Combinatorial Biosynthesis of 3-<i>O</i>-Carbamoylmaytansinol by Rational Engineering of the Tailoring Steps of Ansamitocins
Qingqing Liu, Yu Wang, Xin Xia, Zhongyue Li, Yaoyao Li, Yuemao Shen, Haoxin Wang
Abstract
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 Actinosynnema pretiosum . However, due to the high self-toxicity of AP-3 to A. pretiosum, the yield of AP-3 has been difficult to improve. Herein, a new maytansinoid with much lower self-toxicity to A. pretiosum, 3- O -carbamoylmaytansinol (CAM, 3 ), was designed and generated by introducing the 3- O -carbamoyltransferase gene asc21b together with the N -methyltransferase genes from exogenous maytansinoid gene clusters into the 3- O -acyltransferase gene ( asm19 ) deleted mutant HGF052. Meanwhile, two new shunt products, 20- O -demethyl-19-dechloro- N -demethyl-4,5-desepoxy-CAM ( 4 ) and 20- O -demethyl- N -demethyl-4,5-desepoxy-CAM ( 5 ) 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- O -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.