| Congresso Brasileiro de Microbiologia 2023 | Resumo: 700-1 | ||||
Resumo:The thermophilic fungus Thermothelomyces thermophilus (previously Myceliophthora thermophila) has in its genome a significant number of genes encoding plant cell-wall-degrading enzymes. Since T. thermophilus can be a great source of thermostable proteins involved in the depolymerization of lignocellulosic materials different enzymes have been identified and produced from this fungus. The conversion of lignocellulose into its subunits is a limited step therefore, more studies are needed to improve this process. Recently, oxidative proteins that act directly on lignocellulosic biomass have been discovered and classified as lytic polysaccharide monooxygenases (LPMOs). Filamentous fungi contain large number of genes that codify to LPMOs as T. thermophilus which contains 23 codifying genes to LPMOs from the family AA9 (Auxiliary activity enzymes) and 3 codifying genes to LPMOs from the family AA16. Since T. thermophilus shows this considerable number of LPMOs in the genome and only a small number of them are commonly expressed, heterologous expression is important tool to over express and characterize these enzymes for their biotechnological application. This study aims to amplify and purify the LPMO’s codifying genes from T. thermophilus to be further introduced in Escherichia coli and over express in Aspergillus nidulans A773. Firstly, genes were amplified using specified oligonucleotides for each gene by PCR using T. thermophilus DNA as template. The PCR product was purified using the Wizard SV Gel and PCR Clean-Up System kit. Fragments of the genes were cloned into the plasmid pEXPYR by Gibson Assembly, and the plasmids were expanded in E. coli cells. Positive colonies were confirmed by PCR and cultivated on LB medium containing ampicillin. Colonies were incubated overnight and then, centrifugated to recover the pellet (cell biomass). DNA was extract from the positive colonies using the Wizard Plus SV Minipreps DNA purification system kit. Purified DNAs were introduced into A. nidulans by PEG mediated transformation. Positive colonies were confirmed by the identification of the LPMOs corresponding bands in SDS-PAGE. From the 23 LPM’s codifying genes of the family AA9, 15 were successfully cloned into E. coli from which 8 were introduced in A. nidulans with success. In the case of the LPMO’s codifying genes from family AA16, all of them were successfully introduced in E. coli and A. nidulans. Palavras-chave: Thermothelomyces thermophilus, Heterologous expression, Lytic polysaccharide monooxygenase, Filamentous fungi, Aspergillus nidulans Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2022/04713-0, 2021/06679-1 and 2022/04227-9. | |||||