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Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2010 Jun 22; Vol. 107 (25), pp. 11250-4. Date of Electronic Publication: 2010 Jun 07. - Publication Year :
- 2010
-
Abstract
- 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 wild-type 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.
- Subjects :
- Base Sequence
DNA-Directed RNA Polymerases metabolism
Gene Library
Genetic Engineering
Ivermectin metabolism
Molecular Sequence Data
Mutation
Oligonucleotide Array Sequence Analysis
Promoter Regions, Genetic
Sequence Homology, Nucleic Acid
Transcription, Genetic
Bacterial Proteins genetics
Bioengineering methods
DNA-Binding Proteins genetics
Gene Expression Regulation, Bacterial
Ivermectin analogs & derivatives
Sigma Factor genetics
Streptomyces metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 107
- Issue :
- 25
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 20534557
- Full Text :
- https://doi.org/10.1073/pnas.1006085107