Myceliophthora thermophila as a chassis to the development of an efficient cell factory to secrete enzymes active in lignocellulosic materials

• Grant number: 15/08079-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): June 01, 2015
• Effective date (End): December 31, 2015
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Fernando Segato
• Grantee: Matheus Augusto Raimundo Nogueira da Silva
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil
• Associated research grant: 14/18714-2 - Enzymatic oxidation of sugarcane bagasse: discovery, characterization and new application of oxidative enzymes active in carbohydrates, applied to the enhancement of a fungal cell factory, AP.BIOEN.JP

Abstract

The plant cell wall polysaccharides are an abundant source as renewable raw material and can be used to produce biofuels and other compounds. The enzymatically conversion of these polysaccharides into fermentable sugars still is a slow and high cost process; therefore, a fast and efficient process must be developed. Nowadays, the commercial cocktails had only enzymes whose hydrolysis mechanisms for the lignocellulose compounds are acid-base, which are not much efficient to lysate the crystalline cellulose and lignin. Recently were discovered enzymes that had oxidative mechanisms, which work as auxiliary enzymes, acting directly on the crystalline cellulose, named LPMOs. These enzymes were then added to the available cocktails, improving their performance. Analyzing the transcriptome and secretome of the thermophilic fungus Myceliophthora thermophila grown on sugarcane bagasse, we verified that these microorganism has the hydrolytic and oxidative mechanisms for lignocellulose degradation. Despite the presence of both mechanisms, verified on the experiments that the capacity that M. Thermophila presented on using the compounds of the plant cell wall, when this was grown on sugarcane bagasse in natura as the only nutrients source, is quite lower in comparison with the white rot fungus Ceriporiopsis subvermispora, although the protein secretion machinery of the M. thermophila is more robust. Comparing the genome of M. thermophila and C. subvermispora, was verified on the first one the absence of enzymes, like peroxidases, which are involved on the degradation and modification of lignin, through what we conclude that the secretion of such enzymes on C. subvermispora are the differential for the access of the carbohydrates contained on the biomass. For the development of a more efficient cell factory, not only for the production of enzymes of interest, as also on the biomass degradation, this project aim: 1) The creation of a M. thermophyla mutant with auxotroph on uracil and uridine; 2) Develop a vector for M. thermophyla, which will use a constitutive promoter, using Gibson Assembly Method; 3) Transform M. thermophyla with a manganese peroxidase of C. subvermispora, for combining the synergic oxidative and hydrolytic actions of the both microorganisms in a single cell with thermophilic characteristics and study the effects of these complementation on the degradation of lignocellulosic biomass. (AU)