UV mutagenesis and tolerance adaptive laboratory evolution of Pichia fermentans modulate the membrane- bound xylose uptake transporters (XUT) genes for enhanced xylitol production

Development of a strain improvement strategy is inevitable for the industrial production of commercial chemicals. In this study, a promising yeast, Pichia fermentans NCIM 3638 was selected for metabolic modulation aimed at xylitol (a low-calorie sweetener) production. The strain was subjected to UV mutagenesis followed by sequential LiCl-induced oxidative stress to modulate xylose metabolism for enhanced xylitol production. The resultant mutant strain, P. fermentans KS-MUT9, achieved a maximum xylitol yield of 0.61 g/g xylose, representing a 1.61-fold increase compared to the wild type. Analysis of key enzymes involved in xylose metabolism revealed a 7.47-fold increase in xylose reductase activity (1.27 IU/mg) and a 0.22-fold decrease in xylitol dehydrogenase activity (0.11 IU/mg) in the mutant strain relative to the wild-type, correlating with the enhanced xylitol yield. Molecular investigations using qPCR demonstrated upregulation of the xylose reductase gene (XYL1, 3.89-fold), xylitol dehydrogenase gene (XYL2, 1.91-fold), and a substantial 14.93-fold increase in the xylose uptake transporter gene-4 (XUT4), supporting metabolic rewiring through the adopted strain improvement strategy. Additionally, Sanger sequencing identified six and four nucleotide substitutions in XUT6 and XUT7 of KS-MUT9, respectively. Furthermore, to assess industrial scalability, a mathematical evaluation of the fermentative potential of the mutant strain was conducted to determine critical scale-up kinetic parameters (Xc, Sc, Pc) using unstructured kinetic modeling. The mutant strain developed through UV mutagenesis and LiCl-assisted tolerance adaptive laboratory evolution exhibited a reprogrammed metabolic profile favoring enhanced xylitol production, highlighting its potential for industrial bioproduction without ethical or regulatory concerns.