Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor Species: Use for Extending Genetic Methodologies to Other Members of This Genus

Abstract

The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

“…First, a 2.31-kb DNA fragment containing the targeted insertion region sequences (intergenic space between convergent genes Cbes0863-Cbes0864) in the C. bescii chromosome was amplified using primers DC456 (with KpnI site) and DC457 (with EcoRI site) using C. bescii genomic DNA as a template. The 4.0-kb DNA fragments containing an apramycin resistance-gene cassette, pyrF cassette (30), and the pSC101 replication origin, were amplified from pDCW88 (19) using primers DC081and DC356. The DC081 and DC356 primers were engineered to contain KpnI and EcoRI sites, respectively.…”

A New Class of Tungsten-Containing Oxidoreductase in Caldicellulosiruptor, a Genus of Plant Biomass-Degrading Thermophilic Bacteria

“…The C. bescii replicating shuttle vector pDCW89 (25) was modified via Gibson Assembly (New England BioLabs) to include a His tag and a multiple cloning site from the commercial vector pET24a (Novagen), generating pIMS89. The P. furiosus aor gene (PF0346) and the C. bescii S-layer protein promoter region (200 bp starting immediately upstream of the start of Athe_2303) were amplified from genomic DNA, spliced together using overlap PCR and cloned into pIMS89 to create pIMSAOR (see Fig.…”

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

“…In most cases where strains gained ISCbe4 elements, the existing ISCbe4 elements from the parent strains were retained at the same genome location in the daughter 4) strains. As shown in Table 2, the sister strains MACB1017 to MACB1021 have different numbers of ISCbe4 elements.…”

“…To this end, a genetic system was developed for C. bescii utilizing a uracil auxotrophic mutant background strain and the counterselectable marker pyrF, a gene required for biosynthesis of uracil that also confers sensitivity to 5-fluoroorotic acid (5-FOA) (2,3). Because there was no method for direct selection of a targeted deletion of pyrF in wild-type C. bescii, the initial development of a genetic background strain relied on the selection of random mutants containing deletions in uracil biosynthesis pathway genes (4). This method resulted in strain JWCB005, which has a partial deletion in both the pyrF and pyrA genes (4).…”

“…Because there was no method for direct selection of a targeted deletion of pyrF in wild-type C. bescii, the initial development of a genetic background strain relied on the selection of random mutants containing deletions in uracil biosynthesis pathway genes (4). This method resulted in strain JWCB005, which has a partial deletion in both the pyrF and pyrA genes (4). A more recent development leading to the improvement of C. bescii genetic methodologies was the use of a high-temperature kanamycin resistance gene (htk codon optimized for C. bescii [Cbhtk]) that enables antibiotic resistance to be utilized for gene insertion or deletion (5).…”