Application of the thermostable beta-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions

作者：Jensen Torbjorn Olshoj; Pogrebnyakov Ivan; Falkenberg Kristoffer Bach; Redl Stephanie; Nielsen Alex Toftgaard^*

来源：AMB Express, 2017, 7(1): 169.

DOI：10.1186/s13568-017-0469-z

• Genetic reporter
• Anaerobic genetics
• Thermophile
• Thermostable enzyme

Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that beta-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 degrees 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 degrees C. It showed an important advantage over the conventional beta-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 beta-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species.

• 出版日期2017-9-6