Establishment of an Efficient Polyethylene Glycol (PEG)-Mediated Transformation System in Pleurotus eryngii var. ferulae Using Comprehensive Optimization and Multiple Endogenous Promoters

Qi Zhang,Zhongyang Ding,Sha Xu,Guiyang Shi,Mengye Shen,Lei Chen,Youran Li,Liting Zhao,Jingyun Liu

doi:10.3390/jof8020186

Abstract

Pleurotus eryngii var. ferulae, a fungus of the genus Pleurotus, 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 (cbx) was used to establish a polyethylene glycol-mediated transformation (PMT) system in P. eryngii var. ferulae. 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 Pleurotus sp. (10–40 CFU/μg). Moreover, three endogenous promoters (Ppfgpd1, Ppfgpd2, and Ppfsar1) 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 P. eryngii var. ferulae and provides a method to investigate the underlying molecular mechanisms and secondary metabolic pathway modifications.

Highlights

Pleurotus eryngii var. ferulae, a member of the Pleurotus species, is a tetrapolar heterothallic edible mushroom [1]
This study describes a genetic transformation system for P. eryngii var. ferulae
The original pfsdhb gene was retained while the mutant pfsdhb from pKAB existed independently in the P. eryngii var. ferulae genome for replication and transcription

Summary

Introduction

Pleurotus eryngii var. ferulae, a member of the Pleurotus species, is a tetrapolar heterothallic edible mushroom [1]. Ferulae, a member of the Pleurotus species, is a tetrapolar heterothallic edible mushroom [1]. Ferulae is edible and has several medicinal properties, including anti-tumor, antioxidant, anti-obesity, and immunoregulatory properties [4,5,6,7]. Ferulae secretes large amounts of lignin-degrading enzymes (laccase, manganese peroxidase, and lignin peroxidase) for industrial use [8]. These enzymes can be used to degrade lignin, provide bioenergy, decolor dyes, and treat sewage [9]. Our previous studies confirmed that co-cultivating P. eryngii var. Ferulae with certain fungi can increase the transcription Our previous studies confirmed that co-cultivating P. eryngii var. ferulae with certain fungi can increase the transcription

Methods

Results

Discussion

Conclusion