Ribulokinase and Transcriptional Regulation of Arabinose Metabolism in Clostridium acetobutylicum

Abstract

The transcription factor AraR controls utilization of L-arabinose in Bacillus subtilis. In this study, we combined a comparative genomic reconstruction of AraR regulons in nine Clostridium species with detailed experimental characterization of AraRmediated regulation in Clostridium acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase, AraK, present in all analyzed Clostridium species was identified, which was a nonorthologous replacement of previously characterized ribulokinases. The predicted function of the araK gene was confirmed by inactivation of the araK gene in C. acetobutylicum and biochemical assays using purified recombinant AraK. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of L-arabinose on DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by testing gene expression patterns in both wild-type and araR-inactivated mutant strains during growth in the absence or presence of L-arabinose.

Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13 C Metabolic Flux Analysis

“…A DNA microarray analysis has shown that this gene was strongly induced by arabinose (33). We have found that expression of this xfp gene was regulated by the transcriptional factor AraR, which controls arabinose utilization in C. acetobutylicum (48). Moreover, we have detected the xylulose-5-P phosphoketolase activity in C. acetobutylicum grown on arabinose by using the in vitro enzyme assay (data not shown).…”

Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum

“…Abbreviations are as follows: XynT, sugar/Na + (H + ) simporter; AraE1/AraE2/XylT, sugar-proton symporter; XylA, xylose isomerase [43]; XynB, Beta-xylosidase; XylB, xylulose kinase; Tkt1/Tkt2, transketolase; Tal, transaldolase; AraA1/AraA2, L-arabinose isomerase; AraK, ribulokinase [44]; AraD, L-Ribulose-5-phosphate 4-epimerase; ScrA, beta-glucosides specific PTS IIBCA component; ScrB, sucrase-6-phosphate hydrolase; ScrK, fructokinase; MltF, mannitol-specific PTS IIA component; MtlA, mannitol-specific PTS IIBC component; MtlD, mannitol-1-phosphate 5-dehydrogenase; FruB, 1-phosphofructokinase; Pmi, phosphomannose isomerase; GlvA, maltose-6-phosphate glucosidase; GlgC, glucose-1-phosphate adenylyltransferase; GlgA, starch synthase; LacG, 6-phospho-beta-galactosidase; LacA, galactose-6-phosphate isomerase subunit A; LacB, galactose-6-phosphate isomerase subunit B; LacC, tagatose-6-phosphate kinase; GatY, tagatose-bisphosphate aldolase; GalK, galactokinase; GalT, galactose-1-phosphate uridylyltransferase; GalE, UDP-galactose 4-epimerase; GlpK, glycerol kinase; GlpA, glycerol-3-phosphate dehydrogenase; GanA, arabinogalactan endo-1,4-beta-galactosidase; GlpF, glycerol uptake facilitator protein; GlcK, glucokinase; GlvG, 6-phospho-alpha-glucosidase; GlgC, glucose 1-phosphate adenylyltransferase; GlgA, glycogen synthase; AraT , arabinosides-proton symporter [44]; Arb43, Alpha-L-arabinofuranosidase II precursor [44]; Ptk, phosphoketolase; Rpi, ribose 5-phosphate isomerase; Rpe, aldose-1-epimerase; Epi, arabinose mutarotase; GAP, glyceraldehyde 3-phosphate; F1P, fructose 1-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-biphosphate; G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; C6P, cellobiose 6-phosphate; Xylu5P, xylulose 5-phosphate; DHA, dihydroxyacetone.…”

Combination of RNA sequencing and metabolite data to elucidate improved toxic compound tolerance and butanol fermentation of Clostridium acetobutylicum from wheat straw hydrolysate by supplying sodium sulfide

“…Only an alternative xylose isomerase gene, xylA-II (CAC2610), was identified in C. acetobutylicum (Gu et al, 2010). However, both xylA-II and xylB may be essential for xylose metabolism in C. acetobutylicum because inactivation of either gene resulted in the loss of xylose consumption (Gu et al, 2010;Xiao et al, 2011;Zhang et al, 2012). xylA-II (CAC2610) is required for converting xylose into xylulose during the first step of xylose metabolism, and CAC2610 exhibited 14.97 times stronger expression in +SS than in ÀSS.…”