A C6/C5 Co-Fermenting Saccharomyces Cerevisiae Strain with the Alleviation of Antagonism Between Xylose Utilization and Robustness

During second-generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co-fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co-fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP-treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1-6 showed significantly enhanced tolerance, with a six-fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1-6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M-15. 6M-15 also exhibited increased co-fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g(-1) h(-1)) in high-level mixed sugars (80 g L-1 glucose and 40 g L-1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non-detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH similar to 3.5, 6M-15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g(-1). 6M-15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second-generation bioethanol or other high value-added products from lignocellulosic biomass.