Complete Genome Sequence of the 2,3-Butanediol-Producing Klebsiella pneumoniae Strain KCTC 2242

Abstract

Here we report the full genome sequence of Klebsiella pneumoniae KCTC 2242,consisting of a 5.26-Mb chromosome (57.6% GC%; 5,035 genes [4,923 encoding known proteins, 112 RNA genes]) and a 202-kb plasmid (50.2% GC%; 229 genes [229 encoding known proteins]).

GENOME ANNOUNCEMENT

2,3-Butanediol (2,3-BD) is a glycol widely used as a reagent in a number of chemical syntheses. In addition to its applications in plastics, solvent, and antifreeze preparations, it could also be converted to 1,3-butadiene (for synthetic rubber), diacetyl (flavoring agent), or methyl ethyl ketone (liquid fuel additive) or to precursors of polyurethane (8). Interest in microbial production of 2,3-butanediol has been increasing recently due to the extensive industrial applications of this product (3). This colorless and odorless liquid with a high boiling point and a low freezing point is a potential valuable fuel additive (5). Klebsiella pneumoniae KCTC 2242 is a Gram-negative, nonmotile, encapsulated, facultative anaerobic, rod-shaped bacterium. Although K. pneumoniae is a pathogenic microorganism, much research on 2,3-butanediol production is going on (2, 6). Whole-genome sequencing and transcriptome analysis were carried out not only to compare gene data with that of the already published genes of K. pneumoniae NTUH-K2044 (NCBI accession number AP006725), K. pneumoniae 342 (NCBI accession number CP000964), and K. pneumoniae MGH 78578 (NCBI accession number CP000647) but also to discover the characteristics and genomic level of K. pneumoniae KCTC 2242.

Genomic DNA isolated using an overnight culture of strain KCTC 2242 and a DNeasy blood and tissue kit (Qiagen) was sequenced by 454 GS FLX Titanium pyrosequencing (Roche), following the manufacturer's instructions, with 25× coverage (4). Then, the 501,755 reads generated, with a length of 129,696,091 bp, were assembled using a GS De Novo assembler (version 2.3; Roche). The N50 contig was 146,341 bp in length, and largest contig assembled was 358,873 bp. This assembly generated 67 large contigs (>500 bp), with a length of 5,394,883 bp. A total of 480,845 reads (95.83% of the total) were assembled into 10 scaffolds composed of 99 contigs, with a length of 5,426,912 bp. The N50 scaffold was 2,943,199 bp, and the average length of the scaffolds was 542,691 bp.

The order of scaffolds was determined by a similarity search among published references and confirmed by PCR size checks. Gaps both within and between scaffolds can be closed by long-range PCR (using MG Taq-HF DNA polymerase; Macrogen, Seoul, South Korea) and subsequent Sanger sequencing using an ABI 3730XL capillary sequencer. The sequences from ABI 3730XL sequencing and scaffolds were completely assembled into one circular genome by the use of Phred/Phrap/Consed software (1) and annotated with the Prokaryote Genomes Automatic Annotation Pipeline (PGAAP) (7).

The complete genome is composed of a circular chromosome of 5,259,571 bp (57.6% GC content), which includes 5,035 coding genes, 87 tRNA genes, and 25 rRNA genes. A total of 4,923 coding genes (97.77% of the total) have putative functions assigned on the basis of annotation. Plasmid pKCTC2242 contains 202,852 bp (50.2% GC content; 229 coding sequences [CDS]). A transcriptome experiment using KCTC 2242 and the Roche FLX system was performed to improve its genome annotation. Although small amounts of non-rRNA sequences were obtained from the transcriptome analysis, 6,195 reads (91%) of the 6,746 mapped non-rRNA reads were mapped to 1,169 predicted coding genes.

We found that acetoin production increases rapidly in the stationary phase during production of 2,3-butanediol. We also found that, after the 2,3-butanediol production is examined by transcriptome analysis, the expression level of acetoin reductase increases sharply. As described above, in producing 2,3-butanediol, it is necessary to block the route of succinic acid, lactic acid, and fumaric acid, as well as to inactivate acetoin reductase.

Nucleotide sequence accession numbers.

The complete sequences of the Klebsiella pneumoniae KCTC 2242 main chromosome and of the Klebsiella pneumoniae KCTC 2242 pKCTC2242 plasmid have been deposited in NCBI GenBank (accession numbers CP002910 and CP002911, respectively).