kretzschmaria zonata: from the genome to the application of the enzyme extract for sugarcane bagasse saccharification
Authors:   , Carmo Mariana Regina Almas 
Published: August 2024

Brazil is the largest sugarcane producer in the world and its production is destined for the sugar and ethanol processing industry, which generates tons of lignocellulosic wastes annually. Considering the global need for energy security and environmental concerns, the agro-industrial wastes use as raw materials to generate value-added products is a promising alternative for the sector. Lignocellulosic residues are rich in polysaccharides such as cellulose and hemicellulose, however, they require prior hydrolysis of their chains to be applied in the second-generation ethanol production process. The biomass hydrolysis process consists of the action of several carbohydrate-active enzymes (CAZymes) and it is considered the highest bottleneck in large-scale production, due to the high costs of commercial enzymes. For this reason, the search for new enzymes or enzymes that are more efficient than those currently commercialized has become the subject of research for decades. Overall, filamentous fungi are known to be good producers of plant cell wall degrading enzymes, but phytopathogenic fungi, which secrete several enzymes to infect the host plant and to obtain the nutrients necessary for their survival, have aroused the interest of researchers. Therefore, the present study aimed to sequence the phytopathogen fungus Kretzschmaria zonata genome, to predict the genes encoding CAZymes, to obtain its sugar metabolic model, to prospect the lignocellulolytic enzymes of this fungus grown on different carbon sources such as wheat bran, corn cob, barley and sugarcane bagasse, and to apply the crude enzymatic extract in the saccharification of two sugarcane bagasse varieties, a commercial variety - Ridesa RB 867515 - and a variety of energy cane - C-90176. The size of the assembled genome was 30.15 Mbp and a total of 12,135 protein-coding genes were identified, with 575 predicted CAZymes. The repertoire of predicted CAZymes was broad and more similar to the profile presented by the filamentous fungus Aspergillus niger, which is a Eurotiomycetes, than with the fungus Trichoderma reesei, which is a Sordariomycetes, as well as K. zonata. This suggests that the K. zonata is an interesting source of enzymes for biotechnology. K. zonata possess a complete metabolic network, except for a gene absence in the galacturonic acid catabolic pathway, which suggests that the fungus can potentially grow on different carbon sources. Maximum cellulolytic activity was obtained for the crude extract obtained after fungal growth on corn cob as carbon source, especially for xylanase, 3.5 U/mL. The extract obtained after growth on wheat bran was the only able to induce all the analyzed enzymes and sugarcane bagasse was the carbon source that induced the lowest diversity of enzymes. For the saccharification experiment, the wheat bran enzymatic extract at a concentration of 2.5 FPU/g of sugarcane bagasse showed the maximum release of glucose and xylose in 96 h, corresponding to 3.1 and 2. 1 g/L, respectively. Thus, these results allowed a better understanding of the enzymatic, metabolic and growth profiles of the fungus K. zonata, which can contribute to a more efficiently application of its enzymes in biotechnology processes. Keywords: Phytopathogenic fungi; Enzimatic profile; Sugar metabolic model; Enzimatic hydrolysis.