Combinatorial Biosynthesis of 3--Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins.

Currently, most maytansine-containing antibody-drug conjugates (ADCs) in clinical trials are prepared with DM1 or DM4, which in turn is synthesized mainly from ansamitocin P-3 (AP-3), a bacterial maytansinoid, isolated from . However, due to the high self-toxicity of AP-3 to , the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to , 3--carbamoylmaytansinol (CAM, ), was designed and generated by introducing the 3--carbamoyltransferase gene together with the -methyltransferase genes from exogenous maytansinoid gene clusters into the 3--acyltransferase gene () deleted mutant HGF052. Meanwhile, two new shunt products, 20--demethyl-19-dechloro--demethyl-4,5-desepoxy-CAM () and 20--demethyl--demethyl-4,5-desepoxy-CAM () were identified from the recombinant strain. Furthermore, by screening of liquid fermentation media, overexpression of bottleneck tailoring enzymes and the pathway-specific activator, the titer of CAM reached 498 mg/L in the engineered strain. Since the 3--carbamoyl group of CAM can be removed by chemical cleavage as AP-3 to produce maytansinol, our work suggests that CAM may be a promising alternative to AP-3 in the future development of ADCs.