Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose

Abstract

Background: Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose.

Results: We found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR.

Conclusion: Trichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.

Keywords: Alpha-tubulin; Biomass saccharification enzyme; Carbon catabolite repression; Cellulase; Glucose resistant; Hemicellulase; Trichoderma reesei.

Fig. 1

Secretory protein production by the aabgl1 recombinant strains and parent strain PC-3-7. Fermentation was conducted using 10% (w/v) microcrystalline cellulose and 2% (w/v) beechwood xylan as carbon sources. Data are expressed as mean ± SD of three biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. *p < 0.05

Fig. 2

Colony morphology of PC-3-7 and PC-3-7ΔtubB. a Colonies of PC-3-7 and PC-3-7ΔtubB formed on potato dextrose agar (PDA) plate at 30 °C for 3 days. Scale bar indicates 10 mm. b Colony size of PC-3-7 and PC-3-7ΔtubB incubated on PDA plate. Data are expressed as mean ± SD of three biological replicates

Fig. 3

SEM images of PC-3-7 and PC-3-7ΔtubB. SEM images of T. reesei PC-3-7 (a and c) and PC-3-7ΔtubB (b and d) mycelia grown in liquid culture containing 1% (w/v) glucose for 1 day (a, b) and 1% (w/v) cellulose for 3 days (c, d). Scale bar indicates 10 µm. White arrows indicate the rounded hypha

Fig. 4

Secretory protein production by the tubB-deficient strains and parent strain PC-3-7. a Time course of the amount of secretory protein. Fermentation was conducted using 10% (w/v) microcrystalline cellulose and 2% (w/v) beechwood xylan as carbon sources. Data are expressed as mean ± SD of three biological replicates. b Amount of secreted protein at the end of cultivation. Data are expressed as mean ± SD of three biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. *p < 0.05

Fig. 5

Effects of glucose on protein production, respiration, and sugar uptake of PC-3-7 and PC-3-7ΔtubB. a Secretory protein production, d the concentration of CO₂ in the exhaust gas and f glucose and cellobiose concentration when cultivated in medium with 10% (w/v) microcrystalline cellulose. b Secretory protein production, e the concentration of CO₂ in the exhaust gas and g glucose and cellobiose concentration when cultivated in medium with 10% (w/v) microcrystalline cellulose and 2.5% (w/v) glucose. Blue lines indicate PC-3-7 and orange lines indicate PC-3-7ΔtubB. c Cellulolytic and hemicellulolytic enzyme activities in the culture supernatant after 120 h of cultivation, including pNPGase (the BGL activity), pNPLase (the CBH1 activity), CMCase (the EG activity), pNPXase (the BXL activity), pNPX2ase and xylanase (the XYN activity). In f and g, solid lines indicate glucose and dashed lines indicate cellobiose. Data are expressed as mean ± SD of three biological replicates. For secretory protein production, statistical significance was determined by two-tailed unpaired Student’s t-test. **p < 0.01. The arrows in c and e indicate the time at which cells were collected for RNA-seq analysis

Fig. 6

Normalized expression of major CAZymes in PC-3-7 and PC-3-7ΔtubB. Blue bars indicate PC-3-7 and orange bars indicate PC-3-7ΔtubB. C, cultivated with cellulose; C + G, cultivated with cellulose and glucose. Data are expressed as mean ± SD of three biological replicates

Fig. 7

Scatter plots of gene expression profiles. Scatter plots between PC-3-7_48h_C and PC-3-7_48h_C + G (a) and ΔtubB_48h_C, ΔtubB_48h_C + G (b), PC-3-7_48h_C and ΔtubB_48h_C, and PC-3-7_48h_C + G and ΔtubB_48h_C + G. The x- and y-axis represent RPKM value of all CDSs. The DEGs identified in DEseq analysis are highlighted in orange color

Fig. 8

Sugar consumption by PC-3-7 and PC-3-7ΔtubB Blue lines indicate PC-3-7 and orange lines indicate PC-3-7ΔtubB. a Glucose concentration after 0.2% (w/v) glucose was added. b Xylose concentration after 0.2% (w/v) xylose was added. c Glucose and cellobiose concentrations in the supernatant after 0.2% (w/v) cellobiose was added. Solid lines indicate glucose concentration and dashed lines indicate cellobiose concentration. d Glucose and cellobiose concentrations in the supernatant after 0.2% (w/v) glucose and 0.2% (w/v) cellobiose were added. Solid lines indicate glucose concentration and dashed lines indicate cellobiose concentration. Data are expressed as mean ± SD of three biological replicates