CRISPR\u2013Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

Ann-Kathrin Löbs,Andrew Flores,Ian Wheeldon,Ronja Engel,Cory Schwartz

doi:10.1186/s13068-017-0854-5

Ann-Kathrin Löbs, Andrew Flores + Show 3 more

Open Access

https://doi.org/10.1186/s13068-017-0854-5

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Abstract

BackgroundThe 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 C5, C6, and C12 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.ResultsEnabled 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.ConclusionsNewly designed RNA polymerase III promoters for sgRNA expression in K. marxianus enable a CRISPR–Cas9 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.

Highlights

The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the bio‐ chemical production of fuels and chemicals
Double knockout of ATF1 and ATF2 in S. cerevisiae eliminates the synthesis of the medium-chain ester isoamyl acetate and reduces ethyl acetate production by 50% [11]
Given the fast growth kinetics, thermotolerance to 45 °C, and the high capacity to synthesize ethyl acetate, we selected CBS 6556 as a model K. marxianus strain to further understand the roles of KmADHs and KmATF in ethyl acetate and ethanol biosynthesis

Summary

Introduction

The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the bio‐ chemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C5, C6, and C12 sugars into the volatile short-chain ester ethyl acetate. Double knockout of ATF1 and ATF2 in S. cerevisiae eliminates the synthesis of the medium-chain ester isoamyl acetate and reduces ethyl acetate production by 50% [11]. These enzymes localize to the endoplasmic reticulum and lipid droplets; this has been shown to be critical for high activity [12, 13]. The metabolism of K. marxianus is less well understood than that of S. cerevisiae, and promiscuous Adh activity toward hemiacetal oxidation and the roles of different Adh enzymes in ester and alcohol metabolism are not yet completely understood

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