Background:The thermotolerant yeast Kluyveromyces marxianus 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 5 , C 6 , and C 12 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 Streptococcus pyogenes for use in K. marxianus. The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the KmATF 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 KmAdh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the KmADH2 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 K. marxianus enable a CRISPRCas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically KmADH1-7 and KmATF. KmAdh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate.

Usability is a core construct of website evaluation and inherently defined as interactive. Yet, when analysing first impressions of websites, expected usability, i.e., before use, is of interest. Here we investigate to what extend ratings of expected usability are related to (a) experienced usability, i.e., ratings after use, and (b) objective usability measures, i.e., task performance. Furthermore, we try to elucidate how ratings of expected usability are correlated to aesthetic judgments. In an experiment, 57 participants submitted expected usability ratings after the presentation of website screenshots in three viewing-time conditions (50, 500, and 10,000 ms) and after an interactive task (experienced usability). Additionally, objective usability measures (task completion and duration) and subjective aesthetics evaluations were recorded for each website. The results at both the group and individual level show that expected usability ratings are not significantly related either to experienced usability or objective usability measures. Instead, they are highly correlated with aesthetics ratings. Taken together, our results highlight the need for interaction in empirical website usability testing, even when exploring very early usability impressions. In our study, user ratings of expected usability were no valid proxy neither for objective usability nor for experienced website usability.

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