Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that the transcriptional response after 6h of exposure to wheat straw was very different from the response at 24h of exposure to the same substrate. For example, less than half of the genes encoding carbohydrate active enzymes that were induced after 24h of exposure to wheat straw, were also induced after 6h exposure. Importantly, over a third of the genes induced after 6h of exposure to wheat straw were also induced during 6h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. We show, using proteomics, that carbon-starved cultures indeed release CAZymes with predicted activity on plant polysaccharides. Analysis of the enzymatic activity and the reaction products, indicates that these proteins are enzymes that can degrade various plant polysaccharides to generate both known, as well as potentially new, inducers of CAZymes.

To investigate the anti-Helicobacter pylori activity of 28 strains of Lactobacillus salivarius and 12 other lactobacilli, isolated from different sites and from different geographical regions. An in vitro agar plate diffusion assay was employed to assess the Lactobacillus anti-H. pylori activity. Nine out of 28 L. salivarius strains and 3/12 other Lactobacillus species tested inhibited H. pylori growth. There was no correlation between ecological niche/geographical location of isolation of the lactobacilli and their inhibitory capability. Further studies on strain L. salivarius UCC119 showed that this strain could inhibit growth of 6/6 clinical isolates of H. pylori, five of which were antibiotic-resistant. This inhibition was not due to acid production and was not mediated by a protein, but did require the presence of live cells. Growth inhibition of H. pylori by L. salivarius is strain-dependent and is not linked to any particular environmental niche or geographic location. Strains of L. salivarius showing highest anti-H. pylori activity may be useful as an adjunct in the treatment of strains that are resistant to conventional antibiotics.

Summary: Caffeic acid O‐methyltransferase(COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barleyCOMTorthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with threeCOMTgenes expressed in lignifying tissues.RNAi significantly reduced stemCOMTprotein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount ofp‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in anyRNAi line despite significantCOMTsuppression and altered lignin. The novelCOMTgene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barleyCOMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barleyCOMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.

Trichoderma reesei is used to produce saccharifying enzyme cocktails for biofuels. There is limited understanding of the transcription factors (TFs) that regulate genes involved in release and catabolism of l‐arabinose and d‐galactose, as the main TF XYR1 is only partially involved. Here, the T. reesei ortholog of ARA1 from Pyricularia oryzae that regulates l‐arabinose releasing and catabolic genes was deleted and characterized by growth profiling and transcriptomics along with a xyr1 mutant and xyr1/ara1 double mutant. Our results show that in addition to the l‐arabinose‐related role, T. reesei ARA1 is essential for expression of d‐galactose releasing and catabolic genes, while XYR1 is not involved in this process.

Gaining new knowledge through fungal monoculture responses to lignocellulose is a widely used approach that can lead to better cocktails for lignocellulose saccharification (the enzymatic release of sugars which are subsequently used to make biofuels). However, responses in lignocellulose mixed cultures are rarely studied in the same detail even though in nature fungi often degrade lignocellulose as mixed communities. Using a dual RNA-seq approach, we describe the first study of the transcriptional responses of wild-type strains of Aspergillus niger, Trichoderma reesei and Penicillium chrysogenum in two and three mixed species shake-flask cultures with wheat straw. Based on quantification of species-specific rRNA, a set of conditions was identified where mixed cultures could be sampled so as to obtain sufficient RNA-seq reads for analysis from each species. The number of differentially-expressed genes varied from a couple of thousand to fewer than one hundred. The proportion of carbohydrate active enzyme (CAZy) encoding transcripts was lower in the majority of the mixed cultures compared to the respective straw monocultures. A small subset of P. chrysogenum CAZy genes showed five to ten-fold significantly increased transcript abundance in a two-species mixed culture with T. reesei. However, a substantial number of T. reesei CAZy transcripts showed reduced abundance in mixed cultures. The highly induced genes in mixed cultures indicated that fungal antagonism was a major part of the mixed cultures. In line with this, secondary metabolite producing gene clusters showed increased transcript abundance in mixed cultures and also mixed cultures with T. reesei led to a decrease in the mycelial biomass of A. niger. Significantly higher monomeric sugar release from straw was only measured using a minority of the mixed culture filtrates and there was no overall improvement. This study demonstrates fungal interaction with changes in transcripts, enzyme activities and biomass in the mixed cultures and whilst there were minor beneficial effects for CAZy transcripts and activities, the competitive interaction between T. reesei and the other fungi was the most prominent feature of this study.