Studies On Biosynthesis And Structual Modification Of Cuevanes And The Post-PKS Modification Of Ansatrienins

Cuevaenes is a class of linear polyenes with antibacterial and antitumor activities, which are synthesized by type I polyketide synthases using 3-hydroxybenzoic acid(3-HBA) as starter unit. Previously, cuevaene A-E were isolated from Streptomyces sp. LZ35, and the biosynthetic gene cluster of cuevaenes was identified in strain LZ35, and the chorismate hydrolase Cuv10 has been proved to be responsible for the conversion of chorismate to 3-HBA. However, the post PKS modifications of cuevaenes have not been characterized. Here, we identified that cuv9、cuv18 and cuvl9 are essential for the biosynthesis of cuevaenes, and cuv21 and cuv23 are not required for the formation of cuevaenes through gene deletion and complementation experiments in strain LZ35. Since Cuvl8 has been proposed to be responsible for the hydroxylation of 3-HBA during the elongation of the polyketide chain, we tried to test the activity of Cuvl8 by incubation of Cuvl8 with ACP tethered substrates, however, no positive results had been obtained. In addition, in order to elucidate the catalytic mechanism of CuvlO, we tried to resolve the crystal structure of Cuv 10, but failed. We identified several probable active sites of CuvlO through site-directed point mutations. At last, we replaced the Adenylation domain in the loading module of cuevaenes with that of geldanamycin, and several putative products were identified in the mutant. The isolation and characterization of the products is still undergoing.Ansatrienins is a subfamily of ansamycins with antifungal and antitumor activities, which are synthesized by type I polyketide synthases using 3-amino-5-hydroxybenzoic acid (AHBA) as starter unit. Previously, ansatrienins were isolated from Streptomyces sp. XZQH13 by constitutive expression of the LAL family regulator gene astGl, and as/C and astFl has been proved to be responsible for the addition of the O-(N-cyclohexanecarbonyl)-D-alaninyl side chain. Here, we further characterized the D-alanylation of ansatrienins by PKS gene deletion, complementation of astC and astFl, and point mutation of AstC. Meanwhile, we identified that AstF2 is responsible for the 2-hydroxylation of AHBA in vivo, but the function of AstF2 has not yet been determined in vitro.In conclusion, we identified several uncharacterized genes that are essential for the biosynthesis of cuevaenes, and generated putative cuevaenes derivatives through combinatorial biosynthesis, which set the stage for for the production of novel ansamycins in the future. Moreover, we characterized the D-alanylation mechanism of ansatrienins, and identified the function of AstF2 in vivo, which provide new biological components for the diversification of ansamycins through synthetic biology approaches.