Figure 1.
Cellulase production in N. crassa is regulated by cellobiose induction and CCR.
CCR is decreased in absence of glucose, allowing scouting enzymes to liberate cellobiose from cellulose. Cellobiose (or a derivative) results in activation of the transcription factor CLR1, which induces expression of transporters for cellodextrins, β-glucosidases, and clr-2. Production of the transcription factor CLR2 drives cellulase gene expression. Both intracellular and extracellular β-glucosidase enzymes catalyze conversion of cellobiose to glucose, which can trigger carbon catabolite repression via glucose sensing mechanisms and transcriptional repression by CRE1.
Figure 2.
Deletion of vib-1 abolishes production of cellulases and utilization of cellulosic material.
(A) Growth of WT and Δvib-1 on Avicel after 4 days; growth of WT is indicated by formation of orange mycelia, versus no growth of the Δvib-1 mutant. (B) Cellulase activity from 4-day old culture supernatants from Avicel-grown cultures of WT, the Δvib-1 mutant, the Pvib-1 strain (constitutive expression of vib-1 in a Δvib-1 strain) and the PTrvib1 strain (constitutive expression of T. reesei vib1 in a Δvib-1 strain). Cellulase activity was measured using Avicel as a substrate and represented by the amount of glucose and cellobiose released. The equivalent of glucose from cellobiose was calculated and represented by the light gray bar. (C) The secretomes of strains analyzed in panel B are shown.
Figure 3.
Constitutive expression of clr-2 rescued the cellulase production defect of the Δvib-1 mutant.
(A) Protein concentration and cellulase activity in a Δvib-1 mutant versus a Δvib-1 strain constitutively expressing clr-2 (Pc clr-2; Δvib-1) and WT and a Pc clr-2 strain under Avicel conditions. (B) Expression levels from RNA-seq data of genes encoding major classes of CAZy proteins from WT and Δvib-1 shifted to Avicel versus the Pc clr-2 and Pc clr-2; Δvib-1 strains shifted to minimal media with no carbon source. FPKM (Fragment Per Kilobase per exon per Megabase mapped) for individual genes were averaged between three biological replicates and pooled by CAZy class. (C) Hierarchical clustering of FPKM for 91 Avicel-regulon genes in the Δvib-1 mutant and WT on Avicel (Av) and the Δvib-1, WT, Pc clr-2 and the Pc clr-2; Δvib-1 strains switched to no carbon conditions (Nc). Results are displayed as heat maps with log (FPKM) from minimum (bright blue) to maximum (bright yellow).
Figure 4.
VIB1 is not required for cellobiose sensing or signaling.
(A) Expression levels of two cellulase genes (cbh-1 and gh5-1) were assessed in the Δ3βG; Δvib-1 strain versus WT, and the Δ3βG and Δvib-1 strains after a shift from sucrose VMM to 0.2% cellobiose versus 2% Avicel. Gene expression levels were measured by relative quantitative-PCR using actin as a control and normalized to expression level when cultures were shifted to VMM with no carbon source. (B) CMCase activity after 24 hrs of growth on 2% cellobiose of the Δ3βG; Δvib-1 strain relative to the WT, and the Δ3βG and Δvib-1 strains. Measured enzyme activity in arbitrary unit (AU) was normalized against to the mycelial biomass of each culture.
Figure 5.
Comparative analysis of gene expression between Δvib-1 and WT shifted to media lacking a carbon source (Nc) versus Avicel (Av) revealed potential CCR regulators.
Hierarchical clustering of FPKM for 770 genes that were differentially expressed in the Δvib-1 mutant when shifted to either carbon-free and Avicel conditions. Heat maps showing log (FPKM) from minimum (bright blue∶L) to maximum (bright yellow∶H) revealed three gene sets: A 273-gene set that has a similar expression pattern between the Δvib-1 mutant and WT, although the induction level on Avicel differed; a 173-gene set that displayed a similar expression pattern between the Δvib-1 mutant and WT that is high (H) on Nc and low (L) on Avicel; a 360-gene set consisting of genes that were generally expressed at higher levels in Δvib-1 on Avicel than in other samples/conditions.
Figure 6.
Screen for function of new proteins involved in CCR.
(A) Growth assays of the WT, Δvib-1, Δcre-1, Δcol-26, ΔcreB, and ΔcreD strains on 2-deoxy-glucose (2-DG) when grown on 2% cellobiose VMM for 2 days or on 2% Avicel VMM for 4 days. (B) Effects of col-26 and cre-1 deletions on sensitivity to 2-DG and allyl alcohol. Strains were inoculated and grown in 2% cellobiose VMM with 100 mM allyl alcohol for 40 hrs. For 2-DG sensitivity tests, the strains were inoculated and grown in 2% Avicel with either 0.2% 2-DG or 0.5% 2-DG for 5 days.
Figure 7.
Deletion of col-26 causes defects in glucose sensing/metabolism.
(A) Mycelial biomass of the Δcol-26 mutant relative to WT and the Δcre-1 strains on glucose, fructose, sucrose, or cellobiose. Mycelial biomass was measured at 24 hrs after inoculation. (B) Glucose uptake of WT, Δvib-1, Δcre-1, Δcol-26; Δvib-1 and the Δcre-1; Δcol-26; Δvib-1 mutants was assayed by monitoring glucose remaining in the medium at 5 min, 20 min, and 60 min from cultures of identical biomass.
Figure 8.
Simultaneous deletion of cre-1 and col-26 rescues the phenotype of Δvib-1 on cellulose.
(A) Cellulase activity of culture supernatants after 4-days of growth on Avicel from WT versus Δvib-1, Δcre-1, Δcol-26, Δcre-1; Δvib-1, Δcol-26; Δvib-1 and Δcre-1; Δcol-26; Δvib-1 strains. (B) RT-PCR measurements of clr-2 and cbh-1 expression in the WT versus the Δcol-26, Δcre-1, and the Δcre-1; Δcol-26; Δvib-1 cultures after 5-days of growth on Avicel. Expression levels were normalized to WT. (C) The CMCase activity of Avicel cultures of WT versus Δcol-26, Δcre-1, and the Δcre-1; Δcol-26; Δvib-1 mutants during a time course of growth on Avicel.
Figure 9.
Suppression of cre-1 and col-26 expression by VIB1 plays a role in early inductive and utilization phase during growth on cellulose.
The transcriptional expression of cre-1, vib-1, col-26, clr-2, and cbh-1 were measured by RT-PCR at 4 hrs (A) and 24 hrs (B) after 16 hr sucrose growth cultures were transferred to Avicel conditions. Expression levels were normalized to WT.
Figure 10.
Model for role of VIB1 in regulating glucose sensing/metabolism and CCR under cellulolytic conditions.
In an early encounter of N. crassa to cellulose, “scouting” enzymes induced by carbon starvation act on cellulose to liberate cellulolytic inducers that activate signaling cascades that include activation of CLR1, and subsequent expression of CLR2 and induction of genes encoding cellulases. Efficient cellulolytic induction also requires de-repression of carbon catabolite repression (CCR), as many cellulase genes are not expressed even in the presence of an inducer if a preferred carbon source is available. In the early cellulolytic induction, VIB1 functions to repress CRE1-mediated CCR and glucose sensing/metabolism by COL26, which results in CCR de-repression and productive cellulolytic responses. COL26 also plays a role in repressing cre-1 expression and thus alleviating CCR. As cellulases are produced, glucose is liberated from cellulose and N. crassa transits into the utilization phase, which is associated with reduced transcription of cellulases [44]. Repressive function of VIB1 on col-26 expression may be important for tuning cellular responses for the need to produce sufficient enzymes to liberate simple sugars from cellulose, but without over-activating CCR. VIB1 also likely regulates other factors important for cellulolytic induction.