Mechanism On Laccase Induction During The Interspecific Interaction Between White Rot Fungi

Laccases belong to broad group of enzymes called polyphenol oxidases containing multi copper or iron atoms, its objective substrates represent extensive, unspecific, and it can degrade phenolic and non-phenolic compounds even highly recalcitrant environmental pollutants. Laccases can be applied widely, including paper and pulp industry, dye and textile industry, brewing industry, and have also been used in biomass energy development of biomass pretreatment to remove lignin. Laccases widely exist in plants and fungi and have also been found in bacteria and insects. So far the most studied laccase is fungal laccase secreted by white-rot fungus, it can be induced by interspecific interaction or exogenous compounds, but the induction mechanism is not clear yet. Making clear the mechanism of laccase induction by interspecific interaction can provides the basic knowledge for production of laccase, and can also be benefit for analyzing the mechanism of fungal metabolic regulation when encountering the fungal interaction.This article has selected five typical white rot fungi as the laccase producers, and built and optimized the system of laccase interspecific induction between white rot fungi, and to further verify the mechanism of the laccase-inductive phenomena by analysis of biochemical and physiological behaviors, transcript and metabolic. The main results are as follows: 1. We have screened Trametes versicolor(TV), Pleurotus ostreatus(PO) and Dichomitus squalens(DS) from 5 fungi strains by arbitrary two-species combination co-cultured on the plate, and assayed the laccase activity. Eventually, the laccase activity of TV vs DS combination were 150 folds and 520 folds higher than each strain of TV and DS; TV vs PO combination were 100 folds and 50 folds higher than each strain of TV and PO; PO vs DS combination were 20 folds and 200 folds higher than each strain of PO and DS. We have verified the exogenous compounds, such as Cu2+ different aromatic compounds and lignin derivatives, indicating that each of them has a strong ability of laccase induction. And preliminary explored the effect of different carbon and nitrogen sources on laccase induction. The results revealed that fructose and L-aspargine are the best carbon and nitrogen source for laccase production. Besides, the components of herbaceous plant better than woods for white rot fungi to produce laccase.2. We have confirmed that the hyphae physical contact is not necessary for the laccase interspecific induction by separating the strains with a semipermeable membrane in the co-culture system. And then, the extracellular secretions from the interaction area were verified to have the laccase inductive ability. But extracellular secretions form pure cultivation has no remarkable inductive ability.3. We have found that a series of regulations have taken place in the interactive area compared to each side on biochemical level. When co-cultivated two stains, cells in a state of defense and attack, participated in cells attack, such as hydrolytic enzymes, antibacterial compounds, etc, participated in cells defense, such as hydrophobic protein, REDOX enzyme and some cytochrome, etc, all of them have remarkable increase expression in the level of genetic and metabolic. Carbohydrate, lipid and amino acid metabolism and other metabolic pathways of related gene upregulated, which mean they provide cells with energy and material basis.By overlapping the transcriptome with metabolic changes, the mechanisms of the laccase induction between white rot fungi are as follows:when encountering the interspecific interaction, cellular receptors recognized the heterogeneous cells, and switch on its offensive system, and then produce ROI, RNI, VOCs and some stimulating metabolites which are harmful to cells’growth and development. As a response, the heterogeneous cells switch on its defensive system by producing a lot of laccases, because responsive elements exist in the laccase promoter region involve in these stress responses. Besides, a large number of hydrolases damaging cell’s integrity and interspecific competition for nutrients are also the reasons for the laccase induction. This study provides evidences for explore the mechanism of laccase induction oh the molecular level, and laid the foundation of further identification of the key factor of white-rot fungus laccase induction and the metabolic regulation network. Meanwhile, this article provides reference for studying the microbial interactions.