Title : Cellular and molecular studies on b-glucosidases from hypercellulolytic fungus Talaromyces pinophilus
Abstract:
The burning of fossil fuels contributes significantly to environmental deterioration and climate change. On the other hand, cellulose made from agricultural waste is an environmentally benign and renewable resource that absorbs carbon from the atmosphere. The manufacture of biofuels and other biobased products is made possible by its enzymatic conversion into fermentable sugars. This sustainable approach encourages
economic viability and environmental preservation while supporting effective waste usage.
Important cellulases that hydrolyze cellulose into fermentable sugars by cleaving β-1,4-glycosidic bonds are produced by filamentous fungus. These enzymes, which include cellobiohydrolases, endoglucanases, and β- glucosidases, work together to break down lignocellulose into simple sugars. Although Trichoderma reesei is frequently utilized to produce industrial cellulase, effective biomass breakdown is limited by its poor β-
glucosidase activity. Talaromyces pinophilus NCIM1228 was shown to be an excellent producer of β- glucosidase in this investigation. Developing a better strain for effective lignocellulosic biomass hydrolysis could be facilitated by engineering its intrinsic β-glucosidase for increased thermostability, pH tolerance, and glucose tolerance.
Additionally, as CAZymes function synergistically and β- glucosidase (BGLs) are regulated by multiple factors, my research aims to thoroughly investigate these BGLs and look for their interacting partners. Furthermore, my study aims to unravel the regulatory pathways governing the expression of BGLs in T. pinophilus.
Individual β-glucosidase (BGL) genes were removed one at a time for functional assessment, and their contribution to total BGL activity was evaluated. BGL expression was measured using enzymatic assays and confirmed by RT-PCR after the wild-type strain was cultivated on various carbon sources. Avicel (crystalline cellulose) had the highest expression, while glucose had the lowest. EMSA was carried out in both scenarios to look into regulatory mechanisms, and mass spectrometry was then used to find the transcription factors involved.
This work highlights the varied functional roles of specific β-glucosidases by showing that they contribute differently to total BGL activity. Carbon-dependent regulation was confirmed by the considerable influence of the carbon source on BGL expression, which was significantly repressed in glucose and maximally inducted in Avicel. Additional condition-specific transcription factors involved in controlling BGL expression were discovered through molecular studies. All things considered, these results shed light on the regulatory and functional mechanisms of BGLs and lay the groundwork for logical strain engineering aimed at improving the efficiency of biomass hydrolysis and cellulase production.
