Draft Genome Sequence of Butanol-Acetone-Producing Clostridium beijerinckii Strain G117

Abstract

A recently discovered wild-type strain, Clostridium beijerinckii G117, is unique in producing butanol and acetone but negligible amounts of ethanol, unlike previously identified acetone-butanol-ethanol (ABE)-generating microbes. Here we report the draft genome sequence of strain G117 (5,806,675 bp; GC content, 29.7%) and the novel findings obtained from its genome annotations.T he solventogenic Clostridium species have attracted renewed attention in the scientific field during the past few years due to the search for sustainable sources of energy (5). Among solventogenic microbes, the Gram-positive anaerobe Clostridium beijerinckii NCIMB 8052 is capable of utilizing pentose and hexose sugars to produce acetone, butanol, and ethanol (ABE) without the "glucose repression" effect, which gives it an attractive advantage over another well-known solventogenic organism, Clostridium acetobutylicum (10). Recently, a newly discovered isolate, Clostridium beijerinckii G117, was distinguished as generating acetone and butanol (AB) but negligible ethanol from fermentation of glucose (1a). The 16S rRNA gene of strain G117 shows 99% identity to that of C. beijerinckii NCIMB 8052. The genome sequencing data for C. beijerinckii NCIMB 8052 will facilitate our understanding of the "omics" (e.g., genomics, transcriptomics, and metabolomics) of solventogenic microbes (5, 9). To further our understanding of solventogenesis in this organism, we present a draft genome sequence of C. beijerinckii G117.The genome of C. beijerinckii G117 was sequenced by a wholegenome shotgun strategy using a high-throughput Illumina HiSeq 2000 at the Beijing Genomics Institute (BGI, Shenzhen, China). A total of 1,766,892 reads with a 296-bp insert size, counting up to 523 Mbp, were obtained, providing 90-fold coverage. Genome sequences were assembled by using the SOAPdenovo program (version 1.05) (6), resulting in 178 contigs with an N 50 of 95,461 bp and a total length of 5,806,675 bp for the whole genome. These contigs were assembled into 89 scaffolds with a maximum length of 271,884 bp. Putative protein coding sequences were identified by Glimmer (version 3.02) (2) and analyzed by BLASTP. The functions of predicted protein-coding genes were annotated through comparisons with Kyoto Encyclopedia of Genes and Genomes (KEGG) (3), Clusters of Orthologous Groups (COG) (8), and Swiss-Prot (1) databases. tRNA and rRNA were annotated with tRNAscan-SE 1.21 (7) and rRNAmmer 1.2 (4), respectively.The draft genome includes 5,806,675 bp with a low GC content of 29.7%, and the average nucleotide identity between strain G117 and NCIMB 8052 was determined to be 97.39%. The genome contains 5,262 predicted genes, 5,111 protein-coding sequences (CDSs), 54 tRNA genes, and 20 rRNAs (including 11 5S rRNAs, 3 16S rRNAs, and 6 23S rRNAs). Among the predicted CDSs, 2,687, 2,937, and 2,102 proteins were functionally annotated in the COG, KEGG, and Swiss-Prot databases, respectively. The annotations indicate that a total of 198 CDSs are involved in energy production and conversi...

Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

“…2). In contrast, previous studies exhibited typical sequential utilization, consuming glucose first and then xylose (9,14,24,36,37,38). For instance, when providing a mixture of glucose and xylose (30 g/liter each), strain G117 consumed all of the glucose in the first 48 h, and xylose utilization was initiated only after the glucose was completely consumed (after 48 h), resulting in a total of 6.5 g/liter butanol after 100 h of fermentation.…”

“…To elucidate whether the high xylose utilization is related to a high degree of expression of xylose isomerase in cultured BOH3, gene expression analysis was conducted on strain BOH3 and results were compared to those for the low-xylose-utilizing solventogenic Clostridium sp. strain G117 (producing 5.2 g/liter butanol from 60 g/liter xylose) (9,36). During the acidogenic phase (0 to 20 h), the expression level of xylose isomerase in strain BOH3 increased up to 165-fold, which is 9 times higher than that of strain G117 (Fig.…”

“…The xylose isomerase gene-specific primers used for qPCR were designed based on the xylose isomerase gene sequence of cultured BOH3 and G117, with the forward primers 5=-ATGTTGCAGTTACAGAGGGAGA-3= (strain BOH3) and 5=-CTGTTTTACTAATCCAA GGTATGTTCA-3 (strain G117) and reverse primers 5=-TTTCATCTTGGCTTACCTT GTC-3= (strain BOH3) and 5=-ATCTAGCTCTTTTGTTATTTCA ATTGC-3= (strain G117). Primers for the housekeeping genes [(3R)-hydroxymyristoyl-ACP dehydratase (fabZ) for strain BOH3 (23) and peptidase T (pepT) for strain G117 (36)] are the following: strain BOH3-forward, 5=-AAAT AGAACCAGGGAAAAGAGCA-3=; strain BOH3-reverse, 5=-GCAACAC CACCAAGTTGAGC-3=; strain G117-forward, 5=-TGATGGAG GCGAG GAAGGTG-3=; and strain G117-reverse, 5=-CATTGTATTCTTTGCAGA CCCTGG-3=. The following real-time PCR program was used: 10 min at 95°C, 45 cycles of 10 s at 95°C, and 30 s at 55°C, followed by a meltingpoint analysis (45 to 95°C) after the final PCR step to verify the specificity of amplified products.…”

Direct conversion of xylan to butanol by a wild‐type Clostridium species strain G117

“…The breakdown of xylan polymers into monomers can be achieved through a group of enzymes collectively called xylanases. Draft genome sequence analysis of strain G117 revealed the presence of several xylanase related genes (Wu et al, ), indicating the genetic potential to breakdown xylan into more metabolically tractable monomers (Table ). Direct fermentation strategies were therefore attempted using strain G117 with 60 g/L xylan as the sole carbon source.…”

“…cDNA samples were used as a DNA template for PCR amplification of the adc and Peptidase T ( pepT ) genes. The pepT gene (Wu et al, ) is a housekeeping gene for strain G117 and was used as an internal control to monitor efficiency of cDNA synthesis and PCR amplification. Details of the primers used for the experiments are shown in Table .…”