Characterization of some industrially relevant polysaccharide monooxygenases and related enzymes

INTRODUCTION AND AIMS By 2030, global rice production is expected to increase to meet the demand of a growing world population. However, rice is severely affected by blast disease, caused by the fungus Magnaporthe oryzae, which can reduce total annual rice production by 10-30% and, if not controlled, can cause complete loss of production. In the early stages of the infection process, M. oryzae forms an infection structure called appressorium to break the plant cuticle and it expresses many polysaccharide and lignin degrading enzymes: among them, polysaccharide monooxygenases (PMOs) degrade polysaccharides by an oxidative mechanism and could be important virulence factors for the fungus. The first objective of the project is to identify M. oryzae PMOs and related enzymes active on polysaccharides and lignin essential for pathogenesis on rice. A screening of natural molecules or inhibitor proteins will then be performed to identify those effective in inhibiting the essential fungal enzymes, thus reducing growth and infection rate of M. oryzae. The final aim of this objective is to develop new methods to control rice blast disease in order to increase food production. Rice straw is the main by-product of rice-producing areas and is a potential resource for biofuel production. Worldwide, about 400 million tons of rice straw are annually produced, with about 30-40 million tons/year in Vietnam alone, but more than 95% is burned, resulting in hazardous airborne emissions. Several polysaccharide and lignin-degrading enzymes like PMOs have been applied in the biofuel industry. When mixed to the typical cellulose-hydrolyzing enzymes, PMOs could dramatically change the enzyme technology involved in biomass degradation, reducing biofuel production costs. However, the activity of PMOs on rice straw needs to be further characterized. The second objective of the project is to heterologously express the M. oryzae PMOs and related enzymes to characterize their enzymatic activities on polysaccharides and lignin, with the aim of developing new enzyme mixtures for rice straw degradation to be used in biofuel production. Aim 1: Characterization of the pathogenetic mechanism of Magnaporthe oryzae to develop new rice blast disease control measures Candidate M. oryzae genes encoding PMOs and related polysaccharides and lignin hydrolyzing enzymes have been identified by an in silico analysis of the fungal genome, completely sequenced. Their expression during the infection process, and particularly during appressorium formation, have been characterized by transcriptomic analysis. Reverse genetics approach: the knock-outs of the most expressed genes (two PMOs, two beta-1,3-glucanases, one ligninase, one chitin synthase and one chitinase) are in progress by targeted homologous recombination. Screening of mutants: infection of rice seedlings and plants will be performed to determine the contribution of the fungal enzymes of interest to fungal virulence. The ability of mutants to form appressorium will also be observed by light microscopy. Natural bioactive compounds and protein inhibitors potentially inhibiting the M. oryzae enzymes shown to play important roles for fungal virulence will be identified by in silico simulation of protein structures. A screening by in vitro bioassays for their ability to inhibit or reduce M. oryzae growth and its virulence on rice seedlings and plants will then be performed. Bioassays will also be performed to evaluate the inhibition potential of bioactive compounds and protein inhibitors on appressorium formation. Effective inhibitors of the target enzymes will then be tested on rice field trials. Rice transgenic plants expressing proteins able to inhibit some target enzymes of M. oryzae will be also created. Aim 2: Application of Magnaporthe oryzae PMOs and related enzymes to rice straw degradation and biofuel production Polysaccharide monooxygenases (PMOs) are enzymes secreted by a variety of fungal and bacterial species. They have recently been found to degrade recalcitrant polysaccharides, including chitin, cellulose and starch with an unprecedented oxidative mechanism. Polysaccharides are known to be degraded by hydrolytic enzymes, termed collectively as glycoside hydrolases (GHs). The available structures of PMOs reveal a conserved fold and a highly conserved monocopper active site on a flat protein surface. PMOs likely act directly on the substrate surface, bypassing the energy-intensive step of removing polysaccharide chains from the insoluble substrates required in GHs. The new ends created by PMOs can be subsequently processed by various GHs, including exo- and endo-glucanases, resulting in enhanced overall polysaccharide degradation. Genes encoding for M. oryzae PMOs and related polysaccharides and lignin degrading enzymes, previously identified by transcriptomic analysis, will be obtained from M. oryzae RNA from infected rice tissues and the corresponding enzymes of interest will be heterologously expressed using the Pichia pastoris yeast system. The recombinant PMOs will then be purified and characterized at their mononuclear copper active site by UV/Vis, rRaman, Electron Paramagnetic Resonance (EPR) and X-Ray Absorption (XAS). The recombinant enzymes will also be purified at a larger scale (pilot scale) and tested for their enzymatic activity by bioassays performed on different substrates. Since PMOs, like other glycoside hydrolases, have different activities on different types of materials, the purified recombinant enzymes will be tested alone or in mixture in order to optimize the digestion of rice straw. In addition, optimization of ratios of M. oryzae PMOs and commercialized polysaccharide hydrolysis enzymes for degradation of rice straw will be performed. The final aim is the development of a novel rice straw degrading enzyme technology. PERSPECTIVES The expected results, that is the characterization of the role and enzymatic activities of M. oryzae PMOs and related enzymes in order to identify new methods to control rice blast disease and to develop new enzyme mixtures for rice straw degradation and biofuel production, will be exploited for production of scientific publications, dissemination purposes and transfer of new agricultural and industrial technologies. In particular, the PMOs or polysaccharide hydrolase enzymes identified in the project will be tested at the trial scale at bioethanol production factories; the bioactive compounds identified in the project and able to inhibit or control rice blast disease will be applied in rice field trials. The expected impact and benefits of these activities are enhancing the use of rice straw for bioethanol production, with consequent reduction of dependence on fossil fuels and rice straw burning, and the effective inhibition or control of the rice blast disease, with consequent increase of rice productivity and improvement of rice farmers’ economic conditions. Another expected result of the proposed research is training several students: in particular, visits at the Vietnam and Italian laboratories will enable the training of young researchers and the future development of new research objectives aimed at improving the agricultural and industrial systems of both countries.