Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii
- Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology; University of Georgia
- Univ. of Georgia, Athens, GA (United States). Dept. of Biochemistry and Molecular Biology
- North Carolina State Univ., Raleigh, NC (United States). Dept. of Chemical and Biomolecular Engineering
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. besciistrains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genespyrFA, 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. besciiwhen it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4movement 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 ofC. besciifrom 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 Cbe4 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 inC. besciihas been established.Caldicellulosiruptor besciiis a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology inC. besciihas 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 IS Cbe4 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 Cbe4 elements. Finally, 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.
- Research Organization:
- Univ. of California, Oakland, CA (United States). Regents of University of California
- Sponsoring Organization:
- USDOE Office of Science (SC)
- Grant/Contract Number:
- AC02-05CH11231
- OSTI ID:
- 1465355
- Journal Information:
- Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 14 Vol. 83; ISSN 0099-2240
- Publisher:
- American Society for MicrobiologyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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