Transgressive phenotypes from outbreeding between the Trichoderma reesei hyper producer RutC30 and a natural isolate.

Trichoderma reesei, the main filamentous fungus used for industrial cellulase production, was long considered to be asexual. The recent discovery of the mating type locus in the natural isolate QM6a and the possibility to cross this sterile female strain with a fertile natural female strain opened up a new avenue for strain optimization. We crossed the hyperproducer RutC30 with a compatible female ascospore-derived isolate of the wild-type strain CBS999.97 and analyzed about 300 offspring. A continuous distribution of secreted protein levels was observed in the progeny, confirming the involvement of several mutated loci in the hyperproductive phenotype. A bias toward MAT1-2 strains was identified for higher producers, but not directly linked to the Mating-type locus itself. Transgressive phenotypes were observed in terms of both productivity and secretome quality, with offspring that outperform their parents for three enzymatic activities. Genomic sequences of the 10 best producers highlighted the genetic diversity generated and the involvement of parental alleles in hyperproduction and fertility. IMPORTANCE: The filamentous fungus Trichoderma reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars. The filamentous fungus T. reesei produces cellulolytic enzymes that are essential for the hydrolysis of lignocellulosic biomass into monomerics sugars, which can in turn be fermented to produce second-generation biofuels and bioproducts. Production performance improvement, which is essential to reduce production cost, relies on classical mutagenesis and genetic engineering techniques. Although sexual reproduction is a powerful tool for improving domesticated species, it is often difficult to apply to industrial fungi since most of them are considered asexual. In this study, we demonstrated that outbreeding is an efficient strategy to optimize T. reesei. Crossing between a natural isolate and a mutagenized strain generated a biodiverse progeny with some offspring displaying transgressive phenotype for cellulase activities.

Effect of the res2 transcription factor gene deletion on protein secretion and stress response in the hyperproducer strain Trichoderma reesei Rut-C30.

BACKGROUND: The fungus Trichoderma reesei is one of the most used industrial cellulase producers due to its high capacity of protein secretion. Strains of T. reesei with enhanced protein secretion capacity, such as Rut-C30, have been obtained after several rounds of random mutagenesis. The strain was shown to possess an expanded endoplasmic reticulum, but the genetic factors responsible for this phenotype remain still unidentified. Recently, three new transcription factors were described in Neurospora crassa which were demonstrated to be involved in protein secretion. One of them, RES2, was involved in upregulation of secretion-related genes. The aim of our present study was therefore to analyze the role of RES2, on protein secretion in the T. reesei Rut-C30 strain. RESULT: Deletion of the res2 gene in Rut-C30 resulted in slightly slower growth on all substrates tested, and lower germination rate as well as lower protein secretion compared to the parental strain Rut-C30. Transcriptomic analysis of the Rut-C30 and the Δres2 mutant strain in secretion stress conditions showed remarkably few differences : 971 genes were differentially expressed (DE) in both strains while 192 genes out of 1163 (~ 16.5%) were DE in Rut-C30 only and 693 out of 1664 genes (~ 41.6%) displayed differential expression solely in Δres2. Notably, induction of protein secretion by cultivating on lactose and addition of secretion stress inducer DTT induced many genes of the secretion pathway similarly in both strains. Among the differentially expressed genes, those coding for amino acid biosynthesis genes, transporters and genes involved in lipid metabolism were found to be enriched specifically in the Δres2 strain upon exposure to lactose or DTT. Besides, redox homeostasis and DNA repair genes were specifically upregulated in the Δres2 strain, indicating an altered stress response. CONCLUSION: These results indicate that in the T. reesei Rut-C30 strain, RES2 does not act as a master regulator of the secretion pathway, but it contributes to a higher protein secretion by adjusting the expression of genes involved in different steps of protein synthesis and the secretion pathway.

Genome sequencing and transcriptome analysis of Trichoderma reesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction.

BACKGROUND: The hydrolysis of biomass to simple sugars used for the production of biofuels in biorefineries requires the action of cellulolytic enzyme mixtures. During the last 50 years, the ascomycete Trichoderma reesei, the main source of industrial cellulase and hemicellulase cocktails, has been subjected to several rounds of classical mutagenesis with the aim to obtain higher production levels. During these random genetic events, strains unable to produce cellulases were generated. Here, whole genome sequencing and transcriptomic analyses of the cellulase-negative strain QM9978 were used for the identification of mutations underlying this cellulase-negative phenotype. RESULTS: Sequence comparison of the cellulase-negative strain QM9978 to the reference strain QM6a identified a total of 43 mutations, of which 33 were located either close to or in coding regions. From those, we identified 23 single-nucleotide variants, nine InDels, and one translocation. The translocation occurred between chromosomes V and VII, is located upstream of the putative transcription factor vib1, and abolishes its expression in QM9978 as detected during the transcriptomic analyses. Ectopic expression of vib1 under the control of its native promoter as well as overexpression of vib1 under the control of a strong constitutive promoter restored cellulase expression in QM9978, thus confirming that the translocation event is the reason for the cellulase-negative phenotype. Gene deletion of vib1 in the moderate producer strain QM9414 and in the high producer strain Rut-C30 reduced cellulase expression in both cases. Overexpression of vib1 in QM9414 and Rut-C30 had no effect on cellulase production, most likely because vib1 is already expressed at an optimal level under normal conditions. CONCLUSION: We were able to establish a link between a chromosomal translocation in QM9978 and the cellulase-negative phenotype of the strain. We identified the transcription factor vib1 as a key regulator of cellulases in T. reesei whose expression is absent in QM9978. We propose that in T. reesei, as in Neurospora crassa, vib1 is involved in cellulase induction, although the exact mechanism remains to be elucidated. The data presented here show an example of a combined genome sequencing and transcriptomic approach to explain a specific trait, in this case the QM9978 cellulase-negative phenotype, and how it helps to better understand the mechanisms during cellulase gene regulation. When focusing on mutations on the single base-pair level, changes on the chromosome level can be easily overlooked and through this work we provide an example that stresses the importance of the big picture of the genomic landscape during analysis of sequencing data.