The non-conventional yeast Kluyveromyces marxianus is one of the fastest growing eukaryotes, is thermotolerant to temperatures upward of 50°C, and has the capacity to assimilate a wide range of C5 and C6 sugars. These traits make K. marxianus an attractive host for the industrial production of biochemicals. In previous strain screening experiments, we have also identified a strong phenotype of short and medium chain ester production, including the production of ethyl acetate in grams per liter quantities, and the synthesis of isoamyl and phenylethyl acetate in 10s of miligrams per liter quantities. These acetate esters are produced via an alcohol acetyltransferase (Atf), which condenses acetyl-CoA with the relevant primary alcohol. Knockout of this function leads to the accumulation of the alcohol precursors and represents a valuable starting for their biochemical production. In this work, we focus on refactoring the Shikimate pathway for 2-phenylethanol production, an important precursor in the fragrance industry with market values upwards of $1,000/kg. To do so, we first developed a one-step markerless multigene integration system that can effectively integrate three unique expression cassettes in a single round of strain engineering. We used this new technique to rapidly create a 27-member strain library that varied the expression of Shikimate pathway genes ARO4, ARO7, and PHA2. This refactoring experiment identified an engineered strain with a five-fold increase in 2-phenylethanol production and demonstrated new capabilities in the rapid engineering of K. marxianus.

Shikimate pathway refactoring in the fast-growing yeast Kluyveromyces marxianus drives high titer production of 2-phenylethanol

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.