The Study On Degradation And Mechanism Of Nodularin By Sphingopyxis Sp.

Cyanotoxins pollution caused by cyanobacterial blooms seriously threatens ecological security and human health.Nodularin（NOD）and Microcystin（MC）are the two most widely distributed and abundant cyanotoxins.Due to the special ring structure,the removal of cyanotoxins by conventional techniques is not ideal.Microbial degradation stands out among many emerging advanced methods owing to its low cost and high efficiency.Scholars at home and abroad have comprehensively recognized the importance of Sphingopyxis bacteria and the mlrCABD gene cluster in MC degradation.Previous NOD-related studies have mostly focused on microbial communities,and there is still a lack of systematic studies on NOD degradation by single strain.Based on the above research background,we raised several scientific questions:Can Sphingopyxis sp.degrade NOD?Are new intermediates produced during the degradation process?Is the mlrgene cluster involved in NOD degradation?Are there other degradation mechanisms?Does the toxicity of NOD decrease with degradation?Can Sphingopyxis sp.degrade multiple cyanotoxins simultaneously?Focusing on the above scientific issues,this study took Sphingopyxis sp.m6 as the research object.First,the degradation characteristics and degradation pathways of NOD were investigated using liquid chromatography and mass spectrometry.Second,the degradation mechanism of NOD by Sphingopyxis sp.was systematically studied by mlrgene heterologous expression engineering bacteria,mlrgene knockout bacteria and proteomics technology.Third,the safety of the degradation reaction system was preliminarily evaluated based on basic cytotoxicity indicators and NOD specific targets.Last,the application potential of microbial technology for removal of various cyanotoxins was explored from the levels of bacteria and protease.The main findings of this study are as follows:1.The degradation experiments showed that Sphingopyxis sp.was able to degrade NOD.The NOD biodegradation kinetics is in accordance with the zero-order kinetics model,the degradation rate constant is 0.17 mg/L/d,and the half-life(t_1/2)is 36 h.Single factor experiments showed that the optimal degradation temperature was 30℃.When the temperature was too high（40℃）,Sphingopyxis sp.lost its degradation ability.Under acidic conditions there was not only a lag period before strain m6 started to degrade NOD,but the degradation rate was also inhibited.The degradation rate was obviously promoted under alkaline environment.The degradation rate gradually increased with the increase of NOD concentration.A total of four degradation products,linear NOD,tetrapeptide Glu-Mdhb-Me Asp-Arg,tripeptide Mdhb-Me Asp-Arg and dipeptide Me Asp-Arg,were identified by mass spectrometry.2.The NOD degradation rate was positively correlated with the expression dynamics of the four mlrgenes.The MlrA encoded by mlrA gene is the degradation initiation enzyme,which hydrolyzes the Arg-Adda bond of the cyclic structure to generate linear NOD.Crude MlrA exhibited higher NOD degradation activity and stronger environmental adaptability.The MlrC encoded by mlrC gene first broke the Adda-Glu bond of linear NOD to generate tetrapeptide and Adda,and then hydrolyzed the tetrapeptide to generate tripeptide and dipeptide in turn.Fatty acid metabolism,lipid transport and metabolism,amino acid transport and metabolism pathways may have regulatory effects on NOD degradation.Amino acid permease,dehydratase,acyl dehydratase,and acyl-Co A synthase may be involved in NOD degradation.3.After NOD was degraded,basic cytotoxicity indicators such as relative cell proliferation rate,cell apoptosis rate,cell necrosis rate,and accumulation of reactive oxygen species（ROS）were significantly improved.The activity of protein phosphatases（PP）2A,NOD specifically inhibitory target,was significantly increased.And the secretion of interleukin-6（IL-6）was decreased.It was suggested that the major toxicity of NOD was significantly improved after degradation by Sphingopyxis sp..4.When Sphingopyxis sp.simultaneously degraded NOD and microcystin-LR（MC-LR）,the degradation efficiency is significantly improved.MC-LR can significantly promote the degradation of NOD by stimulating the up-regulation of mlrA gene expression.When MlrA degraded the two toxins at the same time,the degradation efficiency is higher than that of the bacteria,and the degradation of the two toxins does not affect each other.It was suggested that microbial technology has great application potential for the removal of various cyanotoxins at the levels of bacteria and functional enzymes.In conclusion,this study investigated the specific pathway and mechanism of NOD degradation by Sphingopyxis sp.from two levels of gene function and proteomics,and preliminarily discussed its detoxification effect on NOD and its application potential for the removal of various cyanotoxins.It not only enriches and improves the theoretical knowledge of NOD biodegradation pathways and molecular mechanisms,but also provides theoretical basis and technical support for the practical application of microbial degradation of NOD.