Journal article
Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions: Alternative title; Application of the thermostable beta-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Bacterial Cell Factory Optimization, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Department of Biotechnology and Biomedicine, Technical University of Denmark3
Bacterial Synthetic Biology, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that β-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen concentrations.
This protein functions both as a marker, promoting colony color development in the presence of a lactose analogue S-gal, and as a reporter enabling quantitative measurement by a simple colorimetric assay. Optimal performance was observed at 70 °C and pH 6.4. The gene was introduced into G. thermoglucosidans.
The combination of BgaB expressed from promoters of varying strength with S-gal produced distinct black colonies in aerobic and anaerobic conditions at temperatures ranging from 37 to 60 °C. It showed an important advantage over the conventional β-galactosidase (LacZ) and substrate X-gal, which were inactive at high temperature and under anaerobic conditions.
To demonstrate the versatility of the reporter, a promoter library was constructed by randomizing sequences around −35 and −10 regions in a wild type groES promoter from Geobacillus sp. GHH01. The library contained 28 promoter variants and encompassed fivefold variation. The experimental pipeline allowed construction and measurement of expression levels of the library in just 4 days.
This β-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species.
Language: | English |
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Publisher: | Springer Berlin Heidelberg |
Year: | 2017 |
Pages: | 169 |
ISSN: | 21910855 |
Types: | Journal article |
DOI: | 10.1186/s13568-017-0469-z |
ORCIDs: | Pogrebnyakov, Ivan , Falkenberg, Kristoffer Bach and Nielsen, Alex Toftgaard |