Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering

Abstract

Metabolic engineers develop inexpensive enantioselective syntheses of high-value compounds, but their designs are sometimes confounded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. Here, we present a draft genome sequence of G. stearothermophilus NUB3621 and describe inducible and constitutive expression plasmids that function in this organism. These tools will help us and others to exploit the natural advantages of this system for metabolic engineering applications.

Fig. 1

The RAST server (Aziz et al. 2008) was used to annotate the open reading frames of GsNUB3621. Of the 3,832 features that were identified, 47 % fell into known gene categories. The distribution of their functions is shown

Fig. 2

The maximal unique matches index (Coorevits et al. 2011) was used to compare the GsNUB3621 genome sequence with those of other Geobacillus species (namely, Geobacillus sp. WCH70, Geobacillus thermoglucosidasius C56-YS93, Geobacillus kaustophilus HTA426, Geobacillus thermodenitrificans NG80-2, Geobacillus thermoleovorans CCB_US3_UF5, Geobacillus sp. Y4.1MC1, Geobacillus sp. Y412MC52, Geobacillus sp. Y412MC61, Geobacillus sp. C56-T3, Geobacillus sp. GHH01, and Geobacillus thermoglucosidans TNO-09.020). The phylogenetic separation between Geobacillus sp. Y412MC52 and Geobacillus sp. Y412MC61 can only be detected at higher resolutions

Fig. 3

The regulatory region of the sucrose utilization operon is schematized. Triangles denote promoters while circles represent Shine-Dalgarno sequences. The surT gene encodes an antiterminator that is thought to bind the surR region, allowing transcription from the surP promoter. The region was amplified via PCR; the EcoRI site within surT was incompatible with the BioBrick cloning standard, so it was eliminated by site-directed mutagenesis of a single base to effect a synonymous substitution. An NcoI site was created at the surP start codon; the region was cloned into the E. coli/GsNUB3621 shuttle plasmid pNW33N with restriction enzymes NcoI, NspI, and SphI

Fig. 4

The inducible surP promoter (a) and constitutive ribonuclease HIII promoter (b) were used to express reporter proteins alpha-galactosidase (AgaN) and superfolding green fluorescent protein (sfGFP), respectively. a GsNUB3621 were transformed with the empty E. coli/GsNUB3621 shuttle vector pNW33N (blue) or surT-P_surP-agaN-pNW33N (green). The transformants were propagated overnight in modified LB medium, in the presence (solid lines) or absence (dotted lines) of sucrose. The supernatants were reacted with 4-methylumbelliferyl-alpha-D-galactopyranoside. Aliquots were quenched in sodium hydroxide; the alpha-galactosidase activity (increase in fluorescence over time) was measured in a spectrofluorimeter. b GsNUB3621 was transformed with the empty pNW33N vector (blue) or expression vector P_RHIII-sfGFP--pNW33N (green). The transformants were propagated overnight in mLB medium. The cells were harvested by centrifugation, resuspended in buffer, and lysed by lysozyme-catalyzed hydrolysis of their cell walls. The fluorescence spectra were measured; the values were adjusted by subtracting the fluorescence of a blank (fresh mLB)