Engineering of the TetR family transcriptional regulator SAV151 and its target genes increases avermectin production in Streptomyces avermitilis.
Avermectins produced by Streptomyces avermitilis are used commercially for broad-spectrum parasite control in medical, veterinary, and agricultural fields. Our previous comparative transcriptome analysis of wild-type strain ATCC31267 vs. avermectin-overproducing strain 76-02-e revealed that the gene SAV151, which encodes a TetR family transcriptional regulator, was downregulated in 76-02-e. In the present study, we investigated the role of SAV151 in avermectin production. Deletion of SAV151 increased avermectin yield ~1-fold in ATCC31267, and this phenotype was complemented by a single copy of SAV151. Overexpression of SAV151 in ATCC31267 reduced avermectin yield by ~70%. RT-PCR analysis showed that the promoting effect of SAV151 deletion on avermectin production was not due to alteration of ave genes at the transcriptional level. SAV151 negatively regulated the transcription of itself and of the adjacent transcriptional unit SAV152-SAV153-SAV154. In chromatin immunoprecipitation and gel shift assays, purified His6-tagged SAV151 protein bound to the bidirectional SAV151-SAV152 promoter region. SAV151 bound to two palindromic sequences in this region and thereby repressed transcription from both directions. Two of the SAV151 target genes, SAV152 (which encodes a putative dehydrogenase) and SAV154 (which encodes a putative hydrolase), had promoting effects on avermectin production. Our findings provide the basis for a strategy to increase avermectin production by controlling SAV151 and its target genes.
State Key Laboratory of Agro-Biotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China.
Fei
F
He
Wenshuai
W
Liu
Di
D
Sun
Shuai
S
Luo
Zhi
Z
Chen
Ying
Y
Wen
Jilun
J
Li
D000977:Antiparasitic Agents / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D015870:Gene Expression; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D007559:Ivermectin / Q000031:analogs & derivatives* / Q000378:metabolism; D060847:Metabolic Engineering; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
D000977:Antiparasitic Agents; D004269:DNA, Bacterial; D014157:Transcription Factors; D007559:Ivermectin; C019264:avermectin