A novel luciferase-based reporter tool to monitor the dynamics of carbon catabolite repression in filamentous fungi

Abstract

Filamentous fungi with their diverse inventory of carbohydrate-active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE-1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real-time evaluation of CreA/CRE-1-mediated CCR at the transcriptional level, we developed a luciferase-based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE-1-dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE-1-mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours.

FIGURE 1

Construction and comparison of Cre‐dependent CCR reporter with different numbers and types of Cre‐binding sites in Aspergillus niger. A macroscopic luciferase assay was performed for biomass grown on solid medium. The 4× identical Cre‐binding site luciferase reporter construct as well as the spacer construct containing no Cre‐binding sites and the WT strain for control were compared in their luminescence emissions upon 50% glucose induction. (A) Pictures of the luminescence signal were taken after 2.5 h of induction with doxycycline and D‐luciferin in aMM without carbon source and 1.5 h after glucose induction. (B) Illustration depicts the constructed variants of the Cre reporter (created with BioRender.com) tested in (A). Luciferase microtiter plate assay was performed for six different luciferase reporter constructs (C–H) with 2, 4 or 8 alternating and identical Cre‐binding sites. After 6 h of induction with doxycycline and D‐luciferin in aMM without carbon source, different concentrations of glucose (0.01%, 0.03% and 0.5%) were added. Luminescence values were measured after 3.3 h of glucose induction and normalized to the luciferase value under starvation condition with 0.1% glycerol. The WT control strain was used as blank. Different letters indicate statistically significant differences according to an ANOVA test (n = 3), p < 0.05. The strains were tested by inoculation of one transformant in three biological replicates.

FIGURE 2

Comparison of Aspergillus niger and Neurospora crassa reporter strains upon different monosaccharide inductions. Luciferase microtiter plate assay was performed for A. niger (A) and N. crassa (B) transformed with the 8× alternating Cre‐binding site reporter upon starvation and induction with 0.5% glucose. The assay was also performed for A. niger (C) and N. crassa (D) upon starvation and induction with 0.5% fructose, arabinose, xylose, rhamnose and galacturonic acid. The induction condition with 0.5% glucose was also included as a comparison. After 1.5 h/45 min of starvation in medium with doxycycline and D‐luciferin in aMM without carbon source, different monosaccharides were added (dotted line; t = 0). Luminescence values were measured, normalized to the highest measured luciferase value under starvation conditions and blanked with the WT control strain (n ≥ 3). The strains were tested by inoculation of one transformant in three biological replicates. The dash‐dotted line at 4.5 h post‐induction marks a point with relatively stable luminescence values.

FIGURE 3

Phenotypic observation, 2‐DG and CreA luciferase reporter assay comparing Aspergillus niger truncation strains. Comparison of CreA truncation constructs is depicted in (A). Growth and sporulation of A. niger WT, CreA truncation strains CreA(1–345), CreA(1–281), CreA(1–126), CreA(1–115) and ΔcreA on aMM + 2% glucose were compared after 4 days of incubation at 30°C (B). Different letters indicate statistically significant differences according to an ANOVA test (n = 3), p < 0.05. Strains were generated by CRISPR/Cas9‐mediated truncations of the native CreA locus, and three biological replicates per strain were tested. Growth diameter of A. niger WT, CreA truncation strains CreA(1–345), CreA(1–281), CreA(1–126), CreA(1–115) and ΔcreA were compared upon CCR induction with 20 mM 2‐DG on 1% xylose after 7 days of incubation at 30°C (C). The diameters of three colonies per strain (n = 3) were measured and the average was calculated. Luciferase microtiter plate assay was performed for A. niger WT, CreA truncation strains CreA(1–345), CreA(1–281), CreA(1–126), CreA(1–115) and ΔcreA (D–I) transformed with the 8× alternating Cre‐binding site reporter upon starvation and induction with 0.5% glucose. After 1.5 h of induction with doxycycline and D‐luciferin in aMM without carbon source, 0.5% of glucose was added (dotted line). Luminescence values were measured, normalized to the highest luciferase value under starvation conditions and blanked with the WT control strain (n ≥ 3). The strains were tested by inoculation of one transformant in three biological replicates. The dash‐dotted line at 4.5 h post‐induction marks a point with relatively stable luminescence values.

FIGURE 4

CreA luciferase reporter assay in the strain carrying the deletion of the conserved region in CreA. Luciferase microtiter plate assay was performed for Aspergillus niger CreA × (Δ281–345) deletion strain (A) transformed with the 8× alternating Cre‐binding site reporter upon starvation and induction with 0.5% glucose. After 1.5 h of starvation in medium with doxycycline and D‐luciferin in aMM without carbon source, 0.5% of glucose was added (dotted line). Luminescence values were measured, normalized to the highest luciferase value under starvation condition and blanked with the WT control strain (n ≥ 3). The strains were tested by inoculation of one transformant in three biological replicates. The dash‐dotted line at 4.5 h post‐induction marks a point with relatively stable luminescence values. Schematic representation of the CreA deletion strain [CreA × (Δ281–345)] (B). The strain was generated upon CRISPR/Cas9‐mediated deletion of the conserved region.

FIGURE 5

Comparison of Aspergillus nidulans reporter and control strain upon glucose induction. Luciferase microtiter plate assay was performed for three replicates of A. nidulans strains transformed with the 8× alternating Cre‐binding site reporter (A–C) and the control with a spacer sequence (D–F) upon starvation and induction with 0.5% glucose. After 1.5 h of starvation in medium with doxycycline and D‐luciferin in aMM without carbon source, 0.5% of glucose was added. Luminescence values were measured 4.5 h after induction, normalized to the luciferase value under starvation conditions and blanked with the WT control strain (n ≥ 3). Three transformants per construct (A–C, D–F) were tested by inoculation in three biological replicates each. * and ** indicate statistically significant differences according to an ANOVA test, p < 0.05; p < 0.01.