Kinetic transcriptome analysis reveals an essentially intact induction system in a cellulase hyper-producer Trichoderma reesei strain

International audienceBackground: The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour. Results: We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance. Conclusions: This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

A systems biology approach for the improvement of industrial Trichoderma reesei strains

La compréhension de la régulation de la synthèse et sécrétion de cellulases dans le champignon Trichoderma reesei a fortement évolué ces dernières années. Cependant, le coût de production de cellulases reste encore un des principaux problèmes pour la production de bioéthanol à partir de lignocellulose. C’est dans ce contexte que mon projet de thèse s’inscrit, dans l’amélioration de la production de cellulases de T. reesei et son adaptation aux conditions industrielles. D’abord, nous avons réalisé une analyse du transcriptome sur une lignée de souches améliorées dans des conditions proches du procède industrielle avec lactose comme inducteur. Cette étude nous a permis de trouver des gènes spécifiquement régulés dans la souche plus performante, probablement impliqués dans sa grande capacité de production de cellulases. Dans un deuxième temps, nous avons identifié parmi les gènes régulés du transcriptome lesquels impliquées dans la production de cellulases. A cet effet, des souches mutées ont été construites et leur phénotype évalué. Trois de ces gènes mutés ont affecté la production de cellulase et leur fonction ont dévoilé des nouvelles mécanismes régules. Finalement, nous avons exploré les différences d’expression lorsque nous utilisons un hydrolysat de lignocellulose comme inducteur de cellulases à la place du lactose. Une étude du transcriptome dans ces conditions, nous a permis de caractériser les différences de régulation des gènes entre ces inducteurs. En outre, nous avons identifié un groupe des gènes probablement impliqué dans la détoxification qui peuvent être utilisés dans le futur pour développer une souche résistante aux inhibiteurs.A lot of progress had been done in recent years to understand the regulation of synthesis and secretion of cellulases in the fungus Trichoderma reesei. However, the production cost of cellulases still remains one of the most important limiting steps in the production of bioethanol from lignocellulose. It is in this context that my PhD project has been developed: to genetic engineering T. reesei strains to increase its cellulase production and its adaptation to industrial conditions. First, we conducted a transcriptome analysis to an improved lineage of industrial strains during cellulase production following conditions close to the industrial process (lactose as inducer and fed-batch culture in bioreactor). We found specifically regulated genes for the most performant strain, possibly involved in its high cellulase production capacity. Then, we identified which regulated genes from transcriptome were involved in cellulase production. For this purpose mutated strains for highly regulated genes were constructed and their phenotype assessed. Three mutated genes showed to impact cellulase production and their function gave us an insight into new mechanisms being regulated. Finally, we explored the expression differences when we used a lignocellulose hydrolysate as cellulases inducer instead of lactose. A transcriptomic study of cellulases production on lignocellulose hydrolysates and lactose, allowed us to characterize the differences in regulated genes between these inducers. In addition, a group of genes probably related to detoxification was identified and could be used in the future to develop an inhibitor resistant strain

Proximity ligation scaffolding and comparison of two Trichoderma reesei strains genomes

International audienceBackground: The presence of low complexity and repeated regions in genomes often results in difficulties to assemblesequencing data into full chromosomes. However, the availability of full genome scaffolds is essential to severalinvestigations, regarding for instance the evolution of entire clades, the analysis of chromosome rearrangements,and is pivotal to sexual crossing studies. In non-conventional but industrially relevant model organisms, such as theascomycete Trichoderma reesei, a complete genome assembly is seldom available.Results: The chromosome scaffolds of T. reesei QM6a and Rut-C30 strains have been generated using a contactgenomic/proximity ligation genomic approach. The original reference assembly, encompassing dozens of scaffolds,was reorganized into two sets of seven chromosomes. Chromosomal contact data also allowed to characterize10–40 kb, gene-free, AT-rich (76%) regions corresponding to the T. reesei centromeres. Large chromosomal rearrangements(LCR) in Rut-C30 were then characterized, in agreement with former studies, and the position of LCRbreakpoints used to assess the likely chromosome structure of other T. reesei strains [QM9414, CBS999.97 (1-1, re), andQM9978]. In agreement with published results, we predict that the numerous chromosome rearrangements found inhighly mutated industrial strains may limit the efficiency of sexual reproduction for their improvement.Conclusions: The GRAAL program allowed us to generate the karyotype of the Rut-C30 strain, and from there topredict chromosome structure for most T. reesei strains for which sequence is available. This method that exploitsproximity ligation sequencing approach is a fast, cheap, and straightforward way to characterize both chromosomestructure and centromere sequences and is likely to represent a popular convenient alternative to expensive andwork-intensive resequencing projects

The cellulase induction system in Trichoderma reesei strains remains well conserved despite several generations of random mutagenesis and screening

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MOESM2 of Proximity ligation scaffolding and comparison of two Trichoderma reesei strains genomes

Additional file 2. QM6a reassembly sequence in fasta format (7 chromosomes + unassembled scaffolds)

High-quality genome (re)assembly using chromosomal contact data

International audienceClosing gaps in draft genome assemblies can be costly and time-consuming, and published genomes are therefore often left 'unfinished.' Here we show that genome-wide chromosome conformation capture (3C) data can be used to overcome these limitations, and present a computational approach rooted in polymer physics that determines the most likely genome structure using chromosomal contact data. This algorithm—named GRAAL—generates high-quality assemblies of genomes in which repeated and duplicated regions are accurately represented and offers a direct probabilistic interpretation of the computed structures. We first validated GRAAL on the reference genome of Saccharomyces cerevisiae, as well as other yeast isolates, where GRAAL recovered both known and unknown complex chromosomal structural variations. We then applied GRAAL to the finishing of the assembly of Trichoderma reesei and obtained a number of contigs congruent with the know karyotype of this species. Finally, we showed that GRAAL can accurately reconstruct human chromosomes from either fragments generated in silico or contigs obtained from de novo assembly. In all these applications, GRAAL compared favourably to recently published programmes implementing related approaches