Evolutionary Adaptation to Enhance Xylose Metabolism in Thermotolerant <i>Kluyveromyces Marxianus</i>

An efficient conversion of xylose along with glucose is needed for the sustainable production of lignocellulosic biofuels. However, the assimilation of xylose is still a major loophole for economical 2G ethanol production. Evolutionary adaptation is known to provide stability to the strains in a challenging environment. As an extension of an earlier study, the strains Kluyveromyces marxianus NIRE-K1.1 and K. marxianus NIRE-K3.1 (KmNIRE-K3.1) were subjected to secondary adaptation on optimized minimal media (MM) with the aim to enhance xylose utilization for ethanol production. Both the strains were adapted till a saturated improvement in xylose uptake, that is, 54 generations on MM medium containing xylose. Xylose utilization increased from 14.21% to 45.80% and 10.55% to 45.31%, in evolved strains KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Specific xylose reductase activity also increased 2.04- and 3.36-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Xylitol dehydrogenase activity was also increased by 2.82- and 1.35-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Further, a decrease in redox imbalance was also observed in evolved strains, and hence, reduced in xylitol production was observed during growth and fermentation. Xylose uptake rate increased by 2.53- and 1.5-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively with 2.20- and 6.46-folds higher ethanol concentrations, with 2.25- and 5.86-folds higher volumetric productivity, respectively. This study has demonstrated the role of evolutionary adaptation in developing robust yeast strains. KmNIRE-K1.2 and KmNIRE-K3.2 have shown enhanced ethanol production, enzyme activities, and less by-product formation such as xylitol during xylose metabolism.