School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, People's Republic of China.
J Ind Microbiol Biotechnol. 2022 Jul 30;49(4). doi: 10.1093/jimb/kuac011.
A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7'-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%.
阿普拉霉素是一种结构独特的氨基糖苷类抗生素，由暗沟链霉菌（Streptomyces tenebrarius）产生，用于兽医学或治疗沙门氏菌、大肠杆菌和多杀巴斯德氏菌感染。尽管阿普拉霉素早在近 50 年前就被发现，但许多阿普拉霉素的生物合成步骤仍不清楚。在本研究中，我们鉴定了一种 HemK 家族甲基转移酶 AprI，它是阿普拉霉素生物合成途径中的 7'-N-甲基转移酶。生化实验表明 AprI 将去甲基-阿普罗霉素转化为阿普罗霉素。通过 aprI 基因敲除，我们在 aprI 敲除株中鉴定出一种新的氨基糖苷类抗生素去甲基-阿普拉霉素作为主要产物。去甲基-阿普拉霉素是阿普拉霉素产品中的一种杂质。除了去甲基-阿普拉霉素外，碳巴胺妥布霉素也是另一种主要杂质。然而，与去甲基-阿普拉霉素不同，妥布霉素是由暗沟链霉菌中独立的生物合成途径生物合成的。通过 aprI 的过表达和 tobM2 的敲除（tobM2 是妥布霉素生物合成的关键基因），提高了阿普拉霉素的效价和产率。阿普拉霉素的效价从 2227±320mg/L 提高到 2331±210mg/L，而产物杂质去甲基-阿普拉霉素的效价从 196±36mg/L 降低到 51±9mg/L。此外，野生型菌株的碳巴胺妥布霉素效价为 607±111mg/L，工程菌株为零。阿普拉霉素的产率从 68%提高到 87%，去甲基-阿普拉霉素的产率从 1.17%降低到 0.34%。
