Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain <i>Trichoderma asperellum</i> T-1

<div><p><i>Trichoderma asperellum</i>, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of <i>Trichoderma reesei</i>QM6a, <i>T. asperellum</i> T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in <i>T. asperellum</i> T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in <i>T. asperellum</i> T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain <i>T. asperellum</i> T-1. All of the above results provided good references for better understanding <i>T. asperellum</i> T-1 and improving its application for lignocellulose degradation.</p></div

Growth comparison of <i>T. asperellum</i> T-1 (right side in a; b; c) and <i>T. reesei</i> QM6a (left side in a; b; c) on PDA both on the obverse side and the reverse side of the plates.

<p>(a) Cultivation for 1 day; (b) Cultivation for 2 days; (c) Cultivation for 5 days.</p

Biomass and glucose consumption when different concentrations of glucose were used as the carbon sources.

<p>(Biomass: 0.5%: black triangle up; 1.0%: black square; 1.5%: black diamond; 2.0%: black hex; glucose consumption: 0.5%: dotted; 1.0%: short-short; 1.5%: dash-dot; 2.0%: long dash). Error bars denote standard deviations from the mean values of triplicate measurements (n = 3).</p

SDS-PAGE of fermented supernatant representing the protein expression differences when different concentrations of glucose were added at 0 h (a), 36 h and 60 h (b).

<p>SDS-PAGE of fermented supernatant representing the protein expression differences when different concentrations of glucose were added at 0 h (a), 36 h and 60 h (b).</p

Cellulase production comparison of <i>T. asperellum</i> T-1 and <i>T. reesei</i> with wheat straw as carbon source, expressed as the concentration of reducing sugar released from the substrate by cellulase in the enzyme reaction system.

<p>(a) Filter paper activity (FPA) of <i>T. asperellum</i> T-1 (black circle) and <i>T. reesei</i> (diamond); (b) CMCase activity (endoglucanase activity) of <i>T. asperellum</i> T-1 (black circle) and <i>T. reesei</i> (diamond). Error bars denote standard deviations from the mean values of triplicate measurements (n = 3).</p

Enzyme production inhibition in <i>T. asperellum</i> T-1 by glucose addition at different growth periods, shown as the reducing sugar yield in the enzyme reaction system both in FPA (A) and CMCase (B).

<p>(A) FPA and (B) CMCase activity of <i>T. asperellum</i> T-1 with glucose added at different final concentrations (w/v) at 0 h (a), 36 h (b) and 60 h (c) of cultivation (0.5%: triangle up; 1%: square; 1.5%: diamond; 2.0%: hex; control: black circle). Error bars denote standard deviations from the mean values of triplicate measurements (n = 3).</p