Mechanism Of Ochratoxin A Degradation By Brevundimonas Diminuta HAU429

Ochratoxin A（OTA）is one of the most common mycotoxins produced by various fungi such as As-pergillus and penicillium.Ubiquitously found in animal feed-and food-stuffs including wheat,corn,oat,barley,fruit,wine,eggs,dairy products,coffee,cocoa,tea and meat products et al.,OTA poses serious threaten to animals and human health,causing significant economic losses to animal husbandry as well as health service.It was urgent to explore efficient detoxification strategy to eliminate OTA in food and feed stuffs.Due to high efficiency and environment amity features,biological detoxification has been become the most popular strategy for mycotoxin detoxification.However,biological resources for OTA degradation were still insufficient.The mechanisms underlying remined to be expounded.Current work aims at discov-ering new OTA degrading microorganisms and elaborating the degrading mechanisms of responsible en-zymes.Genomic,transcriptomic and genetic engineering analysis were comprehensively employed for in-vestigation of OTA degradation mechanism of Brevundimonas diminuta HAU429,which was firstly iso-lated and identified by our team.The main work and results are demonstrated as follow.Hippuric acid-L-phenylalanine was employed as the sole carbon source in MSM medium to screen OTA degrading bacteria.129 strains of bacteria were collected with degradation rate of OTA（1μg/m L）above 50%within 72 h,among which HAU429 exhibits the most efficient capacity with degradation rate up to 95%.According to morphology and 16S r RNA based phylogenetic construction,strain HAU429 was identified as Brevundimonas diminuta.In addition,cell-free culture supernatant achieved OTA（1μg/m L）degradation rate of 53%,while cell lysate could completely degrade OTA in 48 h.The optimum pH of OTA degradation by culture supernatant and cell lysate were observed at p H 7.0 and 8.0,The temperature profile of cell lysate for degrading OTA showed a remarkably different pattern in comparison with that of culture supernatant.These results indicated that multiple enzymes were involved in OTA hydrolase by HAU429strain.Subsequently,the degradation product by HAU429 was identified by UPLC-TOF-MS and NMR,confirming the hydrolyzation of amide bond in OTA by HAU429.Further,cytotoxicity of OTA and degradation product OTαwere fully evaluated in L-02 cells.OTA significantly decreased cell viability and induced oxidative stress with mitochondria impairment.Besides,OTA incudes apoptosis and disturbed cell cycle of L-02 cells.In contrast,OTαdisplayed no influence on L-02 cells within these terms.Additionally,metabolomics analysis confirmed OTA severely disturbed sev-eral metabolic pathways associated with antioxidant defense systems and cell proliferation etc.While OTαposed almost negligible effects on L-02 cells.These findings supported that the cleavage of L-β-Phe moiety from OTA resulted in the detoxification of its hepatotoxicity.The genome sequence of strain HAU429 showed a single circular chromosome with a length of3,720,044 bp with 66.89%GC content.A total of 3564 genes were annotated in NR database,of which 163genes were considered enable to hydrolase amino bond.By comparing the amino acid sequence with that of reported OTA hydrolases,9 candidate OTA hydrolases were selected for further research.Transcriptomic analysis of HAU429 incubated with OTA and not were conducted,revealing 164 genes significantly up-regulated.Moreover,9 genes up-regulated were found with amino bond hydrolase potential,which may be responsible for OTA degradation.Totally,18 genes were screened as candidate OTA hydrolase for further study.Molecular response of HAU429 after exposure to OTA was also analyzed at the level of transcrip-tome.Collectively,HAU429 enhanced TCA cycle and oxidative phosphorylation to produce more energy to resist oxidative stress and damage by OTA.Besides,m RNA level of peroxidase and DNA repair pathway were upregulated to alleviate oxidative stress and confront OTA-induced DNA damage.There are 18 candidate OTA hydrolase genes（BT1～BT18）cloned,expressed,purified and enzyme activity identified by genetic engineering and substrate OTA degradation experiments.Except for BT16,all candidate genes were successfully heterologous expressed in E.coli Rosseta（DE3）.Three purified recom-binases（BT6,BT7 and BT9）could effectively degrade OTA（1μg/m L）almost completely within 1 h,50min and 12 h,respectively.Through the characterization of the enzymatic properties,the optimum p H was observed at 7.0 for BT7 and BT9,and 8.0 for BT6.BT6 was highly active under neutral and alkaline pH conditions（pH 7.0～9.0）with above 90%of the maximum activity remained.BT6 and BT7 were basically stable after maintaining at p H 7.0～9.0 at 4℃for 12 h.In comparison,BT9 showed wide tolerance of p H,remaining above 75%of its original activity at p H 4.0～9.0 after 12 h.BT9 displayed the highest OTA-hydrolyzing activity at 37℃,while the optimum temperature was observed at 47～52℃for BT6 and at 47℃for BT7.The stability of BT6 under high temperature guarantee possible applications in future processing industry for agricultural products.Ba²⁺,Mg²⁺,Fe²⁺,Ca²⁺,Mn²⁺and Zn²⁺also showed clearly inhibitory effects on BT6 and BT7,whereas Zn²⁺exhibited a strong activation effect on the BT9 activity.The Possible binding mechanisms of three enzymes with OTA were explored by molecular docking,with hydrogen-bond interactions and binding pockets displayed.All these findings enhanced our understanding of microbial OTA degradation and promote the development of enzyme-catalyzed OTA detoxification in food and feed industries.However,much effort is still needed in the future to discover other intracellular OTA hydrolases in strain HAU429,and to clarify the joint degradation mechanism mediated by multiple enzymes.In conclusion,an efficient OTA degradation strain named Brevundimonas diminuta HAU429 was ob-tained in current work,which degraded 95%OTA within 72 h.We further evaluated hepatotoxicity of OTA and OTαon cell viability,oxidative stress,cell apoptosis,cell cycle and metabolome of L-02 cells,indicat-ing transforming OTA into OTαby HAU429 as the detoxification mechanism.By genome and transcrip-tome analysis,three efficient OTA hydrolases were identified.The enzymatic property and catalytic mech-anism were further investigated.Collectively,current work provided solid foundation for application of HAU429 in OTA detoxification.