Identiﬁcation and Molecular Characterization of the Operon Required for L-Asparagine Utilization in Corynebacterium glutamicum

: Understanding the metabolic pathways of amino acids and their regulation is important for the rational metabolic engineering of amino acid production. The catabolic pathways of L-asparagine and L-aspartate are composed of transporters for amino acid uptake and asparaginase and aspartase, which are involved in the sequential deamination to fumarate. However, knowledge of the catabolic genes for asparagine in bacteria of the Actinobacteria class has been limited. In this study, we identiﬁed and characterized the ans operon required for L-Asn catabolism in Corynebacterium glutamicum R. The operon consisted of genes encoding a transcriptional regulator (AnsR), asparaginase (AnsA2), aspartase (AspA2), and permease (AnsP). The enzymes and permease encoded in the operon were shown to be essential for L-Asn utilization, but another asparaginase, AnsA1, and aspartase, AspA1, were not essential. Expression analysis revealed that the operon was induced in response to extracellular L-Asn and was transcribed as a leaderless mRNA. The DNA-binding assay demonstrated that AnsR acted as a transcriptional repressor of the operon by binding to the inverted repeat at its 5 (cid:48) -end region. The AnsR binding was inhibited by L-Asn. This study provides insights into the functions and regulatory mechanisms of similar operon-like clusters in related bacteria.


Introduction
Corynebacterium glutamicum is a non-pathogenic, Gram-positive, and facultative anaerobic bacterium belonging to the class Actinobacteria. It has been used for the industrial production of amino acids, especially L-glutamate and L-lysine [1][2][3]. The annual global production of amino acids increased to 7.0 million tons in 2016 [4] and is expected to grow further. Strains for amino acid production have been routinely developed using mutagenesis and selection processes. Recent advances in systems and synthetic biology have enabled rational metabolic engineering of strains for the production of not only amino acids but also chemicals, alcohols, and proteins (reviewed in [4][5][6]). For a rational design, information pertaining to metabolic pathways, their regulation, and the characteristics of the enzymes involved is highly important.
Among the amino acids highly produced by C. glutamicum, L-lysine belongs to the aspartate family amino acids (L-Lys, L-Met, L-Thr, and L-Ile), which have a broad spectrum of applications, including those as food additives, feed supplements, pharmaceuticals, cosmetics, and polymer materials [4,7]. A number of strains have been developed for the production of the aspartate family amino acids based on findings from random mutagenesis developed for the production of the aspartate family amino acids based on findings from random mutagenesis and omics studies (reviewed in [7]). The precursor L-Asp is formed by aspartate aminotransferase, which catalyzes the amination of oxaloacetate using glutamate as an amino donor. The second pathway for L-Asp formation is the deamination of L-Asn by asparaginase. Endogenous asparaginase in C. glutamicum has been purified and characterized [8]. Aspartate ammonia-lyase (aspartase), which catalyzes the reversible deamination of L-Asp to fumarate and ammonium, is encoded in the genome of C. glutamicum and acts as the third pathway for supplying L-Asp ( Figure  1a). However, a previous study reported no aspartase activity in C. glutamicum [9]. Even though asparaginase and aspartase are encoded in the genome, C. glutamicum has been recognized to be incapable of utilizing L-Asp and L-Asn as a carbon source. Biochemical and genetic studies regarding the bacterial utilization of L-Asn and L-Asp have been reported. Two enzymes, namely asparaginase and aspartase, are required for the use of the amino acids ( Figure 1). Asparaginase deaminates L-Asn to generate L-Asp and ammonia. Aspartase deaminates L-Asp to fumarate, which is metabolized in the TCA cycle. Escherichia coli has one aspartase and two asparaginases; one is a cytoplasmic enzyme with low affinity, while the other is located in the periplasm and has high affinity [10,11]. The periplasmic enzyme AnsB plays a role in the provision of an electron acceptor under anaerobic conditions [12]. In Bacillus subtilis, asparaginase and aspartase are encoded by the ansAB operon, which is induced in response to extracellular L-Asn [13]. As asparaginase releases ammonia from L-Asn, another asparaginase, AnsZ, is regulated in response to nitrogen limitation [14]. Pseudomonas aeruginosa utilizes L-Asn and L-Asp as carbon and nitrogen sources and D-Asn as a nitrogen source. It possesses two asparaginases and one aspartase, which are induced by L-Asn and L-Asp, respectively [15]. Mycobacterium tuberculosis has secretory asparaginase and uses the enzyme to assimilate nitrogen and to resist acid stress in macrophages [16]. In the enteric bacterium Biochemical and genetic studies regarding the bacterial utilization of L-Asn and L-Asp have been reported. Two enzymes, namely asparaginase and aspartase, are required for the use of the amino acids ( Figure 1). Asparaginase deaminates L-Asn to generate L-Asp and ammonia. Aspartase deaminates L-Asp to fumarate, which is metabolized in the TCA cycle. Escherichia coli has one aspartase and two asparaginases; one is a cytoplasmic enzyme with low affinity, while the other is located in the periplasm and has high affinity [10,11]. The periplasmic enzyme AnsB plays a role in the provision of an electron acceptor under anaerobic conditions [12]. In Bacillus subtilis, asparaginase and aspartase are encoded by the ansAB operon, which is induced in response to extracellular L-Asn [13]. As asparaginase releases ammonia from L-Asn, another asparaginase, AnsZ, is regulated in response to nitrogen limitation [14]. Pseudomonas aeruginosa utilizes L-Asn and L-Asp as carbon and nitrogen sources and D-Asn as a nitrogen source. It possesses two asparaginases and one aspartase, which are induced by L-Asn and L-Asp, respectively [15]. Mycobacterium tuberculosis has secretory asparaginase and uses the enzyme to assimilate nitrogen and to resist acid stress in macrophages [16]. In the enteric bacterium Yersinia pseudotuberculosis, aspartase is upregulated under acidic conditions and is involved in acid stress resistance [17]. Thus, carbon and nitrogen sources and environmental stress affect the expression of bacterial asparaginase and aspartase.
In this study, we identified and characterized a gene-cluster encoding asparaginase, aspartase, and L-Asn permease in the genome of C. glutamicum strain R. The gene products conferred the ability to utilize L-Asn as a carbon source. The expression of these genes was induced in response to exogenous L-Asn. The enzymes in this cluster would offer an alternative aspartate supply option for amino acid production.

Bacterial Strains, Plasmids, and Culture Conditions
The bacterial strains and plasmids used in our study are listed in Table 1. For genetic manipulation, E. coli strains were cultivated in lysogeny broth (LB) at 37 • C. For the growth experiments, C. glutamicum strains were precultured overnight at 33 • C in a nutrient-rich medium (A medium) [18] supplemented with 2% glucose. The cells were inoculated into a minimal medium (BTM medium) [19] supplemented with 2% glucose or 20 mM amino acids in 96-well titer plates. The cultivation and growth monitoring were performed using a microplate reader HiTS (Scinics, Tokyo, Japan) as described previously [20]. The optical density (OD) at 600 nm was monitored for 24 h. For expression analysis, C. glutamicum strains were precultured as described above. The cells were resuspended in fresh A medium and inoculated into 100 mL of A medium in a 500 mL flask. The initial OD at 610 nm was 0.5. After 3 h, L-Asn or L-Asp was added at 20 mM. Growth was monitored by measuring the OD at 610 nm. The antibiotic concentrations used for E. coli cultures included 50 µg ampicillin mL −1 and 50 µg kanamycin mL −1 , and kanamycin (50 µg mL −1 ) was used in the case of C. glutamicum.

Construction of Plasmids and Mutants
General recombination experiments, including the transformation of E. coli and plasmid extraction, were performed according to standard protocols [21]. To construct the plasmids for the deletion of the target gene, the flanking regions of the gene were amplified using the primers listed in Table S1. The PCR fragments were fused by overlapping PCR or ligation and then cloned into the sucrose counter-selectable suicide vector pCRB725 [24]. Wild-type C. glutamicum cells were transformed using the constructed plasmids. Kanamycin-resistant clones were screened using sucrose medium, as described previously [24]. The ansA1 gene in the asnA2 deletion mutant or the aspA1 gene in the aspA2 deletion mutant was deleted to construct double-deletion mutants of asparaginase or aspartase genes.

RNA Extraction
For expression analysis, C. glutamicum strains were precultured overnight at 33 • C in A medium supplemented with 2% glucose. The cells were resuspended in fresh A medium and inoculated into 100 mL of A medium in a 500 mL flask (the initial OD at 610 nm was 0.5). After 3 h, L-Asn or L-Asp was added at 20 mM. For RNA stabilization, the sampled culture was mixed with an equal volume of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). After incubation at room temperature, the cells were collected by centrifugation and stored at −80 • C. Total RNA was isolated using NucleoSpin ® RNA (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer's instructions. Purified RNA was treated with DNase I (Takara Bio Inc., Shiga, Japan), precipitated using ethanol, suspended in RNase-free water, and stored at −80 • C until further use.

Determination of Transcriptional Start Site (TSS)
The TSS of the ans operon was determined by 5 -rapid amplification of cDNA ends (RACE) analysis using the SMARTer™ RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA, USA), as described previously [25]. The total RNA extracted from the cells was poly(A)-tailed. cDNA was synthesized using the supplied oligo-dT anchored primer. The cDNA was amplified using the Universal Primer A (supplied with the kit) and gene-specific primer (Table S1). PCR products were cloned into the pGEM-T Easy Vector (Promega Corporation, Madison, WI, USA). Several clones harboring the 5 RACE-PCR product were sequenced to determine the TSS.

Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR)
qRT-PCR was performed using the 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Power SYBR ® Green PCR Master Mix with MuLV Reverse Transcriptase and RNase Inhibitor from the GeneAmp RNA PCR Kit (Thermo Fisher Scientific), as previously described [26]. The primers used are listed in Table S1. The comparative threshold cycle method (Thermo Fisher Scientific) was used to quantify the relative expression, and the relative expression ratios of each gene were normalized using the values for 16S rRNA.

Overexpression and Purification of His-Tagged AnsR
The ansR gene was amplified and cloned into pColdI (Takara) to generate pCRC667. E. coli BL21(DE3) cells were transformed with pCRC667 and grown at 37 • C in LB medium until OD at 600 nm reached 0.5. After cooling and incubating at 15 • C, 1 mM IPTG was added to induce His-tagged AnsR expression. Cells were collected and mechanically disrupted using FastPrep FP120 (Thermo Savant, Waltham, MA, USA). The His-tagged protein was purified with affinity chromatography using Ni-NTA agarose (Qiagen) according to the manufacturer's instructions. The eluted protein was desalinated using a PD-10 column (GE Healthcare Bioscience, Piscataway, NJ, USA) pre-equilibrated with buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , and 1 mM EDTA). The protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) with bovine serum albumin (BSA) as a standard.

Electrophoretic Mobility Shift Assay (EMSA)
DNA fragments encompassing the promoter and intragenic regions of the ans operon were generated by PCR using the primers listed in Table S1 and were used as DNA probes. The DNA probes were incubated with purified His-tagged AnsR in buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 3 mM MgCl 2 , 5 mM CaCl 2 , 0.1 mM EDTA, 0.1 mM DTT, 10% glycerol, and 0.15 mg/mL BSA) at room temperature for 25 min. To examine the effect of effector molecules on AnsR binding, the AnsR was incubated for 15 min with effectors prior to incubation with DNA probes. The reaction mixtures were loaded onto a 5% polyacrylamide gel and electrophoresed in 0.5x Tris-borate-EDTA buffer (89 mM Tris, 89 mM borate, and 2 mM EDTA, pH 8.3). The gel was stained with SYBR ® Green EMSA nucleic acid gel stain (Thermo Fischer Scientific, Waltham, MA, USA) and visualized using LAS3000 (Fujifilm, Tokyo, Japan).

Conservation of the ans Gene Cluster
In the genome of C. glutamicum strain R, we found a gene cluster consisting of four genes (cgR_2807-cgR_2810); the genes encoded a GntR-type transcriptional regulator, asparaginase, aspartase, and permease, respectively (Figure 1b). We designated the genes in the cluster as ansR, ansA2, aspA2, and ansP, respectively. Another asparaginase, AnsA1, and aspartase, AspA1, were encoded by cgR_2025 and cgR_1563, respectively, at different loci in the genome. While ansA1 encoding AnsA1 and aspA1 encoding AspA1 are conserved among all corynebacterial genomes, a homologous gene cluster encoding asparaginase and/or aspartase is not conserved in the type strain ATCC 13032 but is found in the genomes of the following limited species of Corynebacterium: Corynebacterium suranareeae, Corynebacterium deserti, Corynebacterium callunae, and Corynebacterium cyclohexanicum. The function of the genes in the operon has not been investigated in any of the bacteria mentioned above.

Role of Genes in the Operon for L-Asn Utilization
As ansA2 encoded an asparaginase, the utilization of L-Asn as a carbon source was examined. When L-Asn was used as the sole carbon source, strain R was able to grow, but strain ATCC 13032, in which the ans cluster was not encoded, was unable to grow ( Table 2), suggesting the involvement of the gene cluster in L-Asn utilization. We examined the role of these genes in the ans cluster by individually deleting each gene. None of the deletion mutants of ansA2, aspA2, or ansP could grow in minimal medium with L-Asn as the sole carbon source, indicating that the genes in the cluster were essential for L-Asn utilization. In contrast, the deletion mutants of ansA1 and aspA1, which encoded another asparaginase, AnsA1, and aspartase, AspA1, respectively, grew at the same level as that observed for the wild type, showing that these enzymes are not involved in L-Asn utilization.
To examine the role of the gene cluster in the utilization of L-Asn as a nitrogen source, the gene deletion mutants were cultivated in minimal medium containing glucose as a carbon source and L-Asn as a nitrogen source. All deletion mutants, including the ansA1 and aspA1 mutants, grew in the medium, indicating that the asparaginases AnsA1 and AnsA2 functioned equally in utilizing L-Asn as a nitrogen source. Indeed, double-deletion mutants of ansA1 and ansA2 were unable to utilize L-Asn as the nitrogen source (Table 1).

ans Operon Induced upon L-Asn Supplementation
The ans gene cluster was shown to be essential for L-Asn utilization. To examine whether the gene cluster was induced by L-Asn, the expression of the genes was measured before and after the supplementation of L-Asn. The wild-type strain was grown in nutrientrich medium for 3 h, and then L-Asn or L-Asp was supplemented at 20 mM. The expression of the genes in the cluster was highly upregulated after 30 min of L-Asn supplementation ( Figure 2). Supplementation of L-Asp had no effect on the gene expression. These results show that the ans gene cluster was induced in response to L-Asn.
These four genes were similarly upregulated, indicating that they form an operon. RT-PCR analysis using RNA extracted from cells grown in the presence of L-Asn demonstrated that these four genes were transcribed as a polycistronic mRNA. The transcriptional start site (TSS) was determined with RACE-PCR. The TSS was mapped to adenine at position +13 with respect to the initiation codon ATG of the first gene, ansR. As another ATG codon existed at this position, the original annotation was incorrect, and the gene cluster was transcribed as a leaderless mRNA from the adenine of the newly defined initiation codon (Figure 3). The −10 and −35 regions of the σ 70 -type housekeeping sigma factor σ A -dependent promoter [27] were located upstream of the TSS (Figure 3). Table 2. Growth properties of strains on glucose or amino acids as a sole carbon or nitrogen source. Cell growth was monitored using optical density at 600 nm for 24 h. Data of deletion mutants of genes in the ans operon are highlighted with the same color as the corresponding genes in Figure 1. Data of type strain ATCC 13032 are highlighted with gray.

Genotype
Carbon or Nitrogen Source Amino Acids As Glucose L-Asparagine     Expression analysis of the ans operon genes in response to the supplementation of Lasparagine or L-aspartate. RNA was extracted before (−) or 30 min after (+) the amino acid supplementation. Transcript levels of ansR, ansA2, aspA2, and ansP were determined by quantitative reverse-transcription polymerase chain reaction analysis. Transcript levels in the WT before the amino acid supplementation were standardized to 1. Mean values obtained from three independent cultivations are shown with their standard deviations.

ans Operon Repressed by AnsR
The first gene of the operon, ansR, encoded a GntR-type transcriptional regulator. To examine whether the regulator was involved in the L-Asn-responsive expression of the operon, an ansR gene deletion mutant was constructed. The wild type and ansR deletion mutant were cultivated in nutrient-rich medium and supplemented with L-Asn or L-Asp to examine the changes in the expression of the ans operon, as described above. The expression of the operon genes in the deletion mutant was much higher than that in the wild type before the supplementation of amino acids ( Figure 2). The expression levels of the genes in the mutant were maintained for 30 min with L-Asn or L-Asp supplementation. In the wild type, the expression of the ans operon was not altered upon supplementation of L-Asp. These findings indicate that the ans operon was repressed by AnsR. The growth of the ansR deletion mutant on nutrient-rich medium supplemented with L-Asp was comparable to that of the wild type, whereas the mutant reached a slightly higher OD than the wild type in the growth experiments carried out using medium supplemented with L-Asn.

AnsR Directly Repressed the ans Operon
To examine whether AnsR directly represses the ans operon, the binding of AnsR to the promoter region of the operon was investigated using EMSA. A DNA probe encompassing the promoter region (positions −209 and +21 with respect to the TSS) was

ans Operon Repressed by AnsR
The first gene of the operon, ansR, encoded a GntR-type transcriptional regulator. To examine whether the regulator was involved in the L-Asn-responsive expression of the operon, an ansR gene deletion mutant was constructed. The wild type and ansR deletion mutant were cultivated in nutrient-rich medium and supplemented with L-Asn or L-Asp to examine the changes in the expression of the ans operon, as described above. The expression of the operon genes in the deletion mutant was much higher than that in the wild type before the supplementation of amino acids ( Figure 2). The expression levels of the genes in the mutant were maintained for 30 min with L-Asn or L-Asp supplementation. In the wild type, the expression of the ans operon was not altered upon supplementation of L-Asp. These findings indicate that the ans operon was repressed by AnsR. The growth of the ansR deletion mutant on nutrient-rich medium supplemented with L-Asp was comparable to that of the wild type, whereas the mutant reached a slightly higher OD than the wild type in the growth experiments carried out using medium supplemented with L-Asn.

AnsR Directly Repressed the ans Operon
To examine whether AnsR directly represses the ans operon, the binding of AnsR to the promoter region of the operon was investigated using EMSA. A DNA probe encompassing the promoter region (positions −209 and +21 with respect to the TSS) was incubated with the purified AnsR. A shifted band was observed, which increased in an AnsR-amountdependent manner (Figure 4). Therefore, it was concluded that AnsR binds to the promoter region and directly represses operon expression. incubated with the purified AnsR. A shifted band was observed, which increased in an AnsR-amount-dependent manner ( Figure 4). Therefore, it was concluded that AnsR binds to the promoter region and directly represses operon expression. To locate the AnsR binding site, a series of DNA probes encompassing the upstream and intragenic regions of the ansR gene were used in the EMSA. AnsR did not interact To locate the AnsR binding site, a series of DNA probes encompassing the upstream and intragenic regions of the ansR gene were used in the EMSA. AnsR did not interact with the DNA probe encompassing the region between positions −209 and +6 with respect to the TSS, but it bound to the DNA probe encompassing positions +1 and +198 (Figure 4). Combined with these findings, the region between positions +1 and +21 with respect to the TSS is likely to be required for AnsR binding. In this region, the inverted repeat 5 -ACCTGTCTGACAGCT-3 was found at positions +5 and +19 with respect to the TSS. To investigate the involvement of this site in AnsR binding, EMSA was performed with DNA probes containing mutations in the putative binding site ( Figure 5). As the putative binding site was located in the intragenic region, the mutation(s) was/were designed to make synonymous codons, except for probe PM7. A DNA probe containing mutations in the inverted repeat reduced the affinity for AnsR. In contrast, the point mutation of T at the center of the binding site had no effect on the binding. The DNA probe with the inverted repeat that was completely exchanged using several mutations (PM7) had entirely lost its interaction with AnsR. These findings confirm that the inverted repeat was the AnsR-binding site. The finding that AnsR binding to the P1 probe was weaker than its bindings to the P3 and P4 probes ( Figure 4) is likely because the binding site was located at the end of the P1 probe.
To search for metabolites (effector molecules) affecting AnsR binding, AnsR was incubated with L-Asn or L-Asp prior to incubation with the DNA probe. When AnsR was incubated with the DNA probe in the presence of L-Asn, free DNA increased in a dose-dependent manner, indicating that L-Asn inhibited the binding of AnsR to the site ( Figure 6). In contrast, L-Asp had no effect on AnsR binding.  To search for metabolites (effector molecules) affecting AnsR binding, AnsR was incubated with L-Asn or L-Asp prior to incubation with the DNA probe. When AnsR was incubated with the DNA probe in the presence of L-Asn, free DNA increased in a dosedependent manner, indicating that L-Asn inhibited the binding of AnsR to the site ( Figure  6). In contrast, L-Asp had no effect on AnsR binding.  To search for metabolites (effector molecules) affecting AnsR binding, AnsR was incubated with L-Asn or L-Asp prior to incubation with the DNA probe. When AnsR was incubated with the DNA probe in the presence of L-Asn, free DNA increased in a dosedependent manner, indicating that L-Asn inhibited the binding of AnsR to the site ( Figure  6). In contrast, L-Asp had no effect on AnsR binding.

Discussion
In this study, we identified and characterized the ans operon that encodes asparaginase, aspartase, and L-Asn transporter in C. glutamicum. Gene deletion studies have revealed that the genes in this operon are essential for L-Asn utilization as a carbon source. Other asparaginase and aspartase, which are presumably and constitutively expressed, are not involved in L-Asn utilization. Expression analysis demonstrated that the genes in the operon are co-transcribed and are induced by L-Asn. L-Asn-responsive expression is controlled by the transcriptional regulator AnsR, whose DNA-binding activity is inhibited in the presence of L-Asn.
The proteins encoded in the operon showed the highest identity with those in the genomes of bacteria of the genera Arthrobacter, Glutamicibacter, and Rhodococcus, which belong to the class Actinobacteria, and the genus Rhizobium of Alphaproteobacteria. To the best of our knowledge, L-Asn utilization genes have not been demonstrated in the bacteria belonging to the Actinobacteria class described above. Thus, the findings related to the ans operon in C. glutamicum provide insights into the physiological functions and regulation of the genes in related bacteria. For example, Mycobacterium dioxanotrophicus, which belongs to the class Actinobacteria, possesses the AnsR ortholog BTO20_32665. L-Asn permease is encoded directly downstream of this gene, suggesting that the permease is repressed by the regulator, although neither asparaginase nor aspartase gene are found in the flanking region.
The ans operon is repressed by AnsR under normal growth conditions and derepressed in response to the presence of L-Asn. L-Asn-responsive transcriptional regulatory system for the asparaginase gene is prevalent in bacteria. Rhizobium etli contains a similar gene cluster encoding a GntR-type transcriptional regulator (AnsR), L-Asn permease (AnsP), asparaginase (AnsA), and aspartase (AnsB) in that order, in the large plasmid p42e [28,29]. Unlike the ans operon in C. glutamicum, the transcriptional regulator gene is not co-transcribed with the other genes ansPAB [28]. Indeed, the σ 70 -type promoter motif is found between ansR and ansP [28,30]. Although not directly demonstrated, the transcriptional regulator AnsR is involved in L-Asn-responsive induction of this operon [28]. The expression of the operon was not induced by L-Asp. The expression level induced by L-Asn was decreased in the presence of other carbon sources such as glucose and glycerol [31]. The expression of the Rhizobium operon was insensitive to the nitrogen source in the medium as well as oxygen levels. The operon ansAB encoding the asparaginase AnsA and the aspartase AnsB in B. subtilis is also induced in response to the presence of L-Asn. The XRE-family transcriptional regulator AnsR, which is encoded by ansR and divergently transcribed from the operon, represses the ansAB operon and mediates L-Asn-responsive expression [13,14,32]. B. subtilis has another asparaginase, AnsZ, which is positively regulated by the global nitrogen regulator TnrA in response to a nitrogen source [14]. While both asparaginases AnsA and AnsZ liberate ammonia, their expression is regulated by distinct regulators that sense different molecules.
In P. aeruginosa, the L-Asn permease AnsP and asparaginase AnsA are encoded by the ansPA operon, which is induced in response to L-Asn [15]. The Cro/CI family transcriptional regulator AnsR, which is divergently transcribed from ansP, positively regulates this operon. In addition, the aspartate ammonia lyase (aspartase) AspA is encoded in a different genomic locus and is positively regulated by the LysR-type transcriptional regulator AspR in response to extracellular or endogenous L-Asp [15]. In addition to aspA, transporters for acidic amino acids and C4 dicarboxylate, which are induced in response to L-Asp, are required for the amino acid utilization. C. glutamicum is unable to utilize L-Asp as a carbon source, probably because of the lack of a corresponding uptake system.
To the best of our knowledge, C. glutamicum AnsR is the first case of an asparagine catabolic gene repressor whose binding site has been identified. The AnsR binding site is located downstream of the TSS, indicating a roadblock mechanism for repression by AnsR. AnsR belongs to the FadR subfamily of the GntR family, whose consensus binding site is 5 -TNGTNNNACNA-3 [33]. The identified AnsR binding site was the inverted repeat 5 -ACCTGTCTGACAGCT-3 , which partially matched the consensus site. Mutational analysis of the binding site confirmed the importance of the inverted repeat. Highly similar putative binding sites are found in the 5 -region of the AnsR encoding genes in the bacteria of the genera Arthrobacter, Glutamicibacter, and Streptomyces, which carry the orthologous asparagine operon in the genome. This is likely because the six N-terminal residues of orthologous regulators in these bacteria are highly conserved. In the genome of some species of the genus Rhodococcus in the Actinobacteria class, the AnsR encoding gene is divergently transcribed from genes encoding aspartase (AspA), L-Asn permease (AnsP), and asparaginase (AnsA). A putative AnsR binding site was found in the intergenic region between ansR and aspA. Thus, the operon in these bacteria is plausibly controlled by a comparable transcriptional regulatory system.
Aspartase catalyzes not only the deamination of aspartate to fumarate for utilization as a carbon source, but also the amination of fumarate to L-Asp with ammonia ( Figure 1a). Generally, L-Asp is synthesized by aspartate aminotransferase, which catalyzes oxaloacetate amination using L-Glu as an amino donor. The amination reaction of aspartase, which utilizes ammonia instead of L-Glu, could be applied to the production of the aspartate family amino acids. Indeed, to increase L-Asp availability for L-Lys production, E. coli aspartase has been introduced into a L-Lys production strain of C. glutamicum. When fumarate was supplied, L-Lys production was increased [9]. The enzymatic characterization of the aspartases AspA1 and AspA2 is worth investigation for the application.
The ans operon is encoded in limited corynebacterial species and strains. Similarly, the strain-specific genes involved in carbon utilization and outer protein synthesis have been identified in C. glutamicum: β-glucoside [34,35], shikimate [36], phenylacetate [37], and cellular surface protein [38]. Characterization of such strain-specific genes would advance our understanding of the catabolic capability of C. glutamicum and lead to the identification of novel regulatory mechanisms.