Tr-milRNA1 Contributes to Lignocellulase Secretion under Heat Stress by Regulating the Lectin-Type Cargo Receptor Gene Trvip36 in Trichoderma guizhouence NJAU 4742

Background: MicroRNA plays an important role in multifarious biological processes by regulating their corresponding target genes. However, the biological function and regulatory mechanism of fungal microRNA-like RNAs (milRNAs) remain poorly understood. Methods: In this study, combined with deep sequencing and bioinformatics analysis, milRNAs and their targets from Trichoderma guizhouence NJAU 4742 were isolated and identified under solid-state fermentation (SSF) by using rice straw as the sole carbon source at 28 °C and 37 °C, respectively. Results: A critical milRNA, TGA1_S04_31828 (Tr-milRNA1), was highly expressed under heat stress (37 °C) and adaptively regulated lignocellulase secretion. Overexpression of Tr-milRNA1 (OE-Tr-milRNA1) did not affect vegetative growth, but significantly increased lignocellulose utilization under heat stress. Based on the bioinformatics analysis and qPCR validation, a target of Tr-milRNA1 was identified as Trvip36, a lectin-type cargo receptor. The expression of Tr-milRNA1 and Trvip36 showed a divergent trend under SSF when the temperature was increased from 28 °C to 37 °C. In addition, the expression of Trvip36 was suppressed significantly in Tr-milRNA1 overexpression strain (OE-Tr-milRNA1). Compared with the wild type, deletion of Trvip36 (ΔTrvip36) significantly improved the secretion of lignocellulases by reducing the retention of lignocellulases in the ER under heat stress. Conclusions: Tr-milRNA1 from NJAU 4742 improved lignocellulose utilization under heat stress by regulating the expression of the corresponding target gene Trvip36. These findings might open avenues for exploring the mechanism of lignocellulase secretion in filamentous fungi.

Effect of Alkali-Treated Birch Sawdust on the Lignocellulase Secretion and Exo-Polysaccharide Production by Inonotus Obliquus Under Submerged Fermentation and its Lignocellulose Degradation Patterns

The objectives of this study were to investigate the medicinal mushroom Inonotus obliquus on the production of polysaccharides and changes of extracellular lignocellulolytic enzymes during submerged fermentation using alkali-treated birch sawdust as substrate. Meanwhile, in order to explore the degradation mode of lignocellulose in alkali-treated birch sawdust, degradation analysis of three components of lignocellulose was carried out. The fungus process in alkali-treated birch sawdust medium resulted in a higher degradation rate of cellulose, hemicellulose, and lignin of 39.24%, 51.00% and 31.3% after 11 days of submerged fermentation by the mycelium of I. obliquus, respectively. Maximal polysaccharide production and α-glucosidase inhibition rate determined in the alkali-treated birch sawdust medium were 6.93 mg/mL and 55.80%,while they were 4.98 mg/mL and 27.89% in the control. Moreover, high activities of Laccase (51.95 IU/mL) , CMCase (1.35 IU/mL) , FPA (0.50 IU/mL) and β-glucosidase (0.55 IU/mL) were observed in alkali-treated birch sawdust medium, respectively. The results demonstrated that the addition of alkali-treated birch sawdust could promote the yield of active polysaccharides and induce the production of cellulase and xylanase, indicating that alkali pretreatment was conducive to utilization of birch sawdust by I. obliquus.