Jadomycin B, Synthesis The Cyclase Jadd, Jadi Of Gene Knock Addition To Streptomyces Coelicolor Membrane Proteome Analysis

Jadomycin B is a representative angucycline of aromatic polyketide produced by Streptomyces venezuelae ISP5230, catalyzed by type II polyketide synthases. Jadomycin B can inhibit the growth of Gram-positive bacteria, yeast and cancer cell line. Since Han et al. cloned jadomycin biosynthetic gene cluster using actI from S. coelicolor as a probe, studies of jadomycin B biosynthesis was carried out at molecular level. Cyclization is a probing understood phenomenon in the biosynthesis of type Ⅱ aromatic polyketide, various polyketides have different cyclic models, in number and orientation of the aromatic rings. Cyclization of jadomycin B is a model to probe the cyclization mechanism of intramolecular aldol condensation.JadD mutant VS210 (jadD::kan~R) and jadI mutant VS212, VS213 (jadI::kan~R) were constructed by insertional inactivation. In VS210, a 777 bp fragment was deleted from jadD and replaced with 998 bp kanamycin resistant gene kan~R. While in VS212 and VS213, a 273 bp fragment was deleted from jadI and replaced with insertion of kan~R in both orientations. Analyses of metabolites in the fermentation both of these mutant strains with HPLC and LC-MS/MS indicate that JadD and JadI are both involved in the biosynthesis of jadomycin B. However, no novel metabolic intermediates were found in VS210, VS212 and VS213. Above-mentioned results demonstrate that the inserted kan~R may have affected the expressions of downstream genes in the jadomycin B gene cluster. Part Ⅱ Analyses of Streptomyces coelicolor inner membrane proteome by multidimentional protein identification technologyStreptomyces coelicolor is the model species among streptomycetes. Untilnow, proteomic analyses of 5. coelicolor have been conducted using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry method, few integral membrane proteins were identified due to the hydrophobic and low-abundance nature of these proteins. In this work, 154 possible inner membrane proteins from 5. coelicolor were identified using high pH-proteinase K sample preparation method and multidimensional protein identification technology, among them 44 are integral membrane proteins containing at least one transmembrane domain, most peptides and their corresponding proteins were identified for the first time.