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

Abstract

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.

Guttation capsules containing hydrogen peroxide: an evolutionarily conserved NADPH oxidase gains a role in wars between related fungi

“…The open reading frames of genes nox1 (OPB44254), nox2 (OPB36463) and nor1 (OPB40972) were retrieved from the genome of Tgui (NCBI GenBank: LVVK00000000.1). A plasmid with a hygromycin resistance cassette (Derntl et al ., ), a complementary plasmid encoding geneticin resistance (Seiboth et al ., ) and an overexpression plasmid harbouring the T. reesei cdna1 promoter (Uzbas et al ., ) were constructed and directly used for polyethylene glycol (PEG)‐mediated protoplast transformation as described in in Supporting Information S6. Transformants were screened by PCR with a Phire Plant Direct PCR kit (Thermo Scientific, USA) and subjected to three rounds of single‐spore purification.…”

CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a

“…T. reesei strain QM6a Δ tmus53 Δ pyr4 was used as wild type (WT) strain in this study and it was obtained from the Institute of Chemical Engineering (Viena University of Technology, Viena, Austria) [37]. The strain was maintained in MEX medium (3% (w/v) malt extract and 2% (w/v) agar)) at 30°C supplemented with 5 mM uridine for pyr4 auxotrophic selection [38].…”

A Review on the Feedstocks for the Sustainable Production of Bioactive Compounds in Biorefineries

“…This is a very important feature, given that both first and second-generation feedstocks can be used to produce pentoses, hexoses, and even other compounds as starch and glycerol can also be used as substrate. Second, with the current advances of metabolic engineering, well-known microorganisms are being studied in order to develop a heterologous expression of proteins that could synthesize antibiotics and overcome drawbacks as the inhibition by certain compounds, broaden the range of carbon sources or even potentiate the production [55].…”