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<p><i>Phanerochaete chrysosporium</i>, a model white-rot fungus, plays a central role in lignin degradation but has remained recalcitrant to genetic manipulation due to inherently low transformation efficiencies. In this study, we present a novel whisker-assisted transformation method employing potassium titanate whiskers combined with freeze–thaw pretreatment, L-ornithine supplementation, and sonication to enable efficient DNA delivery into <i>P. chrysosporium</i>. Using the Cas9-sGFP construct as a reporter system, transformants were selected on hygromycin and validated through PCR and fluorescence microscopy. Our protocol consistently generated hygromycin-resistant transformants, achieving a reproducible transformation efficiency of ~ 0.26%, outperforming Agrobacterium-mediated transformation and comparable to shockwave-based approaches. The successful integration and functional expression of the sGFP reporter confirmed the stability and reliability of this approach. This study provides the first evidence of whisker-mediated delivery and stable integration of exogenous DNA into the genomic DNA of a white-rot fungus. By providing a simple, cost-effective, and reproducible transformation strategy, this work addresses a longstanding bottleneck in fungal biotechnology. The method provides a scalable alternative for CRISPR-based genome engineering in ligninolytic fungi and unlocks opportunities for metabolic engineering of <i>P. chrysosporium</i>, including targeted gene disruption, overexpression of ligninolytic enzymes, and expression of heterologous biosynthetic pathways for the sustainable production of industrially valuable compounds.</p>

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A potassium titanate whisker-assisted method for genetic transformation of Phanerochaete chrysosporium using a Cas9-sGFP expression vector

  • 期刊论文

  • 发表日期：

    2026年3月31日

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  • Falguni Aggarwal,
  • Simran Jatwani,
  • Rishabh Rajkumar Jadhav,
  • Rachana Yadav,
  • Saurabh Jyoti Sarma,
  • Deepa Khare

摘要

Phanerochaete chrysosporium, a model white-rot fungus, plays a central role in lignin degradation but has remained recalcitrant to genetic manipulation due to inherently low transformation efficiencies. In this study, we present a novel whisker-assisted transformation method employing potassium titanate whiskers combined with freeze–thaw pretreatment, L-ornithine supplementation, and sonication to enable efficient DNA delivery into P. chrysosporium. Using the Cas9-sGFP construct as a reporter system, transformants were selected on hygromycin and validated through PCR and fluorescence microscopy. Our protocol consistently generated hygromycin-resistant transformants, achieving a reproducible transformation efficiency of ~ 0.26%, outperforming Agrobacterium-mediated transformation and comparable to shockwave-based approaches. The successful integration and functional expression of the sGFP reporter confirmed the stability and reliability of this approach. This study provides the first evidence of whisker-mediated delivery and stable integration of exogenous DNA into the genomic DNA of a white-rot fungus. By providing a simple, cost-effective, and reproducible transformation strategy, this work addresses a longstanding bottleneck in fungal biotechnology. The method provides a scalable alternative for CRISPR-based genome engineering in ligninolytic fungi and unlocks opportunities for metabolic engineering of P. chrysosporium, including targeted gene disruption, overexpression of ligninolytic enzymes, and expression of heterologous biosynthetic pathways for the sustainable production of industrially valuable compounds.