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

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.

Current Advances in the Functional Genes of Edible and Medicinal Fungi: Research Techniques, Functional Analysis, and Prospects

“…However, its low conversion efficiency has limited the systematic study of its molecular mechanisms and metabolic control. Therefore, Zhang et al [75] established the PMT conversion method in P. ferulae for the first time and reported that adding 50 µg SS-DNA, 70 µg λDNA, and 0.4 µmol spermidine could maximize the conversion efficiency. An overexpression system containing three different endogenous promoters (P PFGPD1 , P PFGPD2 , and P PFSAR1 ) was established to drive the expression of different genes, promoting the expression of functional genes and novel secondary metabolic pathways.…”

Application of Nanotechnology in Plant Genetic Engineering

“…The popularity of the PEG delivery system lies in the following advantages: conversion efficiency, easy operation, low cost and mild reaction conditions. However, the PEG delivery system is limited by genotype and must be applied to naked protoplasts which are commonly used for transient gene expression [41].…”

An Efficient CRISPR/Cas9 Genome Editing System for a Ganoderma lucidum Cultivated Strain by Ribonucleoprotein Method

“…The mixture was incubated at 37 • C for 15 min to allow RNP complex formation. For PEG-mediated transformation, the following steps were performed referring to the methods described by Yu et al and Zhang et al [22,23]: 100 µL STC containing 10 7 protoplasts, 20 µL RNP complexes, and 50 µL PTC (600 g/L PEG 4000, Sigma, St. Louis, MO, USA; 10 mmol/L Tris-HCl pH 7.5; 50 mmol/L CaCl 2 ) were mixed with 0.006% (final concentration) of Triton X-100 (Biofroxx, Einhausen, Germany) on ice and incubated for 10 min. An amount of 1 mL PTC was gently added and mixed in the transformation system, then incubated at 20 • C for 30 min.…”