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Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576

  • Jiazhen Zheng
  • Yue LiEmail author
  • Hanye Guan
  • Jihui Zhang
  • Huarong TanEmail author
Applied genetics and molecular biotechnology

Abstract

Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with PkasO*. Compared to the yield (1282 mg/L) of Streptomyces fradiae CGMCC 4.576, the engineered strain Sf/pKCZ03 had a 36% enhancement of neomycin production. Quantitative real-time PCR analysis revealed the increased transcription of structural genes (neoE, neoB, neoL, aacC8) and regulatory genes (neoR, neoH) in Sf/pKCZ03. Additionally, under the supplementation of 1 g/L N-acetyl-D-glucosamine and 5 g/L L-glutamine, the yield of engineered strain Sf/pKCZ03 showed 62% and 107% improvements compared to that of the wild-type strain in the original medium, respectively. These findings demonstrated that engineering the antibiotic gene cluster in combination with precursors feeding was an effective approach for strain improvement, and would be potentially extended to other Streptomyces for large-scale production of commercialized antibiotics.

Keywords

Neomycin Streptomyces fradiae Gene cluster Precursor supply 

Notes

Acknowledgments

We are grateful to Professor Yuhui Sun (School of Pharmaceutical Sciences, Wuhan University, China) for kindly providing the plasmid pWHU2653.

Funding

This work was supported by Beijing Natural Science Foundation (Grant No. 5184034) and the National Natural Science Foundation of China (Grant Nos. 31771378, 31800029, and 31470206).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approved

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9597_MOESM1_ESM.pdf (549 kb)
ESM 1 (PDF 548 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  2. 2.College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina

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