Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties.
BACKGROUND
Trichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH). Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general.


RESULTS
We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30) using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor) on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production.


CONCLUSIONS
aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.
Vitikainen
Marika
M
VTT Technical Research Centre of Finland, FI-02044 VTT, Espoo, Finland. marika.vitikainen@vtt.fi
Arvas
Mikko
M
Pakula
Tiina
T
Oja
Merja
M
Penttilä
Merja
M
Saloheimo
Markku
M
D002480:Cellulase / Q000096:biosynthesis*; D055028:Comparative Genomic Hybridization / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics; D023281:Genomics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D017384:Sequence Deletion; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism*
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D002480:Cellulase