Xylose is the second most abundant sugar in cellulosic hydrolysate. Xylose utilization has been extensively studied in both microbes that naturally utilize it, for example Escherichia coli and Scheffersomyces stipitis, as well as in model organisms that have abundant genetic tools such as Saccharomyces cerevisiae. However, in non-conventional microorganisms that possess great industrial potential, only minimal research has been conducted to enable and improve their xylose utilization. In this

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... udy, we chose Yarrowia lipolytica as an example to explore threekey strategies to enable xylose utilization by a non-catabolizing host for the purposes of producing a variety of value-added chemicals. First, we integrated two heterologous genes that are essential for xylose utilization. Introduction of the pathway itself was not enough to enable xylose utilization, so a starvation-enabled adaptation was used to activate the pathway and kick-start xylose catabolism. Through whole genome sequencing, we found the copy numbers of both genes were multiplied during starvation. The resulting strain was able to produce 15g/L lipid from xylose in bioreactors. We then tested several targets within the pentose phosphate pathway that have been shown to improve xylose utilization in S. cerevisiae. Among these, only native xylulose kinase overexpression was shown to effectively improve xylose utilization in Y. lipolytica. Next, we utilized this strain to expand the product portfolio from xylose through the use of a strain mating approach. Specifically, we altered the native mating type within this strain and mated it with three separately engineered strains of Y. lipolytica that overproduce α-linolenic acid, riboflavin or triacetic acid lactone. The mated diploid strains were able to produce all three products from xylose in high quantities. Finally, we turn to the first step of xylose catabolism, transport into the cell, to remove a common bottleneck. To do so, we utilized a series of evolution and selection steps to engineer a xylose [...]