Mutagenesis combined with 2-deoxyglucose is not a suitable tool to select strains of Papiliotrema laurentii less sensitive to glucose catabolite repression

Abstract

Assimilation of major sugars from lignocellulosic biomasses is pivotal for achieving a feasible oil production by oleaginous yeasts in biorefineries. Papiliotrema laurentii UFV-1 is an oleaginous yeast capable of converting lignocellulosic sugars such as glucose and xylose into lipids; however, glucose is assimilated before xylose, impairing high volumetric lipid productivity. To circumvent this drawback, we hypothesized that random mutagenesis combined with 2-deoxyglucose (2DG) selection would be a suitable strategy for selecting strains of P. laurentii UFV-1 less sensitive to glucose repression. First, we determined the growth kinetics parameters of the wild strain in minimum medium with glucose and/or xylose. Then, the yeast was subjected to mutagenesis by ultraviolet irradiation, and mutants were selected in a culture medium containing 2DG. Among the 24 selected mutants, the M17 strain stood out due to its capacity to achieve a higher cell density at the 2DG inhibitory concentration. Surprisingly, both M17 and wildtype strains presented the same xylose and glucose consumption profile. Although M17 grew faster in xylose and preserved the oleaginous phenotype, it could not co-assimilate glucose and xylose. Interestingly, the tolerant strain grew assimilating 2DG and xylose simultaneously, likely incorporating 2DG into its biomass. Otherwise, the wild strain presented arrested growth and only grew after exporting 2DG back to the media. Since carbon catabolite repression and 2DG response mechanisms are poorly studied and remains elusive in Basidiomycota yeasts, we provided cues to guide future studies that will allow a better understanding of the mechanisms involved with 2DG resistance in these yeasts.