Rapid transformation of high cellulase-producing mutant strains of Trichoderma reesei by microprojectile bombardment

Abstract

Intact conidia of three industrially relevant strains of Trichoderma reesei were effectively transformed by particle bombardment. Transformations were carried out individually with plasmids carrying either the fungal amdS or bacterial hph gene as a selectable marker and by cotransformation with both plasmids. Transformant yields with single plasmids were up to 11 stable transformants per mg DNA at the bombardment distance of 6 cm. Mitotic stability of the transformants was 75±100% and the cotransformation ef®ciency averaged 92% when the ®rst selection was performed on hygromycin B plates. The entire procedure could be completed in 1 week with the hph marker.

Transformation of the Mycorrhizal Basidiomycetes,Suillus grevilleiandS. bovinus, by Particle Bombardment

“…The 100% mitotic stability of the 12 transformants is higher than the 60-70% of Asperigillus nidulans 30) achieved by particle bombardment. Hazell et al 18) and Te'o et al 33) also obtained results similar to ours.…”

“…Particle bombardment is technically simpler than PEG/CaCl 2 , electroporation and REMI, since it does not require protoplast isolation. This technique has been used to transform the rust fungus Puccinia graminis, 17) Trichoderma reesei, 18) and Aspergillus nidulans. 19) Ectomycohrrizal fungi, Paxillus involutus 20) and Pisolithus tinctorius, 15) have also been transformed in this manner, but transformation of Suillus spp.…”

Trichoderma reesei RUT-C30 – thirty years of strain improvement

“…However, the technique has been particularly difficult with RUT-C30, seemingly due to a reduced ability of the strain to regenerate its cell wall (Nevalainen et al, 1995;Bergquist et al, 2002). Biolistic bombardment has provided a quick and reliable transformation system of T. reesei in general, whereby a good number of stable transformants are generated (Hazell et al, 2000;Te'o et al, 2002). However, despite several attempts at optimization, including modification of codon usage of heterologous genes for Microbiology 158 Microscopy has been used to visually identify bottlenecks along the secretory pathway of heterologous proteins.…”

Approaches to functional genomics in filamentous fungi

“…In recent years, efficient transformation systems have been developed for a wide range of filamentous fungi (reviewed in [1,4,5]). Successful transformation of fungi has been achieved by: CaCl 2 /polyethylene glycol [6][7][8], electroporation [9-15], particle bombardment [2,6,10,16,17], and Agrobacterium tumefaciens-mediated transformation (AMT) [1].…”