UC Riverside

The threat of climate change and a recent sway in popular opinion towards sustainable energy and chemicals have fueled the field of biotechnology for renewable chemicals production. To achieve process feasibility and compete with fossil-based processes, microbial production units are required to synthesize products at high titers and productivities while utilizing cheap and sustainable substrates. To fulfill these requirements careful selection of the host organism is essential. The emergence of efficient genome editing tools has enabled engineering of, thus far, intractable organisms, and allows for the selection of a host organism based on a desired phenotype that is beneficial for the process. The yeast Kluyveromyces marxianus was chosen because of its natural capacity to produce high amounts of ethyl acetate. Other characteristics such as fast growth kinetics, thermotolerance and the ability to metabolize various carbon sources make this host especially interesting for industrial applications. The development of an efficient CRISPR-Cas9 system in K. marxianus allowed us to interrogate the role of alcohol acetyltransferases and alcohol dehydrogenases in volatile metabolite production. We identified Eat1 as the critical enzyme for acetate ester production, and found that mitochondrial localization of Eat1 is essential for high ester production yields. Overexpression of Eat1 significantly increased ester production indicating that this step is the bottleneck of the reaction. To furthermore increase ester production TCA cycle flux was slowed down through CRISPR interference-mediated knockdown of the TCA cycle and electron transport chain.