bThe Corynebacterium glutamicum ATCC 31831 araBDA operon consists of three L-arabinose catabolic genes, upstream of which the galM, araR, and araE genes are located in opposite orientation. araR encodes a LacI-type transcriptional regulator that negatively regulates the L-arabinose-inducible expression of araBDA and araE (encoding an L-arabinose transporter), through a mechanism that has yet to be identified. Here we show that the AraR protein binds in vitro to three sites: one upstream of araBDA and two upstream of araE. We verify that a 16-bp consensus palindromic sequence is essential for binding of AraR, using a series of mutations introduced upstream of araB in electrophoretic mobility shift assays. Moreover, the DNA-binding activity of AraR is reduced by L-arabinose. We employ quantitative reverse transcription-PCR (qRT-PCR) analyses using various mutant strains deficient in L-arabinose utilization genes to demonstrate that the prominent upregulation of araBDA and araE within 5 min of L-arabinose supplementation is dependent on the uptake but independent of the catabolism of L-arabinose. Similar expression patterns, together with the upregulation by araR disruption without L-arabinose, are evident with the apparent galM-araR operon, although attendant changes in expression levels are much smaller than those realized with the expression of araBDA and araE. The AraR-binding site upstream of araB overlaps the ؊10 region of the divergent galM promoter. These observations indicate that AraR acts as a transcriptional repressor of araBDA, araE, and galM-araR and that L-arabinose acts as an intracellular negative effector of the AraR-dependent regulation.

In Corynebacterium glutamicum ATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression of L-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BS B ) between the divergent operons (araBDA and galM-araR) and two (BS E1 and BS E2 ) upstream of araE. L-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BS B mutation resulted in derepression of both araBDA and galM-araR operons. The effects of BS E1 and/or BS E2 mutation on araE expression revealed that the two sites independently function as the cis elements, but BS E1 plays the primary role. However, AraR was shown to bind to these sites with almost the same affinity in vitro. Taken together, the expression of araBDA and araE is strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the ؊10 or ؊35 region of the galM-araR and araE promoters is less effective in repression. Furthermore, downregulation of araBDA and araE dependent on L-arabinose catabolism observed in the BS B mutant and the AraR-independent araR promoter identified within galM-araR add complexity to regulation of the AraR regulon derepressed by L-arabinose. IMPORTANCECorynebacterium glutamicum has a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. Most C. glutamicum strains are unable to use a pentose sugar L-arabinose as a carbon source. However, genes for L-arabinose utilization and its regulation have been recently identified in C. glutamicum ATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression by L-arabinose and thereby highlights the complex regulatory feedback loops in combination with L-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner. L ignocellulosic biomass from agricultural and agro-industrial residues represents one of the important renewable resources expected to be used for the industrial production of biofuels and biochemicals (1, 2). However, significant amounts of pentose sugars derived from its hemicellulose component are one major technical hurdle in the use for economically feasible bioprocesses. These sugars, such as D-xylose and L-arabinose, are not efficiently utilized by conventional and industrial microorganisms, and improvement of the pentose sugar metabolic pathways by genetic engineering is often limited by preferential utilization of a most abundant and favorable hexose sugar, D-glucose, in lignocellulosic biomass (3).Corynebacterium glutamicum is a Gram-positive actinobacterium with a high GϩC content in its genomic DNA. It has a long history as an industri...

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-L-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-L-arabinofuranoside with k values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.