Engineering xylose catabolism in the yeast Komagataella phaffii

Efficient xylose utilization is crucial for biomass hydrolysate valorization. However, Komagataella phaffii cannot efficiently utilize xylose. Here, we constructed xylose isomerase (XI)-xylulokinase (XK) pathway, nonoxidative pentose phosphate pathway (PPP), and nonoxidative glycolysis (NOG) pathway in K. phaffii to increase cell growth on xylose. Additionally, the bypass pathway of xylose metabolism, the high osmolarity glycerol/mitogen-activated protein kinase (HOG-MAPK) signaling pathway, and possible negative transcription factors were blocked to further promote xylose catabolism. Moreover, adaptive laboratory evolution (ALE) dramatically improved xylose utilization, and six potential targets were identified through multiomics and reverse engineering. The engineered strain exhibited the highest reported specific growth rate μ_max of up to 0.042 h^-1 and lag time of 25.0 h with a biomass yield of 0.366 g dry cell weight/g from sole xylose in minimal media. This strain also showed faster metabolite turnover, efficient free fatty acid (FFA) production and partial amelioration of glucose repression effect from xylose alone. The engineered metabolic plasticity described here will facilitate the regulation of xylose catabolism in other nonnative xylose-consuming yeasts.