A Multiomics Perspective on Plant Cell Wall-Degrading Enzyme Production: Insights from the Unexploited Fungus Trichoderma erinaceum.

Trichoderma erinaceum is a filamentous fungus that was isolated from decaying sugarcane straw at a Brazilian ethanol biorefinery. This fungus shows potential as a source of plant cell wall-degrading enzymes (PCWDEs). In this study, we conducted a comprehensive multiomics investigation of T. erinaceum to gain insights into its enzymatic capabilities and genetic makeup. Firstly, we performed genome sequencing and assembly, which resulted in the identification of 10,942 genes in the T. erinaceum genome. We then conducted transcriptomics and secretome analyses to map the gene expression patterns and identify the enzymes produced by T. erinaceum in the presence of different substrates such as glucose, microcrystalline cellulose, pretreated sugarcane straw, and pretreated energy cane bagasse. Our analyses revealed that T. erinaceum highly expresses genes directly related to lignocellulose degradation when grown on pretreated energy cane and sugarcane substrates. Furthermore, our secretome analysis identified 35 carbohydrate-active enzymes, primarily PCWDEs. To further explore the enzymatic capabilities of T. erinaceum, we selected a ß-glucosidase from the secretome data for recombinant production in a fungal strain. The recombinant enzyme demonstrated superior performance in degrading cellobiose and laminaribiose compared to a well-known enzyme derived from Trichoderma reesei. Overall, this comprehensive study provides valuable insights into both the genetic patterns of T. erinaceum and its potential for lignocellulose degradation and enzyme production. The obtained genomic data can serve as an important resource for future genetic engineering efforts aimed at optimizing enzyme production from this fungus.

Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw.

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of ß-glucosidase, endoxylanase, ß-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.

Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential.

Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.