A CRE1- regulated cluster is responsible for light dependent production of dihydrotrichotetronin in Trichoderma reesei

Abstract

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide). Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.

Fig 1. Light dependent regulation by CRE1 and the CRE1-regulated cluster.

Transcriptome analysis of Δcre1 was done in comparison to QM9414 as wild-type in constant light and constant darkness upon growth on microcrystalline cellulose as carbon source for 72 hours. (A) Venn diagrams showing positive and negative gene regulation in a strain lacking cre1 in light and darkness upon growth on cellulose. (B) Regulation of cluster genes by CRE1 in light and darkness related to the respective wildtype strain under the same conditions. (C) Schematic representation of the cluster genes along with protein IDs as assigned in JGI (http://genome.jgi.doe.gov/Trire2/Trire2.home.html) along with protein designations assigned previously [5, 41, 48].

Fig 2. Secondary metabolite patterns in deletion mutants.

HPTLC (high performance thin layer chromatography) analysis of secondary metabolites secreted by mutant strains in the LCS cluster upon growth on cellulose in light or darkness. Numbers represent protein IDs of genes deleted in the respective analyzed strain. Samples are adjusted to biomass produced and hence represent secondary metabolites produced by equal amounts of biomass. The three panels represent different methods of visualization of the same metabolite patterns: Upper panel: Remission at 366 nm; middle panel: derivatized, remission at 366 nm, lower panel: derivatized, transmission visible light. The analysis of light- and dark samples was done in parallel on the same HPTLC plate and consequently signal strengths in light and dark are comparable. Arrows highlight bands with altered signal strength compared to wildtype. Three biological replicates were analyzed and a representative sample is shown.

Fig 3. Quantitative mass spectrometry analysis in light and darkness.

(A) Quantitative analysis of abundance of trichodimerol (“T”), dihydrotrichotetronin (“D”) and paracelsin B (“P”) in light compared to darkness in wildtype. (B-G) Quantitative analysis of abundance of trichodimerol (B, C), dihydrotrichotetronin (D,E) and paracelsin (F,G) in light (light grey bars) and darkness (dark grey bars) in strains lacking cluster genes upon growth in liquid minimal medium with cellulose as carbon source. Produced metabolites were related to the biomass formed under the respective conditions. Errorbars show standard deviations of two biological replicates. Values with statistically significant difference from wild-type are marked with an asterisk (except for lacking production). Structures show the respective compound.

Fig 4. Transcript levels of cluster genes in a strain lacking TR_102497/YPR2.

Transcript levels of TR_73618, TR_73621, TR_73623 and TR_43701 were determined by quantitative RT-PCR after growth on cellulose in constant darkness (A) or in constant light (B) for 96 hours and are shown relative to the wildtype. Errorbars show standard deviations of at least three biological replicates and three technical replicates. Values with statistically significant difference from wild-type are marked with an asterisk (except when no transcript was detected at all).

Fig 5. Mutual regulatory response of biosynthetic genes to deletions.

The effects of deletions of the biosynthetic genes on transcript levels of TR_73618 (A, B), TR_73621 (C, D) and TR_73623 (E, F) in constant light (LL) or constant darkness (DD) are shown relative to wild-type in darkness. As transcript levels in light are too low to be evaluated next to darkness results (A, C, E), they were also presented separately (B, D, F) with the y-axis showing transcript levels in relation to the wild-type in darkness. Strains were grown in constant light (LL) or constant darkness (DD) on cellulose for 96 hours. Errorbars show standard deviations of at least three biological replicates and three technical replicates. (G, H) Schematic representation of positive and negative feedback of mutual gene regulation in light and darkness. Values with statistically significant difference from wild-type are marked with an asterisk in A,C and E for darkness and in B, D and F for light.

Fig 6. Cellulase regulation by cluster genes.

(A, B) Specific cellulase activity in mutants of the cluster genes upon growth in constant light (A, light grey bars) or in constant darkness (B, dark grey bars) for 96 hours on cellulose related to wild-type. (C, D) Transcript levels of cbh1 in mutants of the cluster genes upon growth in constant light (C, light grey bars) or in constant darkness (D, dark grey bars) for 96 hours on cellulose related to wild-type. Errorbars show standard deviations of at least three biological replicates and two technical replicates. Values with statistically significant difference from wild-type are marked with an asterisk (except for lacking production).