Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426

Abstract

c Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using ␤-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed highlevel expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and ␣-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus.

Thermoadaptation-Directed Enzyme Evolution in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

“…The plasmid pGKE70 ( Fig. 1) was constructed to integrate genes into the trpE locus (GK2204) and to force gene expression under the control of the P gk704 promoter (29). To construct pGKE70, the P gk704 fragment was excised from pGAM48 (29) and the trpE internal sequence (positions 157 to 1360) was amplified using primers 2204F and 2204R.…”

“…Recently, we have studied genetic tools for the aerobic, Grampositive, Bacillus-related thermophile Geobacillus kaustophilus HTA426 with the aim of using the strain for biotechnological applications and biological studies of the genus Geobacillus (29)(30)(31)(32). In these studies, HTA426 cells efficiently produced descendants constitutively resistant to the antibiotics rifampin (Rif) and streptomycin (Str).…”

Plasmid Curing is a Promising Approach to Improve Thermophiles for Biotechnological Applications: Perspectives in Archaea

“…Genetic tools are available for this strain [10][11][12][13][14][15][16]. The whole genome sequence has been determined [17], showing that G. kaustophilus HTA426 harbors the circular plasmid pHTA426 (Figure 1).…”

“…A bgaB expression cassete under the control of the P gk704 promoter was integrated at the gk0707 locus in G. kaustophilus using pGAM48-bgaB, as described previously [15]. G. kaustophilus MK244 and MK633 were subjected to this process to generate strains MK244 bgaB and MK633 bgaB , respectively.…”

“…The bgaB expression cassete was then cloned in the BamHI site of the resulting plasmid to yield pGKE25-bgaB for replacing the gkp09 gene in pHTA426 with the bgaB cassete. The pGAM48-bgaB plasmid [15] was used to integrate a bgaB expression cassete under the control of the P gk704 promoter at the gk0707 locus in the G. kaustophilus chromosome. The pUCG18T plasmid [12] was used to assess the transformation eiciency of G. kaustophilus.…”