Genome Mining Of Antibiotics In Halophilic Archaeon Haladaptatus Paucihalophilus

Antibiotics have been rarely discovered in archaea which live in extreme environment,even the structures of some known compounds such as halocin have not been elucidated.We discover a class II lanthipeptide synthetase gene HapM and four precursor peptide genes(HapA1,HapA2,HapA3 and HapA4)with analysis of genome sequence of Haladaptatus paucihalophilus.The yield of this compound is too low to been identified,although with large fermentation scale.In order to get these four compounds,we clone the gen HapM and four precursor peptide genes(HapA1,HapA2,HapA3 and HapA4)into vector p RSFduet-1 using molecular cloning approach,and heterologously express them in E.coli BL21(DE3).We obtain four compounds with affinity chromatography and desalination.After removal of the leader peptide of the precursor peptide by trypsin in vitro,we purify and determine the molecular weight of the four compounds by HPLC,LC-MS,MALDI-TOF.Meanwhile the bioassay has been performed and the results indicate that compound HapA2 and HapA3 have good resistance to MRSA(Methicillin-resistant Staphylococcus aureus.HapM is identified as a bifunctional enzyme feature with N-terminal dehydratase and C-terminal cyclase.We heterologously express this enzyme and proceed to study its catalysis mechanism.Antibiotics are discovered in archaea indicates the antibiotics exist in the early stage of life evolution,which provide information for exploring the origin of life and antibiotics.HapA2 and HapA3 not only can be act as precursor for drug development in the future,but also provide more compounds with good activity and structural diversity for drug screening.Functional analysis of the class II lanthipeptide synthetase HapM,will enrich the catalytic mechanism of this kind enzyme,and guide the combinatorial biosynthesis of these compounds significantly.