AraR, an l -Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

Abstract

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...

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“…l ‐arabinose utilization in most C glutamicum strains require the heterologous expression of l ‐arabinose catabolism genes from E. coli, except for C. glutamicum ATCC 31831, which has a functional gene cluster for l ‐arabinose utilization, including arabinose transporter ( araE ), arabinose isomerase ( araA ), ribulokinase ( araB ), and ribulose 5‐phosphate 4‐epimerse ( araD ) (Fig. ) . The heterologous expression of l ‐arabinose catabolism genes from E. coli resulted in the production of 5.44 g L −1 L ‐glutamate, 8.04 g L −1 L ‐lysine, 11.8 g L −1 L ‐ornithine, and 4.36 g L −1 L ‐arginine (Table ) …”

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“…This identification is based on changes in their affinity for binding to target operon sequences, alterations in their interactions with other cellular components, or shifts in their capability to form dimer, trimer, or tetramer structures (Ottilie et al 2022 ; Phaneuf et al 2020 ). araR as a GntR family member regulates of carbon catabolism not only of B. subtilis but also other microorganisms (Correia et al 2014 ; Kuge et al 2015 ). Low DNA binding affinity to the arabinose operon was caused by a non-synonymous nucleotide substitution location in araR H226R close to the arabinose binding site on the C-terminal domain (Franco et al 2006 ).…”

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“…Subsequently, C. glutamicum ATCC31831 with the ability to grow on L-arabinose was discovered by random screening. Analysis of the genome of this strain suggested that genes associated with arabinose utilization were included in the araBAD operon, which is negatively controlled by the transcription factor AraR (Kuge et al, 2015). In addition, simultaneous utilization of L-arabinose and D-glucose in C. glutamicum ATCC31831 indicated that carbon metabolism repression was ineffective against arabinose in this strain (Kawaguchi et al, 2009).…”