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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0469-z) contains supplementary material, which is available to authorized users.

Potential Use of Bacillus coagulans in the Food Industry

“…Production of this enzyme from microorganisms allows for higher yields and a greater technological advantage compared to other sources. β-galactosidase also could be produced by several yeast ( Kluyveromyces lactis and Kluyveromyces marxianus ), mold ( Aspergillus niger and Aspergillus oryzae ), and bacteria ( B. circulans , E. coli , Lactobacillus bulgaricus , Lactobacillus thermophile , Geobacillus stearothermophilus ), with the exception of B. coagulans [ 72 , 73 ]. α-galactosidase is an important enzyme in the food industry, and is used many applications, especially in sugar production, biotechnology, and medicine.…”

Evolving methods for rational de novo design of functional RNA molecules

“…The complex analytic method and considerable fluctuations of the β-galactosidase enzymatic activity makes GFP -despite a longer protein folding time -more suitable for functional testing. However, both reporter are perfectly suited for quantification [80,81]. Auxotrophic and resistance markers can be tested in replica-plating, allowing for rapid selection of positive/functional constructs but a quantitative analysis of these markers is difficult.…”

GeobacillusBacteria: Potential Commercial Applications in Industry, Bioremediation, and Bioenergy Production

“…β-Galactosidases are also objects for molecular applications in thermophiles and under anaerobic conditions. The enzyme from G. stearothermophilus functions both as a marker, when it cleaves thermostable dye, 3,4-cyclohexenoesculetin β-d-galactopyranoside (S-gal) to black product, and as a reporter enabling quantitative measurement by a simple colorimetric assay [117].…”