The G-protein Coupled Receptor GPR8 Regulates Secondary Metabolism in Trichoderma reesei

Abstract

Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in Δgpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.

Keywords: SOR cluster; Trichoderma reesei; cellulase; chemical communication; chemosensing; heterotrimeric G-protein pathway; light response; secondary metabolism.

FIGURE 1

Functional category analysis of genes differentially regulated in Δgpr8 in darkness. (A) Functions of genes < 2-fold (p-value < 0.01) up-regulated in Δgpr8 in constant darkness upon growth on cellulose. (B) Functions of genes down-regulated in Δgpr8 in constant darkness upon growth on cellulose. Selected categories highly relevant for T. reesei physiology are shown.

FIGURE 2

Transcript levels of cre1 and SOR cluster related genes in Δgpr8 in darkness on glucose. (A) Schematic representation of the SOR cluster of T. reesei with protein IDs as listed on JGI (http://genome.jgi.doe.gov/Trire2/Trire2.home.html) along with gene designations. SOR7/TR_73631 was named SOR4 in Derntl et al. (2017a). (B) Transcript levels of cre1 and SOR cluster assigned genes in Δgpr8 relative to wild type grown on 1% (w/v) glucose in darkness (DD). Transcriptional changes shown are non-significant (p-value > 0.05) and were analyzed in three biological replicates.

FIGURE 3

Regulatory relationships between strains lacking gpr8 and those lacking ypr2. (A) Principal component analysis (PCA) of datasets for Δgpr8 and Δypr2 upon growth in constant darkness (DD) or constant light (LL) on cellulose. (B) Consistent and contrasting regulatory overlaps between Δgpr8 and Δypr2 upon growth in constant darkness on cellulose. Dominant functional categories of the overlapping genes are given for every comparison.

FIGURE 4

Heatmap of secondary metabolite synthase coding genes. CytP450 (cytochrome P450), NRPS (non-ribosomal peptide synthase), PKS (polyketide synthase) and TPS (terpene synthase) coding genes of wild type, Δgpr8 and Δypr2 strains grown in light (LL) or darkness (DD) on cellulose are shown. A highresolution version of this figure comprising IDs of these genes is shown in Supplementary Material, Image 1.

FIGURE 5

Secondary metabolite production in Δgpr8 and in Δsor7/TR_73631. Mass spectrometric analysis and quantification with internal standards of culture supernatants of Δgpr8 (A) or Δsor7 (B) upon growth on cellulose in constant darkness (DD) relative to wild type. Values are normalized to produced biomass under these conditions. Error bars show standard deviations. Statistical significance was calculated using Student’s T-Test in R Studio (compare means, ggpubr v0.3.0). ** = p-value < 0.01; * = p-value < 0.05.

FIGURE 6

Schematic representation of the regulatory impact of GPR8. Gpr8 transcript abundance is positively regulated by CRE1 in constant darkness (DD) and negatively by ENV1 in constant light (LL) upon growth on cellulose. Considering our results on gene regulation in the absence of GPR8 on glucose and cellulose in light and darkness, we conclude that the signal transmitted by GPR8 has a light- and carbon dependent relevance in T. reesei. Regulation by GPR8 is indirect and can be expected to involve the pathways for signal transmission depicted here as examples to be investigated. Signal transmission through such a cascade leads to the regulatory output: GPR8 positively impacts regulation of ypr2, but only partially shares its target genes upon growth on cellulose in darkness. Sor7 transcript levels are positively influenced by GPR8 as well and SOR7 impacts production of several secondary metabolites, in addition to trichodimerol, for the biosynthesis of which it is essential. The presence of SOR7 also positively influences cellulase regulation through an as yet unknown mechanism contributing to the balance of secondary metabolism and enzyme production. The effect of GPR8 on secondary metabolism is assumed to be in part, but not exclusively mediated by its role in regulation of ypr2 and sor7.