Find in Library

<i>Pleurotus eryngii</i> var. <i>ferulae</i>, a fungus of the genus <i>Pleurotus</i>, efficiently degrades lignin, especially during co-cultivation with other fungi. However, low transformation efficiency and heterologous gene expression restrict systematic studies of the molecular mechanisms and metabolic control of natural products in this mushroom. In this study, the homologous resistance marker carboxin (<i>cbx</i>) was used to establish a polyethylene glycol-mediated transformation (PMT) system in <i>P. eryngii</i> var. <i>ferulae</i>. Optimization of the transformation process greatly improved the number of positive transformants. In particular, we optimized: (i) protoplast preparation and regeneration; (ii) screening methods; and (iii) transformation-promoting factors. The optimized transformation efficiency reached 72.7 CFU/μg, which is higher than the average level of <i>Pleurotus</i> sp. (10–40 CFU/μg). Moreover, three endogenous promoters (P<i>_pfgpd1</i>, P<i>_pfgpd2</i>, and P<i>_pfsar1</i>) were screened and evaluated for different transcription initiation characteristics. A controllable overexpression system was established using these three promoters that satisfied various heterologous gene expression requirements, such as strong or weak, varied, or stable expression levels. This study lays the foundation for recombinant protein expression in <i>P. eryngii</i> var. <i>ferulae</i> and provides a method to investigate the underlying molecular mechanisms and secondary metabolic pathway modifications.