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

Abstract

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 C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA, and the second (MACB1018) is based on the targeted deletion of pyrE, making use of a kanamycin resistance marker. Importantly, an active insertion element, ISCbe4, was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4 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 C. bescii 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 ISCbe4 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 C. bescii. Furthermore, a facile approach for assessing genomic stability in C. bescii has been established.IMPORTANCE Caldicellulosiruptor bescii 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 C. bescii 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 (pyrFA) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 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 ISCbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies.

Genus-Wide Assessment of Lignocellulose Utilization in the Extremely Thermophilic Genus Caldicellulosiruptor by Genomic, Pangenomic, and Metagenomic Analyses

“…Thus far, Caldicellulosiruptor bescii is the only species in the genus that has been shown to have a tractable genetic system, and it has been successfully used to study and improve the features of this bacterium (8,(11)(12)(13)(14)(15). Recently, a kanamycin antibiotic selection marker was developed that significantly improves the ability for genetic manipulation of C. bescii (16) and facilitated development of genetically stable strains for metabolic engineering (17).…”

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction

“…In fact, the large repetitive sequences within this locus led to errors in genome assembly of the GDL in the initial C. bescii genome sequence (10). This was recently corrected and resulted in the relocation of the Athe_1859 to -1857 genes between Athe_1867 and Athe_1866 (11). Transcriptomic and proteomic analyses of highly cellulolytic Caldicellulosiruptor species showed that the GDL is highly transcribed and expressed during growth on both crystalline cellulose and switchgrass (3,12).…”

Lignocellulose solubilization and conversion by extremely thermophilic Caldicellulosiruptor bescii improves by maintaining metabolic activity

“…The most studied from this genus is Caldicellulosiruptor bescii that cometabolizes five‐carbon (C5) and six‐carbon (C6) sugars (Blumer‐Schuette et al, ), thereby rapidly and extensively converting lignocellulose‐derived complex carbohydrates, such as arabinan (C5), mannan (C6), galactan (C6), xylan (C5), and cellulose (C6), into fermentation products (Zurawski et al, , ). C. bescii has already been engineered to produce ethanol from switchgrass (Chung, Cha, Guss, & Westpheling, ), and further improvements in the genetics toolbox for this bacterium (Lipscomb, Conway, Blumer‐Schuette, Kelly, & Adams, ; Williams‐Rhaesa et al, ) bode well for developing strains with enhanced carbohydrate degradation capacity (Conway et al, ) and increased levels of metabolically engineered products (Williams‐Rhaesa et al, ). The complex structure and content of plant biomass exacerbates the microbial degradation of the carbohydrate content present as cellulose and hemicellulose, even at low biomass loadings.…”