Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One of the strains, G. thermoglucosidasius C56-YS93, was then chosen to develop a toolbox for controlled gene expression over a wide range of levels. It includes a library of semi-synthetic constitutive promoters (76-fold difference in expression levels) and an inducible promoter from the xylA gene. A library of synthetic in silico designed ribosome binding sites was also created for further tuning of translation. The PxylA was further used to successfully express native and heterologous xylanases in G. thermoglucosidasius. This toolbox enables fine-tuning of gene expression in Geobacillus species for metabolic engineering approaches in production of biochemicals and heterologous proteins.

Versatile DNA assembly standards and compatible, well-characterized part libraries are essential tools for creating effective designs in synthetic biology. However, to date, vector standards for Gram-positive hosts have limited flexibility. As a result, users often revert to PCR-based methods for building the desired genetic constructs. These methods are inherently prone to introducing mutations, which is problematic considering vector backbone parts are often left unsequenced in cloning workflows. To circumvent this, we present the ProUSER2.0 toolbox: a standardized vector platform for building both integrative and replicative shuttle vectors forBacillus subtilis. The ProUSER2.0 vectors consist of a ProUSER cassette for easy and efficient insertion of cargo sequences and six exchangeable modules. Furthermore, the standard is semicompatible with several previously developed standards, allowing the user to utilize the parts developed for these. To provide parts for the toolbox, seven novel integration sites and six promoters were thoroughly characterized in B. subtilis. Finally, the capacity of the ProUSER2.0 system was demonstrated through the construction of signal peptide libraries for two industrially relevant proteins. Altogether, the ProUSER2.0 toolbox is a powerful and flexible framework for use in B. subtilis.

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.