Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens.
Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens.
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DOI：
10.1016/j.ymben.2013.06.003
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发表时间：
2013-09
影响因子：
8.4
通讯作者：
T. Zhu;Xueqing Cheng;Yuntian Liu;Z. Deng;Delin You
中科院分区：
文献类型：
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作者：
T. Zhu;Xueqing Cheng;Yuntian Liu;Z. Deng;Delin You
金霉素(CTC)是抗生素四环素(TC)家族的重要成员，能抑制细菌中蛋白质的合成，广泛应用于临床治疗、动物饲料和水产养殖。以前的工作已经错综复杂地报道了金黄色链霉菌随机突变株的中间体生物合成CTC的过程，而关键的氯化反应仍不清楚。我们在一个工业生产者中进行了遗传操作，其中约15.0g/L的四氯化碳和1.2g/L的总胆固醇，发现ctcP(一个依赖于FADH2的卤代酶基因)的氯化是四氯化碳生物合成过程中最后一个低效步骤。首先，ΔctcP菌株在不添加KbR的情况下，积累了约18.9g/L的清洁TC，并取消了四氯化碳的生产。随后，CtcP对绝对TC(4S)而不是TC(4R)表现出底物立体特异性，其下限为0.51±0.01min−1，而它可以卤化几个TC类似物。因此，我们设计了一种过表达ctcPinS的策略。我们预计，我们的工作将为微生物中新的TC衍生物提供酶进化和菌株工程方面的生物技术潜力。
Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants ofStreptomyces aureofaciensand the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0 g/l CTC predominated along with 1.2 g/l TC, and discovered that chlorination byctcP(an FADH2-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcPstrain accumulated about 18.9 g/l “clean” TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with lowkcatof 0.51±0.01 min−1, while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression ofctcPinS. aureofaciensand improved CTC production to a final titer of 25.9 g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.