Biosynthesis Of Ansacarbamitocins In Amycolatopsis Sp. CP2808

Maytansinoids, a group of macrolactam ansamycin antibiotics, have excellent cytotoxicity as promising anti-tumor agents. Compared with other antibody-targeted conjugates, maytansinoids-antibody can well reduce side-effects and improve the cell-killing efficiency. The biosynthesis of ansamitocin is investigated as the typical case of maytansinoids.New antibiotic ansacarbamitocin in Amycolatopsis sp. CP2808 was similar to ansamitocin but its further study was stopped because of low fungi-killing activities. Nevertheless its glycoside and ester-chain derivatives brought new light to the modification of maytansinoids. The carbamoyl-modification at C3 position has the same structure with maytansine DM1, whose antibody-conjugates are already applied in the phase II clinical trial. Therefore, this study tried to find out the origins of these structural differences between the biosynthetic pathway of ansamitocin and that of ansacarbamitocin. Through the screening of the genomic library and chromosome walking, a 108.5 kb sequence covering the whole gene cluster was analyzed. This cluster contains 6 genes related to AHBA biosynthesis, 6 post-PKS modification genes and 2 possible regulation genes.Compared to the biosynthetic gene cluster of ansamitocin, the new one has two carbamoyltransferase genes, acm21a and acm21b, and one AHBA synthase gene. However, there are two AHBA synthase genes and only one carbamoyltransferase gene existing in ansamitocin gene cluster. These differences led to further studies. Post-PKS modification genes acm21a, acm21b and glycosyltransferase gene acm25 were interrupted through Redirect technology. The detection of fermentation products shows that carbamoyltransferase Acm21b is responsable for the same reaction catalyzed by Asm21, and the glycosyltransferase Acm25 adds the glucose moiety after four steps of post-modifications.The involvements of the two AHBA synthase genes, asm24 and asm43, in ansamitocin biosynthesis were investigated in A. pretiosum ssp. ATCC31565. These two genes were interrupted individually, and the fermentation broth of the mutants was analyzed. It is shown that asm24 interrupted mutant has higher yield than the asm43 mutant, suggesting that asm43 has more contribution to the production of ansamitocins. Furthemore, an asm24 and asm43 double mutant has been constructed for subsequent investigation.This study found out the biosynthetic gene cluster of ansacarbamitocins, briefly suggested its biosynthetic pathway and Acm21b might be responsble for the C-3 modification of ansacarbamitocins. These results will be an elementary step for the transformation of ansamycins and the increasing of yield.