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The state-of-the-art procedure for gene insertions into Trichoderma reesei is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes asl1 (encoding an enzyme of the l-arginine biosynthesis pathway), the hah1 (encoding an enzyme of the l-lysine biosynthesis pathway), and the pyr4 (encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in T. reesei. By employing goxA of Aspergillus niger as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.

Novel Strategies for Genomic Manipulation of Trichoderma reesei with the Purpose of Strain Engineering