A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

Caldicellulosiruptor bescii, an anaerobic gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose-degrader known. It is of great biotechnological interest as it efficiently deconstructs non-pretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counter-selected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic toolkit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cb htk ) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii . These strains showed resistance to kanamycin at concentrations >50 μg mL -1 , whereas wild-type C. bescii was sensitive to kanamycin at 10 μg mL -1 . In addition, placement of the Cb htk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii , generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counter-selectable marker, like pyrF , and was used together with Cb htk in the Δ pyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. Importance Caldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78{degree sign}C and it has the ability to efficiently deconstruct non-pretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms as only a limited number of antibiotics are stable above 50°C and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work we have developed a selection system for C. bescii using the antibiotic kanamycin and show that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism.