| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1045-1 | ||||
Resumo:The microbial fermentation of simple sugars obtained from plant biomass degradation as building blocks for the production of high-added value products is a key step in the circular economy. However, because of the recalcitrance of plant biomass a high amount of carbohydrate-active enzymes (CAZymes) are necessary to obtain monosaccharides. Then, both a mild pretreatment step and partial enzymatic degradation of plant biomass have been suggested as an alternative approach to obtain oligosaccharides instead of monomers from biomass. Filamentous fungi are the major CAZymes producers in nature, and with the implementation of CRISPR/Cas systems is possible to engineer the fungus at the genome level in order to produce customized enzymatic cocktails, which can not only show it is advantageous to the process but also helps to understand the fungal genetic plasticity to adapt to different conditions. The aim of this work was to engineer an Aspergillus niger strain to produce a tailor-made cellulolytic cocktail to partially degrade pretreated sugarcane straw. Initially, A. niger prtT∆ strain was constructed resulting in a mutant with lower extracellular protease production. The prtT∆ background was then used to construct different strains by deleting exoenzyme genes involved in monosaccharide formation such as cellobiohydrolases and beta-glucosidases. The application of the cocktail produced by A. niger bglA∆ resulted in higher production of cellobiose from sugarcane straw compared to the reference strain. To further improve oligosaccharide production, two cellobiohydrolases were deleted additionally, as well as one beta-xylosidase gene, resulting in the Quintuple∆ strain (prtT∆; bglA∆; cbhA∆; cbhB∆; xlnD∆). The secretome of this mutant resulted in higher production of cellotriose and low levels of xylobiose showing that the strategy of deleting exoenzyme genes is an efficient approach to produce tailor-made enzymatic cocktails. The secretome of the mutants and the reference strain were analyzed by mass spectrometry showing that the mutants secrete different CAZyme profiles, employing different strategies to degrade lignocellulosic biomass. Interestingly, other BGLs and CBHs were secreted in order to compensate for the lack of the deleted enzymes (bglA, cbhA, cbhB). Palavras-chave: CRISPR/Cas9, Aspergillus niger, CAZymes, Cello-oligosaccharides, secretome engineering Agência de fomento:FAPESP, CAPES, CNPq, FAEPEX/PRP/UNICAMP | |||||