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.

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

“…Although several thermophilic Bacillus species have been described to be genetically accessible (11,24,29,31,(37)(38)(39), often laborious protocols, such as protoplast transformation, are required. Moreover, for each species, highly variable transformation efficiencies have been reported for a limited number of strains, suggesting that genetic accessibility is very strain specific.…”

Genetic engineering of Geobacillus spp.

“…Unfortunately, strain NUB3621-R and mutations that led to such phenotype have never been described in the literature (Personal communication with prof. D. R. Zeigler). Recently, Blanchard et al (2014) presented a draft genome sequence of strain NUB3621, which makes it an even more attractive host for genetic engineering.…”

“…Due to its wide growth temperature range, geobacilli, when cultivated at the proper temperature, can serve as a host for the expression of proteins derived not only from thermophiles but also from mesophiles (Blanchard et al, 2014;Couñago and Shamoo, 2005). Several applications of Geobacillus spp.…”

“…However, Geobacillus spp. have some advantages: (1) geobacilli are not fastidious and they can be cultivated on various substrates, (2) they form endospores which allow them to survive unfavorable conditions and facilitate their storage and transportation (Blanchard et al, 2014), and (3) the high growth rate allows high cell densities to be reached and consequently the high production of the required products (Suzuki et al, 2013a).…”

Geobacillus stearothermophilus NUB3621R genetic transformation by electroporation

“…R-M systems are responsible for modification of their host DNA, and for degradation of xenogeneic DNA, which is not modified or modified in different way. However, the G. steraotehrmophillus NUB3621R strain was considered as lacking in a functional restriction-modification system (Blanchard et al, 2014;Wu, Welker, 1989). Still, our results imply that NUB3621R is able to distinguish its own DNA from the foreign one and this leads to the lower transformation efficiency using the plasmid DNA extracted from other bacterial strains.…”