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

is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes , and the second (MACB1018) is based on the targeted deletion of , making use of a kanamycin resistance marker. Importantly, an active insertion element, IS, was discovered in when it disrupted the gene for lactate dehydrogenase () in strain JWCB018, constructed in the JWCB005 background. Additional instances of IS movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and IS elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in Furthermore, a facile approach for assessing genomic stability in has been established. is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes () have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and IS insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these IS elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene have a stable genome structure, making them preferable for future metabolic engineering studies.