(256d) Metabolic Engineering of the High Native Capacity of Kluyveromyces Marxianus to Synthesize Ethyl Acetate

The yeast Kluyveromyces marxianus is a promising candidate for chemicals biosynthesis. Its natural capacity to produce short and medium chain volatile esters and ethanol at high rates, along with rapid growth kinetics at temperatures upwards of 45 °C make it especially interesting as platform for biotechnological chemicals production. The lack of synthetic biology tools along with limited knowledge about the metabolism, especially ester biosynthesis pathways, of K. marxianus, have thus far hampered the development of K. marxianus as a platform for ester production. We have developed an efficient CRISRP-Cas9 genome-editing tool that allows for efficient genomic disruptions and integrations in K. marxianus. This system was applied to elucidate the function of alcohol acetyl/acyltransferase and alcohol dehydrogenaseses (Adh) in ethyl acetate production. In contrast to prior claims, the alcohol acetyltransferase Atf has found to only marginally contribute to bulk ethyl acetate formation. Our results show that the newly found Eat1 (ethanol acetyltransferase) is responsible for the formation of bulk ethyl acetate in K. marxianus. Furthermore, Adh2 activity is essential for ethanol and subsequent ethyl acetate production. In contrast to Atf1 in S. cerevisiae, Eat1 is localized to the mitochondria, allowing for high ethyl acetate production through utilization of the mitochondrial acetyl-CoA pool that is fostered by rapid growth kinetics. To further increase ethyl acetate production in K. marxianus we developed a CRISPR interference (CRISPRi) system to knock down electron transport chain and/or TCA cycle enzyme expression to free available Acetyl-CoA thus shuttling flux from respiration towards ethyl acetate production.