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Component Optimization of Neomycin Biosynthesis via the Reconstitution of a Combinatorial Mini-Gene-Cluster in Streptomyces fradiae.
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2020-08-29 , DOI: 10.1021/acssynbio.0c00281
Jiazhen Zheng 1, 2 , Yue Li 1 , Hanye Guan 1, 2 , Junyue Li 1, 2 , Dong Li 1 , Jihui Zhang 1 , Huarong Tan 1, 2
Affiliation  

Neomycin, a multicomponent aminoglycoside antibiotic, is mainly utilized in livestock husbandry and feed additives in animals. The antimicrobial potency of the main product neomycin B is higher than that of its stereoisomer neomycin C. However, the content of neomycin C as an impurity in the high-producing strain is relatively high, and its isolation or removal from neomycin B is quite difficult, which influences the widespread application of neomycin. In this work, the essential genes responsible for neomycin biosynthesis were evaluated and overexpressed to reduce the content of neomycin C. Among them, neoG and neoH are two novel regulatory genes for neomycin biosynthesis, aphA is a resistance gene, neoN encoding a radical SAM-dependent epimerase is responsible for the conversion of neomycin C to B using SAM as the cofactor, and metK is a SAM synthetase coding gene. We demonstrated that the reconstitution and overexpression of a mini-gene-cluster (PkasO*-neoN-metK-PkasO*-neoGH-aphA) could effectively reduce the accumulation of neomycin C from 19.1 to 12.7% and simultaneously increase neomycin B by ∼13.1% in the engineered strain Sf/pKCZ04 compared with the wild-type strain (Sf). Real-time quantitative polymerase chain reaction analysis revealed the remarkable up-regulation of the neoE, neoH, neoN, and metK genes situated in the mini-gene-cluster. The findings will pave a new path for component optimization and the large-scale industrial production of significant commercial antibiotics.

中文翻译:

通过在 Streptomyces fradiae 中重组组合微型基因簇来优化新霉素生物合成的成分。

新霉素是一种多组分氨基糖苷类抗生素,主要用于畜牧业和动物饲料添加剂。主要产品新霉素B的抗菌效力高于其立体异构体新霉素C,但高产菌株中作为杂质的新霉素C含量较高,从新霉素B中分离或去除相当困难,影响了新霉素的广泛应用。在这项工作中,对负责新霉素生物合成的必需基因进行了评估和过表达,以降低新霉素 C 的含量。 其中,neoGneoH是新霉素生物合成的两个新型调控基因,aphA是一种抗性基因,neoN编码自由基 SAM 依赖性差向异构酶的基因负责使用 SAM 作为辅因子将新霉素 C 转化为 B,metK是 SAM 合成酶编码基因。我们证明了微型基因簇(P kasO * - neoN - metK -P kasO * - neoGH - aphA)的重建和过表达可以有效地将新霉素 C 的积累从 19.1% 减少到 12.7%,同时增加新霉素 B与野生型菌株 (Sf) 相比,工程菌株 Sf/pKCZ04 中的 13.1%。实时定量聚合酶链反应分析揭示了neoEneoH的显着上调neoNmetK基因位于微型基因簇中。这些发现将为成分优化和重要商业抗生素的大规模工业生产铺平道路。
更新日期:2020-09-20
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