From model organism to industrial workhorse: analysis of genes in Aspergillus nidulans and disruption of cre2 for Trichoderma reesei strain improvement.

Abstract

Carbon catabolite repression is a regulatory system whereby an organism can sequentially utilise carbon sources based on their available energy. This system results in the repression of genes encoding enzymes responsible for the utilisation of poorer carbon sources when preferable ones are available. Carbon catabolite repression has been extensively studied in the filamentous fungus Aspergillus nidulans. Repression is mediated via CreA, a zinc finger DNA binding protein, which is in turn, either directly or indirectly, regulated by an ubiquitination / deubiquitination system involving CreB, CreC and CreD. Previous work demonstrated that the A. nidulans genome contains a CreD homologue, ApyA, and that both of these proteins interact with an ubiquitin ligase, HulA. This relationship was proposed to be similar to Rod1p and Rog3p and their interaction with the ubiquitin ligase Rsp5p in Saccharomyces cerevisiae. Both apyA and hulA were targeted for disruption to facilitate phenotypic analysis and the study of epistatic interactions. Deletion of hulA was shown to be lethal in an A. nidulans haploid, but viable as a heterozygote in an A. nidulans diploid. The only detectable phenotypes of this deletion in a heterozygous diploid were increased sensitivity to molybdate and acriflavine. A strain containing a disruption of apyA did not demonstrate any detectable phenotypes, however, the apyA disruption allele showed epistatic interactions with mutations in creB, creC and creD. The disruption of apyA partially suppressed the phenotype of sensitivity to allyl alcohol in the presence of glucose displayed by strains containing mutations in creB and creC. However, the level of suppression exhibited by the disruption of apyA was not as strong as that shown by the creD34 mutation. A strain containing mutations in both creD and apyA demonstrated severe morphological deficiencies on minimal media as well as stronger resistance to acriflavine than creD34 alone, and resistance to molybdate. Bioinformatic analysis of CreD and CreD‐like proteins, including ApyA, from sequenced members of the Aspergilli and Rod1p, Rog3p and related proteins from members of Saccharomycetes suggested that the arrestin-like proteins, a group to which these belong, are subject to frequent gene duplication events. The number and range of sequenced fungal genomes also allowed a bioinformatic examination of the conservation of proteins involved in the carbon repression mechanisms across the fungal kingdom. A homologue of CreA was identified only within the members of Ascomycota that were examined, but putative homologues of CreB and CreC were identified across the fungal kingdom. The Saccharomycetes were an exception to this as a CreC homologue was not indentified and the CreB homologue was highly divergent or absent. The filamentous fungus, Trichoderma reesei is an important source of cellulases for use in the textile and alternative fuel industries. Previous studies have suggested a benefit for the manipulation of carbon catabolite repression for strain improvement, as the industrially significant strain RUTC-30 contains a mutation in cre1, the T. reesei creA homologue. The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was shown to be functional in carbon repression through complementation of a creB mutation in A. nidulans. This gene was targeted for disruption in T. ressei as disruption in A. nidulans leads to carbon derepression of some systems without the severe morphological effects of strains containing creA mutations. A T. reesei strain containing a cre2 disruption exhibited phenotypes similar to the A. nidulans creB mutant strain on solid media and had elevated cellulase levels.