Periodic Reporting for period 1 - FuelingLPMO (LPMO regulation by (poly)phenolics-active enzymes in Ascomycota)
Período documentado: 2021-10-01 hasta 2023-09-30
This project addresses the challenge of converting lignocellulosic biomass, e.g. non-edible agro-industrial wastes and wood, into sustainable biofuels, green chemicals and materials via enzyme technology. A class of microbial enzymes termed lytic polysaccharide monooxygenases (LPMOs) plays a central role in the depolymerization of polysaccharides that compose lignocellulosic biomass, such as cellulose, in Nature and in industrial settings. However, the exact mechanism employed by LPMOs to depolymerize plant polysaccharides is still not fully understood. New knowledge on the LPMO reaction mechanisms and its interplay with other oxidative enzymes involved in the degradation of plant biomass is required to provide the basis for the development of efficient enzyme technologies for lignocellulosic biomass refining. Enzyme-based biorefineries will have important role in the transition from a fossil-based economy to a more sustainable biomass-based economy. The overall objective of this research project is to contribute with a more detailed understanding of the network of enzyme-catalyzed redox reactions involved in lignocellulose degradation by fungi, building on the hypothesis that such understanding can be used to design powerful enzyme systems for efficient biomass conversion into bioproducts.
This project generated important pieces of evidence corroborating the paradigm that peroxygenase dominates over monooxygenase activity in lytic polysaccharide monooxygenase (LPMO) reactions on cellulose. The project led to the discovery that diphenolic compounds (dihydroxybenzenes) generated by the activity of fungal short polyphenol oxidases (PPOs) on lignin-derived phenols can prime but not fuel LPMO reaction and strongly indicates that H2O2 is necessary for cellulose degradation by LPMO.
The mechanism behind the role of PPOs as partner enzymes for LPMOs on cellulose degradation was investigated in depth using MtPPO7 and NcAA9C as a model enzymes and guaiacol as a model lignin-derived compound. It was demonstrated that MtPPO7 can prime NcAA9C by converting guaiacol (unable to activate NcAA9C) into 3-methoxycatechol, which can efficiently reduce the copper ion in the active site of NcAA9C. MtPPO7 catalytic products, however, cannot fuel LPMO reaction on cellulose since no H2O2 is generated in such a cascade reaction system. MtPPO7 do not generate H2O2 and MtPPO7 catalytic products do not generate H2O2 by auto-oxidation nor induce LPMO to generate H2O2 (via oxidase side-activity). The addition of exogenous hydrogen peroxide (H2O2) is required for LPMO activity. The discovery that 3-methoxycatechol (and other diphenolic compounds produced by PPOs) down to stoichiometric amounts (relative to LPMO concentration) can prime but not fuel LPMO reactions corroborates the paradigm that peroxygenase activity dominates over monooxygenase in LPMO reactions. The discovery of LPMO-priming agents with a low propensity to generate H2O2 offers a method for managing LPMO catalysis by controlled H2O2 supply, thus reducing the risk of enzyme inactivation typically observed when excess H2O2 accumulates in the reaction.
One peer-reviewed paper was published in ChemSusChem journal with Open Access (https://doi.org/10.1002/cssc.202300559(se abrirá en una nueva ventana)) and one short article was published in Dansk Kemi, a popular science magazine for chemists and chemical engineers in Danish language (https://ipaper.ipapercms.dk/TechMedia/DanskKemi/2023/?page=22(se abrirá en una nueva ventana)).
The mechanism behind the role of PPOs as partner enzymes for LPMOs on cellulose degradation was investigated in depth using MtPPO7 and NcAA9C as a model enzymes and guaiacol as a model lignin-derived compound. It was demonstrated that MtPPO7 can prime NcAA9C by converting guaiacol (unable to activate NcAA9C) into 3-methoxycatechol, which can efficiently reduce the copper ion in the active site of NcAA9C. MtPPO7 catalytic products, however, cannot fuel LPMO reaction on cellulose since no H2O2 is generated in such a cascade reaction system. MtPPO7 do not generate H2O2 and MtPPO7 catalytic products do not generate H2O2 by auto-oxidation nor induce LPMO to generate H2O2 (via oxidase side-activity). The addition of exogenous hydrogen peroxide (H2O2) is required for LPMO activity. The discovery that 3-methoxycatechol (and other diphenolic compounds produced by PPOs) down to stoichiometric amounts (relative to LPMO concentration) can prime but not fuel LPMO reactions corroborates the paradigm that peroxygenase activity dominates over monooxygenase in LPMO reactions. The discovery of LPMO-priming agents with a low propensity to generate H2O2 offers a method for managing LPMO catalysis by controlled H2O2 supply, thus reducing the risk of enzyme inactivation typically observed when excess H2O2 accumulates in the reaction.
One peer-reviewed paper was published in ChemSusChem journal with Open Access (https://doi.org/10.1002/cssc.202300559(se abrirá en una nueva ventana)) and one short article was published in Dansk Kemi, a popular science magazine for chemists and chemical engineers in Danish language (https://ipaper.ipapercms.dk/TechMedia/DanskKemi/2023/?page=22(se abrirá en una nueva ventana)).
These results provide new knowledge that can be exploited for the development of novel enzyme technologies for the biomass refining industry. This can have implications in the development of the biomass-based circular economy concept and is in line with the current global demand for biomass-derived next-generation fuels, chemicals and materials.