TY - JOUR
T1 - Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
T2 - Alternative title; Application of the thermostable beta-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
AU - Jensen, Torbjørn Ølshøj
AU - Pogrebnyakov, Ivan
AU - Falkenberg, Kristoffer Bach
AU - Redl, Stephanie Maria Anna
AU - Nielsen, Alex Toftgaard
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Genetic reporter
KW - Anaerobic genetics
KW - Thermophile
KW - Thermostable enzyme
U2 - 10.1186/s13568-017-0469-z
DO - 10.1186/s13568-017-0469-z
M3 - Journal article
C2 - 28875485
SN - 2191-0855
VL - 7
JO - A M B Express
JF - A M B Express
IS - 1
M1 - 169
ER -