(424d) Shikimate Pathway Refactoring in the Non-Conventional Yeast Kluyveromyces Marxianus Enables High Titer Production of 2-Phenylethanol

The non-conventional yeast Kluyveromyces marxianus is a promising microbial host for chemical biosynthesis because it is one of the fastest growing eukaryotes, is thermotolerant to temperatures upwards of 50°C, and has the capacity to assimilate a wide range of C5, C6 and C12 sugars. Previously, we have identified a strong phenotype of short and medium chain alcohol production, including the production of 2-phenylethanol (2-PE), an important compound in the fragrance industry with market values upwards of $1,000/kg. Aromatic compounds are almost exclusively biosynthesized from the Shikimate pathway. In this work, we focus on refactoring this pathway to extend the native capacity of K. marxianus to produce 2-PE. To do so, we first developed a one-step markerless gene integration system that can simultaneously integrate up to three unique expression cassettes in a single round of strain engineering. The refactoring experiment was enabled by the creation of a set of synthetic promoters derived from three K. marxianus genes TEF3, PGK, and TDH3 for high, medium and low expression, respectively. The refactored pathway library contains 27 strains with varied expression combinations of ARO4^K221L, ARO7^G141S and PHA2 integrated at the ABZ1 locus in the genome. Analysis of the library identified an engineered K. marxianus strain with a 4.5-fold increase in 2-PE production, reaching 684.24 ± 72.95 mg/L in shake flask experiment with 25 mL YPD medium containing 2% glucose. Integration of one copy of ARO10 driven by the synthetic TEF3 promoter into this high-producing strain further increased titers to about 2 g/L in fed-batch culture. The best strain for 2-PE biosynthesis from the refactored Shikimate pathway library can also be used as a yeast chassis for the biosynthesis of other phenylalanine-derived compounds, and the multigene integration tool advances the ability for rapid engineering of K. marxianus.