TY - JOUR

T1 - The construction of a library of synthetic promoters revealed some specific features of strong Streptomyces promoters

A1 - Seghezzi,Nicolas

A1 - Amar,Patrick

A1 - Købmann,Brian

A1 - Jensen,Peter Ruhdal

A1 - Virolle,Marie-Joëlle

AU - Seghezzi,Nicolas

AU - Amar,Patrick

AU - Købmann,Brian

AU - Jensen,Peter Ruhdal

AU - Virolle,Marie-Joëlle

PB - Springer

PY - 2011

Y1 - 2011

N2 - Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the −35 and −10 consensus promoter sequences were completely randomized and some variability was introduced in the −35 (position 6) and −10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 μg ml−1). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the −10 box, the −10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters.

AB - Streptomyces are bacteria of industrial interest whose genome contains more than 73% of bases GC. In order to define, in these GC-rich bacteria, specific sequence features of strong promoters, a library of synthetic promoters of various sequence composition was constructed in Streptomyces. To do so, the sequences located upstream, between and downstream of the −35 and −10 consensus promoter sequences were completely randomized and some variability was introduced in the −35 (position 6) and −10 (positions 3, 4 and 5) hexamers recognized by the major vegetative sigma factor HrdB. The synthetic promoters were cloned into the promoter-probe plasmid pIJ487 just upstream of the promoter-less aphII gene that confers resistance to neomycin. This synthetic promoter library was transformed into Streptomyces lividans, and the resulting transformants were screened for their ability to grow in the presence of different concentrations of neomycin (20, 50, and 100 μg ml−1). Promoter strengths varied up to 12-fold, in small increments of activity increase, as determined by reverse transcriptase-PCR. This collection of promoters of various strengths can be useful for the fine-tuning of gene expression in genetic engineering projects. Thirty-eight promoters were sequenced, and the sequences of the 14 weakest and 14 strongest promoters were compared using the WebLogo software with small sample correction. This comparison revealed that the −10 box, the −10 extended motif as well as the spacer of the strong Streptomyces promoters are more G rich than those of the weak promoters.

KW - aphII

KW - Promoter strength

KW - Streptomyces

KW - Synthetic promoters

KW - Neomycin

U2 - 10.1007/s00253-010-3018-0

DO - 10.1007/s00253-010-3018-0

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 2

VL - 90

SP - 615

EP - 623

ER -