Engineered Biosynthesis And Directed Accumulation Of Pimar Icin Der Ivatives With Reduced Cytotoxicity

Pimaricin is an important polyene antifungal antibiotic that binds ergosterol and extracts it from fungal membranes,and widely used as food preservatives,veterinary medicaments,and for the treatment of fungal keratitis.But the therapeutic values of pimaricin are undermined by the relatively low antifungal activity and severe side effects caused by its binding to cholesterol in mammalian membranes.Therefore,nextgeneration pimaricin derivatives with an improved therapeutic index are highly desirable.In this study,three pimaricin derivatives(12-decarboxy-12-methyl pimaricin(1),4,5-desepoxy-12-decarboxy-12-methyl pimaricin(2),and 2-hydro-3-hydroxy-4,5-desepoxy-12-decarboxy-12-methyl pimaricin(3))were produced by the mutant strain of S.chattanoogensis L10,in which the P450 monooxygenase gene scnG has been inactivated.In vivo and in vitro analysis of P450 monooxygenase gene scnD demenstrated that the derivative 1 was converted from derivative 2 via C4-C5 epoxidation catalyzed by ScnD,indicating that the accumulation of derivative 2 in scnG mutant is caused by the reduced activity of ScnD.Furthermore,inactivation of pimaricin DH12 domain resulted in specific accumulation of derivative 3,suggesting that derivative 3 might be produced due to the incomplete activity of DH12 domain affected by the gene scnG inactivation.In vitro analysis of ScnG also showed that ScnG could not catalyze the exocyclic carboxylation of pimaricin glycosides 4 or 5,indicating that the exocyclic carboxylation catalyzed by ScnG might occur during polyketide chain extension,rather than the first step of post-PKS modifications.Compared with pimaricin,derivative 1 displayed a 2-fold increase in antifungal activity and a 4.5-fold decrease in hemolytic toxicity,and derivative 2 had comparable antifungal activity and drastically reduced cytotoxicity,whereas derivative 3 showed nearly no antifungal and hemolytic activities.Therefore,while derivatives 1 and 2 could become promising substitutes of pimaricin,derivative 3 was a side-product in scnG mutant.The large-scale production and purification of derivatives 1 and 2 are complicated by the accumulation of side-product 3 in scnG mutant.Considering the production of side-product 3 might be resulted from the partial activity of DH12 domain,improvement of the corresponding dehydratase activity might reduce or eliminate the accumulation of 3.Accordingly,the DH12-KR12 didomain within the pimaricin PKS was swapped with the fully active DH11-KR11 didomain.As predicted,the mutant was not able to produce 3,but accumulates 1 and 2 in high yields.Moreover,the effect of the flanking linker regions on domain swapping was evaluated.It was found that retention of the DH12-KR12 linker regions was more critical for the processivity of hybrid PKSs.Furthermore,high-yield production of derivatives 1 or 2 was subsequently obtained by overexpressing the scnD gene and its partner scnF and by disrupting the scnD gene,respectively.On the basis of directed accumulation of derivatives 1 or 2,we try to further improve their yield via the rational design of pimaricin TE domain.The linear polyketide chain intermidate 6 was obtained by site-directed mutagenesis of TE domain under the guidance of TE domain homologous modeling.And in vitro analysis system of pimaricin TE domain was established with SNAC derivative of 6 as substrate,which provides the opportunity for further yield improvement of derivatives 1 and 2 via the rational design of pimaricin TE domain.In this work,three new pimaricin derivatives were obtained via genetic engineering.To the best of our knowledge,this is also the first report on the concurrent elimination of undesired polyketide metabolites and overproduction of desired products by improving the catalytic efficiency of a DH domain using a di-domain swapping strategy.Moreover,the less toxic pimaricin derivatives(1 and 2)can be further developed into antifungal agents for potential clinical application.