Evolutionary engineering and mutagenesis as alternatives to improve Spathaspora passalidarum for second-generation ethanol production

The pretreatment step required for second-generation ethanol production releases inhibitory compounds that are harmful to yeasts, impacting the use of the hemicellulosic fraction. A proven-effective strategy to overcome this bottleneck of the process is to enhance yeast robustness and resistance to inhibitors. This study aimed to enhance the performance of the yeast Spathaspora passalidarum to the hemicellulosic hydrolysate (HH) obtained from the acid pretreatment of sugarcane bagasse using evolutionary engineering (EE) and mutagenesis. Initially, the tolerance to inhibitors and the HH were analyzed to determine the media composition for EE. The parental strain was subjected to three EE strategies and mutagenesis with ultraviolet (UV) light or ethyl methanesulfonate (EMS). During the inhibition tests, acetic acid had the most significant negative impact on yeast performance, with no growth observed at concentrations exceeding 1.5 g/L. From the adopted strategies, three strains with improved fermentative potential were obtained: JY5102 (from the EE strategy 3), JY5102UV (resulting from the UV light mutagenesis of JY5102), and JY5102EMS (resulting from the EMS mutagenesis of JY5102). The strains were used to ferment the HH, and the mutated strains, JY5102UV and JY5102EMS, stood out with Y_EtOH (from glucose and xylose consumption) of 0.39 ± 0.00 and 0.42 ± 0.01 g/g, respectively. This resulted in increased productivity by 1.6-fold and 1.5-fold, respectively, compared to the parental strain. The improvement in ethanol production may be related to adaptations and mutations associated with xylitol production in both strains, JY5102UV and JY5102EMS, resulting in a 30 % and 60 % decrease in Y_Xylitol, respectively.