Lytic Polysaccharide Monooxygenases Drive Lignin Degradation By Generating Reactive Oxygen Species

Lytic polysaccharide monooxygenases are widely distributed and abundant in genomes of fungi and bacteria.When an exogenous electron donor and oxygen is present,the active center of LPMO can form a Cu-oxygen intermediate,oxidatively breaking cellulose or hemicellulose.However,the lignin resistance barrier usually limits the binding of the enzyme to the polysaccharide.When the binding of LPMO to the polysaccharide is blocked,the Cu-oxygen intermediate will release the active oxygen and produce H₂O₂,which is a key factor in the biodegradation of lignin.If LPMO could involved in lignin degradation,it is of great significance for the lignin biodegradation and the remove of limiting factors of carbon cycle.However,the relationship between LPMO and lignin degradation is still poorly understood.This paper is aimed at revealing the scientific problems that LPMO involved in the biodegradation of lignin and its mechanism.On this basis,this study reveals the relationship between LPMO and lignin-degrading and-modifying peroxidase?LDMPE?by functional comparative genomics.Moreover,the pathways and mechanisms involved in the degradation of lignin by LPMO were elucidated in vitro,and the function of LPMO in lignin degradation was verified in vivo.The main results of the research are as follows:?1?It reveals the universal co-occurrence between LPMO and lignin-degrading and-modifying peroxidase?LDMPE?from fungi and bacterial.Genes encoding LPMO were found in 88.7%of genomes of 71 lignocellulose-degrading strains by functional genomic comparison analysis.The co-occurrence of LPMO and lignin-degrading and-modifying enzyme?LDME?in the genome is ubiquitous and mainly occurs between LPMO and LDMPE.The co-occurrence of LPMO and lignin-degrading and-modifying enzyme?98.2%?was ubiquitous in the genomes,which occurred mainly between LPMO and LDMPE accounted for 83.9%,mainly in the genomes of white rot fungi.The bacteria showed a 46.7%co-occurrence of LPMO and LDME,mainly occurred in LPMO and the key bacterial LDMPE-dye-decolorizing peroxidase?DyP?,mainly in the genomes of actinomycetes.There was a strong correlation between LPMO and LDMPE in the fungal and bacterial genomes?r>0.6?,especially in white-rot fungi?r=0.809?and actinomycetes,indicating that there may be a correlation between LPMO and LDMPE at the genome level.?2?It reveals the mechanism of lignin degradation in vitro driven by a fungal LPMO.PoLPMO9A from Pleurotus ostreatus BP3 was successfully expressed.The electron donors of PoLPMO9A were studied.Based on it,It showed PoLPMO9A could involve in the lignin degradation by driving lignin-degrading peroxidase reaction and improving the Fenton reaction induced by the quinone-hydroquinone redox cycle in vitro.H₂O₂generated by LPMO was preferentially utilized for lignin degradation reaction by PsVP.The LPMO-driven peroxidase reaction could degrade lignin monomer,dimer and lignin macromolecule.The total relative abundance of lignin derivatives of alkali lignin and natural lignin decreased by 17.1%and 6.3%after PoLPMO9A-PsVP treatment.On the other hand,the LPMO-mediated Fenton reaction mechanism was a semiquinone-driven Fenton mechanism,which led to a hydroxyl radical by promoting H₂O₂ production and Fe³⁺reduction rate.The level of H₂O₂ and the reduction of Fe³⁺were related to the species of hydroquinone,the concentration of hydroquinone and LPMO.The hydroxyl radical level of the LPMO-mediated Fenton reaction was 2.77 times higher than that of the control in the presence of 500?m DBQH₂.In vitro studies have shown that LPMO can be used as an auxiliary enzyme to involve in the degradation of lignin by driving the LDMPE reaction and enhancing the Fenton reaction induced by quinone-hydroquinone redox cycle.?3?It confirms the function of LPMO in lignin degradation in vitro and in vivo.Streptomyces coelicolor M145?ScM145?was used to confirm the function of LPMO in the degradation of lignin in vitro and in vivo.The relative transcription levels of Sclpmo10B and Sclpmo10D were significantly higher than that of others,which increased3-11 times in the presence of lignin.The recombinant Sc M145-?LPMO10B?+?and ScM145-?LPMO10D?+?with homologous overexpression of Sc LPMO10B and Sc LPMO10D were successfully constructed.In vitro experiments showed that LPMO from ScM145 was also involved in lignin degradation by driving the LDMPE reaction and the Fenton reaction pathway,similar to the reaction pathway mediated by white rot fungus LPMO.Compared with the wild-type ScM145,ScM145-?LPMO10B?+?and ScM145-?LPMO10D?+?significantly enhanced the degradation of lignin.The structural abundance of lignin derivatives decreased by 25.4%and 31.0%,respectively,which was 2.65 and3.23 times higher than that of wild-type ScM145.At the same time,the iron reduction ability of the overexpressing strain and the total phenol content released by lignin depolymerization were also significantly increased.It shows LPMO plays an important role in the lignin degradation.It also indicates that the lignin degradation pathway driven by LPMO is universal for lignin-degradating strains on the level of phylum.In summary,this study demonstrates for the first time the function and mechanism of LPMO in lignin biodegradation.The research not only expands the biological function of LPMO,but also clarifies the new role of LPMO in lignocellulose decay,which further lays the theoretical foundation for further clarifying the mechanism of microbial lignocellulose degradation.