adh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by Atf. Adh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate.