Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi

Abstract

It is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available for Neurospora crassa to investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCE In fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.

Keywords: Neurospora crassa; cellulose/hemicellulose signaling; competitive inhibition; filamentous fungi; plant cell wall degradation.

FIG 1

Characterization of GH2-1. (A) CMCase activity of enzymes secreted into WT culture supernatants after 3 days of growth in 1% (wt/vol) powdered biomass (Miscanthus [M], chestnut [Ch], oak [O], locust [L], pine [P], cedar [Ce], spruce [S], and fir [F]). (B) Substrate specificity assay of GH2-1 using ρNP-β-d-mannopyranoside (ρNP-β-Man), ρNP-β-d-cellopyranoside (ρNP-β-CB), ρNP-β-d-glucopyranoside (ρNP-β-Glc), and ρNP-α-d-mannopyranoside (ρNP-α-Man) as the substrates. (C) Contour plot for GH2-1 activity, at different combinations of temperatures and pHs in parallel, using ρNP-β-Man as the substrate. (D) β-Mannopyranosidase activity of the cytosolic protein extracts of the WT, Δgh2-1, Δ3βG, and Δqko (the Δ3βG strain crossed to Δgh2-1) strains after growth in 1% (wt/vol) Avicel with 1× Vogel’s salts for 3 days. The bars and lines in the bar and line graphs, respectively, in the figures are the mean values of the biological replicates, and error bars in all figures are standard deviations (SDs) (n = 3). Different uppercase letters indicate differences within data groups that are significantly different (Tukey test, P values of <0.05 were considered significant).

FIG 2

High mannan content is inhibitory for cellulase activity. (A) CMCase activity of enzymes secreted into the Δgh2-1 culture supernatants after 3 days of growth in 1% (wt/vol) powdered biomass (Miscanthus [M], chestnut [Ch], oak [O], locust [L], pine [P], cedar [Ce], spruce [S], and fir [F]). The inhibition is indicated as a percentage relative to the inhibition on Miscanthus (uninhibited; 0%) and fir (highest inhibition; 100%), and the mannan content is calculated from the compositional analysis of the biomass sources used (see Fig. S1A in the supplemental material). (B) CMCase activity of the WT and Δgh2-1 cultures after growth in 1% (wt/vol) bacterial cellulose (BC) with the addition of 0.03% (wt/vol) glucomannan (GM) and mannan (Mn) or mannobiose (MB). (C) 2D-[¹H¹³C]-HSQC spectra for the anomeric region of the extracted intracellular sugars of the mycelia of both WT and the Δgh2-1 strains after growth in 2% (wt/vol) Avicel for 24 h after transfer. β-Glcp, glucose as part of β-1,4-polymer; β-Manp, mannose as part of the β-1,4-polymer; α/β-ManpR, reducing end α/β-mannopyranosyl. (D) CMCase activity of the WT, Δgh2-1, and gh2-1-comp cultures after growth in 1% (wt/vol) Avicel with 1× Vogel’s salts for 3 days. Different lowercase and uppercase letters indicate differences within data groups that are significantly different (Tukey test, P values < 0.05 were considered significant).

FIG 3

Cello- and mannodextrins compete intracellularly, and the inhibition is independent of CCR by CRE-1. (A) CMCase activity of culture supernatants of the indicated strains after growth for 3 days in 1% (wt/vol) Avicel. (B) Viscosity of the culture supernatant of the indicated strains 8 h after transfer to 1% (wt/vol) glucomannan. Different lowercase and uppercase letters indicate differences within data groups that are significantly different (Tukey test, P values < 0.05 were considered significant).

FIG 4

Cello- and mannodextrins compete at the level of sugar uptake. (A) Mycelial weight (dry weight) of the indicated strains after growth for 3 days in 1% (wt/vol) glucomannan (indicated as a percentage of the weight of the WT). (B) Residual mannobiose in the supernatant of the indicated strains at the indicated times after transfer to the uptake solution (100 μM mannobiose). (C) Residual sugars in the culture supernatants of S. cerevisiae heterologously expressing CDT-1 or CDT-2 transporters, 30 min after transfer to the 100 μM uptake solutions (cellobiose [CB] or mannobiose [MB], or both disaccharides simultaneously). Different lower- and uppercase letters indicate differences within data groups that are significantly different (Tukey test, P values < 0.05 were considered significant).

FIG 5

Mannan addition is inhibitory to cellulase production in T. reesei and M. thermophila as well. (A and B) CMCase activity of culture supernatants of M. thermophila WT strain (A) and N. crassa WT and T. reesei RUT-C30 (B) after 3 days growth in 1% (wt/vol) Emcocel with or without the addition of 0.05% (wt/vol) glucomannan (GM). Different lower- and uppercase letters indicate differences within data groups that are significantly different (Tukey test, P values < 0.05 were considered significant).

FIG 6

A model of the induction (A), inhibition (B), and relief of inhibition (C) of cellulase production in N. crassa. After the degradation of cellulose and glucomannan by cellulases (endo- and exo-acting glucanases [orange]) and mannanase (blue), respectively, (gluco)mannodextrins outcompete cellodextrins extracellularly at the level of transport by the MFS-type transporter CDT-1. Intracellularly, cellodextrins and (gluco)mannodextrins are further cleaved into the corresponding glucose and mannose monomers by the action of the intracellular β-glucosidase (GH1-1) and β-mannosidase (GH2-1), respectively. (A) In the case of an intracellular balance between cello- and mannodextrins, an unknown signaling cascade will lead to the activation of the upstream transcription factor CLR-1, which induces expression of the downstream transcription factor CLR-2, which then evokes the major cellulolytic and mannanolytic responses. (B) In the Δgh2-1 deletion strain, undigested (gluco)mannodextrins accumulate in the cytosol, disrupting the intracellular balance of signaling molecules and outcompeting the positively inducing cellodextrins, in a way that the fungus is unable to determine the “adequate” amount of cellulase enzymes to be produced, eventually causing a reduced cellulase production. (C) When gh1-1 is deleted in the Δgh2-1 background (Δgh2-1 Δgh1-1 strain), the accumulating mannodextrins can be counterbalanced by the larger amount of undigested cellodextrins present in the cytosol, which reinforce the induction of the cellulolytic response and relieve the inhibition.