Establishment of High-Efficiency Screening System for Gene Deletion in Fusarium venenatum TB01.

Sheng Tong,Wuxi Chen,Wenyuan Zhou,Yuanxia Sun,Kexin An,Demao Li,Qinhong Wang

doi:10.3390/jof8020169

Abstract

Genetic engineering is one of the most effective methods to obtain fungus strains with desirable traits. However, in some filamentous fungi, targeted gene deletion transformant screening on primary transformation plates is time-consuming and laborious due to a relatively low rate of homologous recombination. A strategy that compensates for the low recombination rate by improving screening efficiency was performed in F. venenatum TB01. In this study, the visualized gene deletion system that could easily distinguish the fluorescent randomly inserted and nonfluorescent putative deletion transformants using green fluorescence protein (GFP) as the marker and a hand-held lamp as the tool was developed. Compared to direct polymerase chain reaction (PCR) screening, the screening efficiency of gene deletion transformants in this system was increased approximately fourfold. The visualized gene deletion system developed here provides a viable method with convenience, high efficiency, and low cost for reaping gene deletion transformants from species with low recombination rates.

Highlights

Academic Editor: Sotiris AmillisF. venenatum, identified from more than 3000 soil organism samples from across the world, has been successfully used as a food-based cell factory [1,2,3]
Many genetic transformation systems have been reported in filamentous fungi, such as Beauveria bassiana, Myceliophthora thermophila, Trichoderma reesei, and Aspergillus niger, and Agrobacterium-tumefaciens-mediated transformation (ATMT)
We developed a PEG-mediated protoplast transformation system in F

Summary

Introduction

Academic Editor: Sotiris AmillisF. venenatum, identified from more than 3000 soil organism samples from across the world, has been successfully used as a food-based cell factory [1,2,3]. The mycoprotein produced by it offers a better nutritional balance than animal- and plant-derived protein, including a reduction in saturated fat, an increase in dietary fiber, and richness in essential amino acids [4]. Compared to the single-cell protein, which is based on the cultivation of protein-producing microorganisms, including bacteria, yeasts, and microalgae, the mycoprotein is more flavorful and has a meat-like texture [2,5]. It has the potential to partially or entirely substitute animal- and plant-derived protein foods that are facing insufficient protein supply due to the aggravation of population growth and environmental pollution [8,9,10]. Many genetic transformation systems have been reported in filamentous fungi, such as Beauveria bassiana, Myceliophthora thermophila, Trichoderma reesei, and Aspergillus niger, and Agrobacterium-tumefaciens-mediated transformation (ATMT)

Methods

Results

Conclusion