Biosynthetic Mechanism Of Swainsonine In Metarhizium Robertsii

Swainsonine（SW）,a poisonous indolizidine-alkaloid mycotoxin to livestocks,was first determined in locoweeds.Recent studies have demonstrated that SW is actually a fungal-endophyte-derived secondary metabolite after culture purification.SW can inhibit the lysosomal α-mannosidase activity,which leads to the occurrence of livestock locoism.On the other hand,it has shown that SW can be more active to suppress the golgi α-mannosidase II activity.Thus,the metablite exhibits immunomodulatory and antitumor effects and has potential applications for pharmaceuticals.Based on the isolation and chemical synthesis of the intermediates,the biosynthetic pathway of SW was once proposed in the ascomycete fungus Rhizoctonia leguminicola.It was later found that SW could also be produced by the entomopathogenic Metarhizium species.Based on our previous acquisition of the genome information of eight Metarhizium species,the biosynthetic mechanism and biological function potential of SW in Metarhizium were investigated and reported in this thesis.Based on the analysis of SW structure,we speculated that the alkaloid might be putatively biosynthesized by a polyketide synthase（PKS）-non-ribosomal peptide synthetase（NRPS）hybrid enzyme.The predicited gene MAA₀8622 of M.robertsii was therefore deleted and the comparative mass spectrum（MS）analysis confirmed that this hybrid enzyme gene is indispensible for SW biosynthesis.There are seven genes within this cluster:FeⅡ/α-ketoglutarate-dependent dioxygenase SwnH2（MAA₀8623）,PKS-NRPS hybrid enzyme SwnK（MAA₀8622）,short-chain dehydrogenase SwnR（MAA₀8621）,short-chain dehydrogenase SwnN（MAA₀8620）,amino acid/polyamine transporter SwnT（MAA₀8619）,FeⅡ/α-ketoglutarate-dependent dioxygenase SwnH1（MAA₀861 8）and transcriptional regulator SwnA（MAA₀8617）.Subsequently,the biosynthetic pathway of SW was elucidated by serial gene deletions,intermediate isolations and strucuture identifiactions,and heterologous gene expression.We found that four genes within this gene cluster are directly related to SW biosynthesis.The hybrid enzyme SwnK is responsible for the condensation of pipecolic acid and malonyl-CoA to form the epimers（1S）-and（1R）-1-hydroxyindolizidines.SwnH2 catalyzes the oxidation of（1R）-1-hydroxyindolizidine to（1 S,2R）-1,2-dihydroxyindolizidine,which will be further oxidized by SwnH1 to（1S,2R,8R）-1,2,8-trihydroxyindolizidine,the end product SW.SwnN is proposed to catalyze the reduction of 1-oxoindolizidine（the non-reduced product released by SwnK）to two 1-hydroxyindolizidines.By comparing the intracellular and extracellular SW contents between the wild type and ΔswnT mutant,we found that SwnT serves as an importer of precursor.In addition,a non-clustered lysine cyclodeaminase SwnL（MAA₀2105）was found to involve in the production of the precursor pipecolic acid for SW.Comparative analysis of the SW-biosynthetic gene cluster among eight Metarhizium species indicated that the cluster is not present in the genomes of the basal species M.album and M.rileyi.Intriguingly,MS examination of SW production indicated that M.guizhouense,even containing the gene cluster,cannot produce SW.RT-PCR analysis demonstrated that the swnK-like gene was not expressed by M.guizhouense as did by other five species grown in the same condition.In addition,the genome surveys indicated that the conservative SW-biosynthetic gene cluster is present in the genomes of some human pathogenic fungi that have never been reported with the ability to produce SW,an indicative of the divergent evolution of the gene cluster.Previous reports have demonstrated that SW has no insecticidal activity.To test this,insect bioassays were performed by using both topical infection and injection methods against the fifth-instar larvae of silkworm.The estimation and comparison of the median lethal time（LT50）values revealed that there was no significant difference between the wild type and△swnK.Further RT-PCR analysis indicated that,relative to the growth in an artificial liquid culture,the expression of swnK was not substantially up-regulated by the fungus during infection and in vivo propagation（i.e.the formation of hyphal bodies）within insect hemocoel.Futhermore,WGA staining of fungal mycelia showed that the biosynthesis of SW is non-essential for fungal colonization of plant roots.It is unexpected to find that no SW will be produced by Metarhizium during the formation of endophytic relationship with maize,the result supports the safe application of Metarhizium as mycoinsecticides.In conclusion,we fully elucidated the mechanism of SW biosynthesis in Metarhizium species.The results of this study will benefit the future effort to produce SW hyperproduction strain by genetic engineering,and the development of the strategies to reduce the harmful effect of S W to animal husbandry.