Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis.
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.
Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, China.
Ying
Y
Zhuo
Wenquan
W
Zhang
Difei
D
Chen
Hong
H
Gao
Jun
J
Tao
Mei
M
Liu
Zhongxuan
Z
Gou
Xianlong
X
Zhou
Bang-Ce
BC
Ye
Qing
Q
Zhang
Siliang
S
Zhang
Li-Xin
LX
Zhang
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D057005:Bioengineering / Q000379:methods*; D004268:DNA-Binding Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005818:Genetic Engineering; D007559:Ivermectin / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000235:genetics*; D013302:Streptomyces / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012808:Sigma Factor; C071941:HrdB protein, Streptomyces; D007559:Ivermectin; C019264:avermectin; D012321:DNA-Directed RNA Polymerases