Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 25, Pages 11250-11254Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1006085107
Keywords
precision engineering; RNA polymerase; overproduction
Categories
Funding
- National 863 Project [2006AA09Z402, 2007AA09Z443]
- Key Project for International Cooperation [2007DFB31620]
- National Natural Science Foundation of China [30560001, 30600001, 30700015]
- National Key Technology RD Program [2007BAI26B02]
- CAS [KSCX2-YW-R-164]
- Important National Science & Technology Specific Projects [2008ZX09401-05, 2009ZX09302-004]
- Hundred Talents Program
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Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wildtype strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by sigma(hrdB) in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.
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