The use of Agrobacterium-mediated insertional mutagenesis sequencing to identify novel genes of Humicola insolens involved in cellulase production.

Abstract

A transfer DNA (T-DNA)-tagged mutant library of Humicola insolens was screened for mutants with altered cellulase production using the plate-clearing zone assay. Three selected mutants (5-A7, 5-C6, and 13-B7) exhibited significantly depressed FPase, CMCase and xylanase activities compared with the wild-type strain upon shake-flask fermentation, while the pNPCase and pNPGase activities of the three mutants were relatively higher than those of the parental strain. Combined with the results of SDS-PAGE and mass spectrometry, we suggest that expression of the CMCases Cel6B, Cel7B, CMC3, and XynA/B/C was reduced in the mutant strains. Twelve putative T-DNA insertion sites were identified in the three mutants via Agrobacterium-mediated insertional mutagenesis sequencing (AIM-Seq). Bioinformatics analysis suggested that a putative dolichyl pyrophosphate phosphatase, two hypothetical proteins encoding genes of unknown function, and/or nine intergenic fragments may be involved in cellulase and hemicellulase production by H. insolens. This provides promising new candidate genes relevant to cellulase production by the fungus, which will be crucial not only for our understanding of the molecular mechanism underlying cellulase production, but also for strain improvement.