Gene Targeting by RNAi-Mediated Knockdown of Potent DNA Ligase IV Homologue in the Cellulase-Producing Fungus Talaromyces cellulolyticus

Abstract

The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

RNAi silencing: A tool for functional genomics research on fungi

“…The first transformants showed a decrease of 96.3 % in transcript levels compared to the parental strain; they were used for allowing the procedure of Knockout with the ligD target gene. The double transformants permitted the development of strains able to homologously recombine, without modifying the protein expression of the mutants compared to the parental strain (Hayata et al, 2014). These results suggest that previous RNAi silencing of genes of the NEHJ system in fungi, could allow the use of Knockout technology most efficiently.…”