The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1

Abstract

Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.

A Novel Thermostable GH3β-Glucosidase fromTalaromyce leycettanuswith Broad Substrate Specificity and Significant Soybean Isoflavone Glycosides-Hydrolyzing Capability

“…The optimal temperature of Bgl3B (65°C) was the same as the β -glucosidase from T. thermophiles [ 9 ], but lower than Bgl3A (75°C) and Cel3a (71.5°C) from T. emersonii [ 23 ]. What is more, Bgl3B had better thermal stability than most β -glucosidases from thermophilic T. piceus [ 9 ], Myceliophthora thermophile [ 24 ], and Humicola insolens [ 25 ]. In addition, Bgl3B was highly resistant to various metal ions and reducing agents but showed susceptibility to Fe 3+ and Ag + .…”

Trichoderma-Inoculated Miscanthus Straw Can Replace Peat in Strawberry Cultivation, with Beneficial Effects on Disease Control

“…are resistant to mycoparasitism by Trichoderma and therefore can rapidly colonize the empty niche created by Trichoderma- induced fungal death in the rhizosphere. Interestingly, the lignocellulose degrading genus Humicola is also known to be involved in disease suppression (Ko et al, 2011 ; Yang et al, 2014 ; Xu et al, 2016 ), which could further explain the increased expression of genes encoding anti-fungal enzymes like chitinase 1 and 2 and phenylalanine ammonia lyase in the strawberry leaves.…”

Engineering the GH1 β-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose

“…Filamentous fungi of the genus Humicola are excellent producers of various cellulolytic enzymes, including several β-glucosidases [ 38 ]. To date, an intracellular GH1 β-glucosidase (Bglhi) [ 14 ], an extracellular GH3 β-glucosidase (BglHi2) [ 39 ] and three heterologous GH3 β-glucosidase isoenzymes from Humicola insolens expressed in Pichia pastoris (HiBgl3A, HiBgl3B and HiBgl3C) [ 40 ] have been characterized.…”