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<p>Development of a strain improvement strategy is inevitable for the industrial production of commercial chemicals. In this study, a promising yeast, <i>Pichia fermentans</i> 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 evolved mutant strain, <i>P. fermentans</i> KS-MUT9, achieved a maximum xylitol yield of 0.61&#xa0;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&#xa0;IU/mg) and a 0.22-fold decrease in xylitol dehydrogenase activity (0.11&#xa0;IU/mg) in the mutant strain relative to the wild-type, consistent with the enhanced xylitol production. Molecular investigations using qPCR demonstrated upregulation of the xylose reductase gene (<i>XYL1</i>, 3.89-fold), xylitol dehydrogenase gene (<i>XYL2</i>, 1.91-fold), and a substantial 14.93-fold increase in the xylose uptake transporter gene-4 (<i>XUT4</i>), supporting metabolic rewiring through the adapted strain improvement strategy. Additionally, Sanger sequencing identified six and four nucleotide substitutions in <i>XUT6</i> and <i>XUT7</i> 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-biomass, Sc-substrate, Pc-product) 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.</p> Graphical abstract <p></p>

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Ultraviolet mutagenesis and tolerance adaptive laboratory evolution of Pichia fermentans modulate the membrane-bound xylose uptake transporter genes for enhanced xylitol production

  • 期刊论文

  • 发表日期：

    2026年5月22日

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  • Ramalingam Kayalvizhi,
  • Samuel Jacob

摘要

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 evolved 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, consistent with the enhanced xylitol production. 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 adapted 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-biomass, Sc-substrate, Pc-product) 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.

Graphical abstract