Metabolic Engineering of Lachancea kluyveri with Enhanced Glucose and Xylose Co-utilization for Lignocellulosic Biofuels

Lignocellulose biorefinery provides a sustainable supply of fuels and value-added compounds. However, the major limitation of its application is the inefficient co-utilization of glucose and xylose by the model yeast. Here, we report for the first time on the metabolic engineering of non-model industrial yeast Lachancea kluyveri for co-utilization of glucose and xylose sustainably using lignocellulosic feedstock. Co-utilization in L. kluyveri was achieved by heterologous expression of XYLl, XYL2, and XYL3 and overexpression of TAL1 and TKL1, which eliminated the imbalanced cofactor specificity, reduced xylitol accumulation, and inadequately formed xylulose-5-phosphate. Further, this is the first report of the development of a robust genome engineering platform for diploid L. kluyveri that facilitated gene modification in both alleles of this yeast. Here, the genes were optimally expressed under P_TEF1, the strongest promoter identified among the other three native promoters P_TPI1, P_PGK1, and P_TDH3. Moreover, the engineered strain was used for metabolic flux analysis using our previously developed L. kluyveri genome-scale metabolic model (iPN730), which suggested the diversion of carbon flux toward competing pathways that can be targeted to achieve further strain improvement. During batch fermentation in a bioreactor, the recombinant host utilized 3.85% glucose and 2% xylose, producing an ethanol titer and yield of 23.65 g/L and 0.42 g/g sugar, respectively, with a maximum sugar consumption rate of 1.57 g/L/h. Further, fermentation using biomass hydrolysate in a bioreactor resulted in the complete consumption of xylose at a rate of 0.33 g/L/h without any xylitol accumulation. Altogether, this work represents the creation of an efficient glucose-xylose-co-utilizing diploid L. kluyveri strain that can be used in lignocellulosic biorefineries.