Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius

Genetic engineering of Geobacillus spp.

“…Moreover, Blanchard et al (2014) employed the regulatory region of the sucrose utilization operon, surT-P surP , for inducible expression of the α-galactosidase gene in G. stearothermophilus NUB3621R (the source of promoter and gene is the same strain) and showed that α-galactosidase activity can be enhanced 5-fold. Another group of researchers identified the cellobioseinducible promoter P βglu (from G. thermoglucosidasius NCIMBB 11955) , which was shown to also be activated by other sugars (glucose, cellobiose) (Bartosiak-Jentys et al, 2013). This promoter, and others that can be induced by more than one specific sugar, can be advantageous for the continuous high expression of genes in such cases when cells are grown with a feedstock containing many carbohydrates (BartosiakJentys et al, 2013).…”

“…The KNT-ase TK101 may be used as a selection marker for the transformed cells in both E. coli and Geobacillus spp. (Bartosiak-Jentys et al, 2013). Nevertheless, the use of the TK101 selection system results in remarkably lower transformation efficiencies of E. coli Kananavičiūtė et al, 2014).…”

“…However, pUCG18, as well as another vector, pBST22 (both contain the same pBST1 replicon), has one drawback, since it is unstable under nonselective conditions (Liao and Kanikula, 1990;. Recently, pUCG18 was reduced in size by eliminating all non-essential sequences (including ampicillin resistance gene) and this led to an improved transformation efficiency of G. thermoglucosidasius NCIMB 11955 (Bartosiak-Jentys et al, 2013).…”

“…Several positively regulated promoters putatively inducible by various mono-or di-saccharides, such as maltose, cellobiose, saccharose, galactose, and myo-inositol, have been described (Suzuki et al, 2013a;Bartosiak-Jentys et al, 2013;Blanchard et al, 2014). Suzuki et al (2013a) investigated various promoters of G. kaustophilus HTA426 and identified two strong promoters.…”

“…In all cases, ETM composed of 10% glycerol and was supplemented with 0.5 M of trehalose or with 0.5 M of sorbitol and 0.5 M of mannitol and an electric field strength of 22-25 kV/cm was applied. The most effective transformation, resulting in 2.8 × 10 5 CFU of transformants/μg of plasmid DNA was reached for strain NCIMB 11955, using the plasmid pUCG3.8 ( Bartosiak-Jentys et al, 2013). However, there are no published data describing the influence of various other factors, such as the composition of media, growth phase and other electroporation parameters on the transformation of these Geobacillus strains.…”