Date Published: September 6, 2017
Publisher: Springer Berlin Heidelberg
Author(s): Torbjørn Ølshøj Jensen, Ivan Pogrebnyakov, Kristoffer Bach Falkenberg, Stephanie Redl, Alex Toftgaard Nielsen.
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.
Economically feasible production of biofuels and biochemicals using microbial cell factories is becoming an increasingly important challenge in the transition towards a sustainable society. Development and optimization of suitable production microorganisms is essential to meet this challenge. Direct engineering of the metabolic pathways of these microorganisms is a recognized method for improving properties and performances. Tuning gene expression to perform metabolic optimization rather than substantial overexpression or inactivation of genes is thus far more appreciated.
The thermostable β-galactosidase from G. stearothermophilus was initially cloned and expressed under control of the Plac promoter in E. coli.
Application of bgaB as a versatile genetic reporter has been proven in mesophilic and thermophilic facultative aerobe bacteria and in mouse embryos in the presence of oxygen (Kishigami et al. 2006; Schrogel and Allmansberger 1997; Suzuki et al. 2013; Yuan and Wong 1995). Focusing on the applications of the bgaB gene under thermophilic and anaerobic conditions, we initially expressed it in E. coli. Optimal conditions for its activity were determined to be 70 °C and pH 6.4. The BgaB protein has previously been characterized by Chen et al. (2008) and Dong et al. (2011), aiming at applications in the dairy industry and by Yuan and Wong (1995) and Schrogel and Allmansberger (1997) who apply bgaB as a reporter gene. In the study by Chen et al. (2008) the optimal conditions for the enzyme were found to be pH 7.0 and 70 °C. Despite the coherence between temperature optima, the pH optimum in the present study (pH 6.4) slightly deviates from that data. Unlike the study by Chen et al. (2008), all activity measurements in our study were performed directly on the cell lysate. Dong et al. (2011) utilized His-tagging of the protein for purification and found pH optimum to be 7.0. It is possible that the addition of the affinity tag may affect protein function and pH optimum, as it has previously been observed for other proteins (Thielges et al. 2011). Additionally, both studies determined pH optimum at 55 °C, while optimal temperature for this enzyme’s activity is 70 °C, the significant influence by the temperature on the activity of BgaB was also shown (at lower temperatures) by Welsch et al. (2012). The study by Schrogel and Allmansberger (1997) test cell extract and found pH optimum coherent to this study, despite that the temperature of the assay was 55 °C. Half-life of the BgaB protein was not assessed in this study, but it has been reported to be 120 h at 60 °C and 9 h at 70 °C (Chen et al. 2008). This and the temperature profile support the application of BgaB as a marker/reporter for organisms growing at higher temperatures (<75 °C). Source: http://doi.org/10.1186/s13568-017-0469-z