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.

中文翻译：

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通过安丝菌素剪裁步骤的合理工程组合生物合成 3-O-氨基甲酰美登醇

目前，临床试验中大多数含美登素的抗体药物偶联物（ADC）都是用 DM1 或 DM4 制备的，而 DM1 或 DM4 主要由安丝菌素 P-3 (AP-3) 合成，安丝菌素 P-3 是一种细菌美登木素生物碱，从Actinosynnema pretiosum中分离出来。然而，由于AP-3对A. pretiosum具有较高的自毒性，导致AP-3的产量一直难以提高。本文通过引入3 -O-氨基甲酰基转移酶基因asc21b以及来自外源的N-甲基转移酶基因，设计并产生了一种对A. pretiosum具有较低自毒性的新美登木素生物碱3- O-氨基甲酰基美登醇(CAM, 3 )。美登木素生物碱基因簇进入 3- O-酰基转移酶基因 ( asm19 ) 缺失的突变体 HGF052。同时，两个新的分流产品20- O-去甲基-19-去氯-N-去甲基-4,5-去环氧-CAM( 4 )和20 -O-去甲基-N-去甲基-4,5-去环氧-CAM( 5 )从重组菌株中鉴定出。此外，通过液体发酵培养基的筛选、瓶颈定制酶和途径特异性激活剂的过表达，工程菌株中CAM的效价达到了498 mg/L。由于 CAM 的 3- O-氨基甲酰基可以通过化学裂解作为 AP-3 去除以产生美登醇，因此我们的工作表明 CAM 可能是未来 ADC 开发中 AP-3 的有前途的替代品。