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Apr 24, 2012 - 2006), benzyl alcohol, 2,4-dihydroxybenzoate, 3,5-dihy- droxytoluene (Shen et al. ..... nol (Nordin et al. 2005), 2,4,6-trichlorophenol (Louie et al.
Appl Microbiol Biotechnol DOI 10.1007/s00253-012-4139-4

MINI-REVIEW

Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium? Xi-Hui Shen & Ning-Yi Zhou & Shuang-Jiang Liu

Received: 18 February 2012 / Revised: 24 April 2012 / Accepted: 24 April 2012 # Springer-Verlag 2012

Abstract With the implementation of the well-established molecular tools and systems biology techniques, new knowledge on aromatic degradation and assimilation by Corynebacterium glutamicum has been emerging. This review summarizes recent findings on degradation of aromatic compounds by C. glutamicum. Among these findings, the mycothiol-dependent gentisate pathway was firstly discovered in C. glutamicum. Other important knowledge derived from C. glutamicum would be the discovery of linkages among aromatic degradation and primary metabolisms such as gluconeogenesis and central carbon metabolism. Various transporters in C. glutamicum have also been identified, and they play an essential role in microbial assimilation of

Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4139-4) contains supplementary material, which is available to authorized users. X.-H. Shen State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi 712100, China N.-Y. Zhou Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China S.-J. Liu State Key Laboratory of Microbial Resources at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China S.-J. Liu (*) Environmental Microbiology Research Center at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China e-mail: [email protected]

aromatic compounds. Regulation on aromatic degradation occurs mainly at transcription level via pathway-specific regulators, but global regulator(s) is presumably involved in the regulation. It is concluded that C. glutamicum is a very useful model organism to disclose new knowledge of biochemistry, physiology, and genetics of the catabolism of aromatic compounds in high GC content Gram-positive bacteria, and that the new physiological properties of aromatic degradation and assimilation are potentially important for industrial applications of C. glutamicum. Keywords Corynebacterium glutamicum . Aromatic compounds . Degradation and assimilation . Transport . Regulation

Introduction Corynebacterium glutamicum is a fast growing, aerobic, and non-pathogenic Gram-positive soil bacterium. It was isolated in an effort to screen for L-glutamate-producing bacteria (Kinoshita et al. 2004; Udaka 1960). Since its discovery, C. glutamicum has been widely investigated and applied in industrial production of various amino acids and vitamins (Hermann 2003; Leuchtenberger et al. 2005; Becker et al. 2009), and recently of bio-based chemicals such as succinate (Okino et al. 2008a), lactate (Okino et al. 2008b), ethanol (Inui, et al. 2004; Sakai et al. 2007), 1,4-diaminobutane (Schneider and Wendisch 2010), 1,5-diaminopentane (Mimitsuka et al. 2007), pyruvate (Wieschalka et al. 2012), and isobutanol (Blombach et al. 2011). Due to its industrial importance, the genomes of several strains of C. glutamicum have been sequenced (Kalinowski et al. 2003; Ikeda and Nakagawa 2003; Yukawa et al. 2007; Lv et al. 2011, 2012). After genomic data mining, genetic clusters potentially

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encoding aromatic degradation were identified, and this observation invoked our interest in the degradation of aromatic compounds by C. glutamicum. The knowledge of microbial degradation of aromatic compounds, as well as its significance and importance for understanding the geobiochemical cycles and for application in removal of such aromatic pollutants from various environments, have been well summarized (Harwood and Parales 1996; Díaz 2004; Cao et al. 2009; Fuchs et al. 2011). Although C. glutamicum has been used as a model bacterium for fermentative production of various amino acids and vitamins, the knowledge of aromatic degradation and assimilation by this bacterium had been rarely explored until recently. This review is aimed at summarizing the recent findings of new physiology of C. glutamicum on aromatic compound degradation and assimilation, transport, and regulation.

C. glutamicum assimilates diverse aromatic compounds It is known that C. glutamicum utilizes less carbohydrates than Escherichia coli and had to be engineered for utilization of glycerol (Rittmann et al. 2008), arabinose (Kawaguchi et al. 2007), xylose (Kawaguchi et al. 2006), or starch (Tateno et al. 2007). In contrast, its ability to utilize aromatic compounds is impressive. C. glutamicum grows on the following aromatic compounds: benzoate, phenol (Shen et al. 2004), 3-hydrobenzoate, gentisate (Shen et al. 2005b), protocatechuate, vanillate, 4-hydroxybenzoate, 4-cresol (Shen and Liu 2005; Qi et al. 2007), resorcinol (Huang et al. 2006), benzyl alcohol, 2,4-dihydroxybenzoate, 3,5-dihydroxytoluene (Shen et al. 2005a), naphthalene (Lee et al. 2010b), vanillin, ferulic acid (Merkens et al. 2005), cinnamate, caffeate, and 4-coumarate (unpublished data). These compounds and their derivatives are channeled into central carbon metabolic pathways, as discussed in the following sections. C. glutamicum was not able to grow on L-phenylalanine, L-tyrosine, and L-tryptophan as carbon sources (Zhao et al. 2011). Exploration of the genome indicated that their degradative pathways for these aromatic amino acids in C. glutamicum were incomplete. Homologs of well-known genes for aromatic amino acid degradation in E. coli, e.g., tyrB, the aromatic amino acid aminotransferase gene; tnaA, the tryptophanase gene; and hmgA, the homogentisate 1,2dioxygenase gene, are missing from the C. glutamicum genome (Ikeda and Nakagawa 2003; Kalinowski et al. 2003; Zhao et al. 2011). Although not reported, this might be a very important reason that C. glutamicum had been repeatedly screened out as an aromatic amino acid producer (Ikeda and Nakagawa 2003).

Peripheral pathways leading to aromatic-ring cleavage in C. glutamicum Bacteria have developed two completely different strategies to degrade aromatic compounds depending on the presence or absence of oxygen (Díaz 2004; Carmona et al. 2009; Fuchs et al. 2011). In the aerobic catabolism of aromatics, structurally diverse aromatic compounds are metabolized through different peripheral pathways to some common intermediates such as catechol, protocatechuate, and gentisate that are subsequently cleaved by ring-cleavage dioxygenases and finally channeled into the central carbon metabolism (Díaz 2004; Cao et al. 2009; Fuchs et al. 2011). The ring-cleavage dioxygenases, catechol 1,2-dioxygenase, protocatechuate 3,4-dioxygenase, gentisate 1,2dioxygenase, and hydroxyquinol 1,2-dioxygenase, were functionally identified in C. glutamicum strain ATCC13032 (Shen et al. 2004, 2005a, b; Shen and Liu 2005; Merkens et al. 2005). Recently, a phenylacetyl-CoA ring-cleavage pathway has been identified in C. glutamicum AS1.542 and strain R, but not in ATCC13032 (unpublished result). Based on genome data mining and experimental results, various peripheral and central pathways have been mapped in C. glutamicum (Fig. 1 and Table S1). The following paragraphs focus on the peripheral pathways leading to protocatechuate. Other peripheral pathways for converting resorcinol, phenol, benzoate, 3-hydrobenzoate, or naphthalene will be discussed in the sections of individual pathways for each compound. The peripheral pathways leading to protocatechuate were explored and experimentally confirmed for various phenylpropenoids such as vanillin, vanillate, ferulate, cinnamate, 4-coumarate, and caffeate in C. glutamicum. Putative vanillin dehydrogenase gene (vdh) is identified based on sequence identity, but has not been experimentally confirmed. The genes (vanA, vanB) encoding vanillate demethylase in C. glutamicum that catalyzes the conversion of vanillate to protocatechuate were functionally identified (Merkens et al. 2005). VanAB of C. glutamicum is highly similar to the VanABs from other bacteria (Priefert et al. 1997; Segura et al. 1999; Kalinowski et al. 2003). Studies with Pseudomonas species revealed that the metabolism of ferulate proceeded via a CoA-dependent, non-β-oxidative peripheral pathway (Venturi et al. 1998; Mitra et al. 1999; Overhage et al. 1999; Jiménez et al. 2002), enzymes homologous to the pseudomonad feruloyl-CoA synthetase (Fcs), and enoyl-CoA hydratase/aldolase (EcH) were identified in Fig. 1 Pathways for the catabolism of aromatic compounds in C.„ glutamicum. A question mark indicates that the enzyme encoding such biochemical step is unknown. The five central aromatic intermediates, i.e. protocatechuate, catechol, gentisate, hydroxyquinol and phenylacetylCoA, are shown in bold

Appl Microbiol Biotechnol 4-cresol

OH

?

H 3C

O

4-hydroxybenzyl alcohol OH



p-coumarate

O

-

?



feruloyl-CoA O ACo S

Fcs

-

OH 4-hydroxybenzoate

OH

OOC

PobA -

Ech

OH OH

-

COO

OOC

O

H benzoate diol BenD

OH

H

OH

OH Vdh

-

OCH3

-

-

Fcs

-

COO

OH

O

maleylpyruvate

-

COO

HO

CatB γ-carboxyCOO muconolactone O

OOC

COO O COO

-

PcaB

O

NagI

CatA

β-carboxyCOO cis,cis-muconate COO cis,cis-muconate

OOC

Ech

OH

HO

PcaGH

Vdh

-

COO OH

gentisate

OH VanAB

NahG

OH

catechol

OCH3

3-hydroxybenzoate

OH

OH dehydroshikimate

OH

HO

OH

QuiC

OOC

-

COO

BenABC

OH

QuiA

protocatechuate

vanillate OOC

benzoate

OH

QuiB -

?

-

COO

OH

OOC

dehydroquinate

Vdh

-

?

shikimate OH

OCH3

vanillin O



OOC

O

Ech

OH

caffeate

-

HO

OCH3

O

OH

QuiA

OH

O

naphthalene

OH

Fcs

4-hydroxy benzaldehyde OHC

benzyl alcohol CH2 OH

OOC

OH HOH2 C

ferulate

quinate OH

HO

-

O

COO

O

OH

NagL -

O

-

COO O fumarylpyruvate

muconolactone

resorcin OH

-

COO

HO

PcaC 1,2,4-trihydroxybenzene

OH

OH

NCgl1113/ NCgl2951

-

COO

O

-

COO

-

NCgl1112/ NCgl2307 PcaIJ

O

OH

Nagk β-ketoadipate enol-lactone

PcaD

COO COO

3-hydroxycis,cis-muconate

CatC

COO

O

-

-

COO COO

OH NCgl1111 COO

O

maleylacetate

OH

OH

-

COO

-

COO

OOC

pyruvate

COSCOA -

COO

OH 2,4-dihydroxybenzoate

-

fumarate

O

O

+

β-ketoadipate

β-ketoadipyl-CoA

PcaF O

3-hydroxyadipyl-CoA

3-oxoadipyl-CoA

O

O

S CoA PaaF

-

PaaH -

O

PaaJ

COO

O

2,3-dehydroadipyl-CoA

TCA cycle

+ SCO A

O

acetyl-CoA phenylacetaldehyde

S CoA

O

O -

COO PaaG/J

SCO A

succinyl-CoA

-

O

COO

HO

S CoA

O

S CoA

S CoA O

O

PaaZ

O

PaaG

O

O S CoA

S CoA

PaaABCDE

PaaK

NH2

O

O O



PadA

H MaoA

-

COO

3-oxo-5,6-dehydro suberyl-CoA

2-oxepin-2(3H)ylideneacetyl-CoA

ring 1,2-epoxyphenyl acetyl-CoA

phenylacetyl-CoA

phenylacetate

phenylethylamine

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a

PcaR

pcaK

pcaR

pcaJ pcaI

pobA

pcaF

pcaD

C

pcaO

pcaB

pcaG pcaH

PcaO

protocatechuate

ADP

b

BenR

catC

catA

catB

benA

DtxR

RipA

ferulate vanillate

c

resorcinol

quinate shikimate

nahG

RolR

ncgl1111

ncgl1112

ncgl1113

QsuR

qsuR

g

nagT

nagR

rolR

f

vanK

vanB

NagR

nagI

nagK

e

benE

iron

vanA

gentisate 3-hydroxybenzoate

nagL

benK

benR

D

VanR

vanR

d

C

B

qsuA

qsuB

D

C

PaaR

paaT

paaK

paaR

I

paaA

B

C

D

E

G

J

F

H

paaZ

Fig. 2 Genetic organization and regulatory mechanisms of pathways for degradation of aromatic compounds in C. glutamicum. a Regulation of the pca and pob clusters by PcaR and PacO acting on the pcaI, pcaH, pcaF, and pobA promoters. b Regulation of the ben and cat operons by BenR acting on benA, benK, and catA promoters. The expression of CatA was also regulated by RipA repressor. c Negative regulation of the van cluster by a PadR-type regulator VanR. d The nag cluster for gentisate and 3-hydroxybenzoate degradation is positively regulated by NagR, which acts on the nagI and nagT promoters. e Regulation of the hydroxyquinol pathway genes (ncgl1111–ncgl1113) is exerted by a TetR-type repressor RolR. f Positive regulation of the

qsu cluster by a LysR-type regulator QsuR. g The paa cluster encoding a TetR-type regulator PaaR which may be involved in the regulation of paa gene expression. The regulatory genes and regulators are indicated in gray. The horizontal thin arrows represent transcripts produced after specific induction. Thick arrows indicate activation effect and blunt bars indicate repression effect. Functions of regulatory genes identified experimentally are indicated in solid lines, whereas functions assumed but not verified are indicated in broken lines. The names of each substrate as signal molecules in Figs. 1 and 2 are shown in the same color

the genome of C. glutamicum. In addition, the pseudomonad enzymes (Fcs, Ech, and Vdh) also converts 4-coumarate and caffeate to protocatechuate (Venturi et al. 1998; Mitra et al. 1999). As observed in our lab, C. glutamicum was able to grow on 4-coumarate and caffeate (unpublished data), but the enzymes converting 4-coumarate and caffeate to protocatechuate in C. glutamicum is still unknown.

Shikimate, 4-cresol, 4-hydroxylbenzoate, and quinate are also converted into protocatechuate (Fig. 2). The conversion of 4-cresol to 4-hydroxybenzoate was catalyzed by 4-cresol methylhydroxylase (PchCF) and NAD-dependent 4hydroxybenzylalcohol dehydrogenase (PchA) in Geobacter metallireducens (Peters et al. 2007; Johannes et al. 2008; Carmona et al. 2009). Conversion of 4-cresol into 4-

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hydroxybenzoate was observed in C. glutamicum (Shen and Liu 2005); however, detection of 4-cresol methylhydroxylase and PchA activities in 4-cresol-grown cells of C. glutamicum failed. Blast searches for putative genes for PchCF and PchA in C. glutamicum genome were not successful. In a proteomic study, two proteins were specifically induced when C. glutamicum was grown with 4-cresol as carbon source. The genes, namely ncgl0525 and ncgl0527 (Table S1), which putatively encoded novel reductase and dehydrogenase, were found to be essential for C. glutamicum growing on 4-cresol (Qi et al. 2007). So far, the catalytic reaction(s) driven by NCgl0525 and NCgl0527 are still unclear. The conversion of 4-hydroxybenzoate into protocatechuate is catalyzed by 4-hydroxybenzoate 3-hydroxylase, encoded by pobA (ncgl1032), in C. glutamicum. This 4hydroxybenzoate 3-hydroxylase is unique in that it prefers NADPH to NADH as a co-substrate, although its sequence is similar to other 4-hydroxybenzoate 3-hydroxylases that prefer NADH as a co-substrate (Huang et al. 2008). C. glutamicum used quinate and shikimate as carbon source for growth (Teramoto et al. 2009). The genetic cluster (qsuABCD) involved in the conversion of quinate/shikimate to protocatechuate was mapped and functionally characterized (Teramoto et al. 2009) (Figs. 1 and 2). Interestingly, the three proteins QsuD (quinate/shikimate dehydrogenase), QsuC (dehyroquinate dehydratase), and QsuB (dehyroshikimate dehydratase) in C. glutamicum were not homologous to their counterparts such as QuiA, QuiB, and QuiC of Acinetobacter species (Elsemore and Ornston 1994). Instead, the QsuB, QsuC, and QsuD of C. glutamicum were more phylogenetically close to the fungal enzymes QutB, QutE, and QutC (Teramoto et al. 2009).

The discovery of mycothiol (MSH)-dependent gentisate pathway and the MSH-dependent maleylpyruvate isomerase C. glutamicum utilizes 3-hydroxybenzoate, gentisate, and naphthalene as carbon source for growth via the gentisate pathway (Shen et al. 2005a; Lee et al. 2010b) (Fig. 1). In Pseudomonas species, the gentisate pathway for aromatic compound(s) degradation is glutathione (GSH) dependent (Zhou et al. 2001) and is featured by a GSH-dependent maleylpyruvate isomerase. A cluster of six genes was mapped on genome and was involved in the gentisate pathway in C. glutamicum (Shen et al. 2005b; Yang et al. 2010) (Fig. 2 and Table S1). The ncgl2918 is essential to gentisate and 3-hydroxybenzoate assimilation. Based on its genetic location and the previous understanding of gentisate pathway, it was deduced that this gene encoded a maleylpyruvate isomerase. Indeed, maleylpyruvate isomerase activity was determined with cellular lysate of C. glutamicum.

Surprisingly, this activity was not dependent on the addition of GSH, which was clearly different from the known GSHdependent maleylpyruvate isomerase (Zhou et al. 2001). Instead of GSH occurring in many Gram-negative bacteria, MSH is the major low molecular weight thiols in high GC content Gram-positive bacteria such as Mycobacterium, Streptomyces, Rhodococcus, and Corynebacterium. The physiological roles of MSH were believed to be equivalent to those of GSH in Gram-negative bacteria (Newton et al. 2008; Jothivasan and Hamilton 2008). The purified Ncgl2918 took MSH molecules as co-factor and its maleylpyruvate isomerase activity was dependent on the presence of MSH molecules. In addition, MSH gene mutants of C. glutamicum lost the ability to grow on gentisate and 3hydroxybenzoate but retained the ability to assimilate 4hydroxybenzoate, benzoate, phenol, and resorcinol, supporting the existence of an MSH-dependent gentisate pathway. It was reported that an mshC (an essential gene for the biosynthesis of MSH) deficient mutant of Rhodococcus jostii strain RHA1 failed to grow when gentisate and 3-hydroxybenzoate were used as carbon source, suggesting that MSH is also involved in the gentisate assimilation in this strain (Dosanjh et al. 2008). Very recently, the MSH-dependent catabolic gene cluster involved in gentisate, naphthalene, and 3hydroxybenzoate catabolism has been identified in Rhodococcus strain NCIMB12038 (Liu et al. 2011). The purified MSH-dependent maleylpyruvate isomerase (MDMPI) is a monomer of 34 kDa. Its apparent Km and Vmax values for maleylpyruvate were determined to be 148.4 μM and 1,520 μmol min−1 mg−1, respectively (Feng et al. 2006). The crystal structure of MDMPI from C. glutamicum was determined at a resolution of 1.75 Å. The crystal structures reveal that the MDMPI of C. glutamicum contains a C-terminal domain possessing a novel folding pattern (αβαββα fold) and an N-terminal divalent metal (Zn2+)-binding domain. The C-terminal domain is necessary for the enzyme activity and structural stability. Furthermore, site-directed mutagenesis revealed that the Arg222 residue at the C-terminal domain was necessary for MDMPI activity (Wang et al. 2007). The discovery of a mycothiol-dependent maleylpyruvate isomerase (MDMPI) in C. glutamicum introduced a new category of maleylpyruvate isomerase, and constitutes significant progress on the way to understanding both the gentisate pathway of aromatic assimilation and the MSH physiology (Rawat and Av-Gay 2007).

The catechol and hydoxyquinol pathways in C. glutamicum Some aromatic compounds including phenol, benzyl alcohol, and benzoate are degraded via catechol. Phenol is converted to catechol by a phenol hydroxylase that was

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putatively encoded by ncgl2588 (Shen et al. 2005a). Two steps are needed to convert benzoate to catechol: The first step is catalyzed by a benzoate dioxygenase complex, encoded putatively by ncgl2320, ncgl2321, and ncgl2322 in C. glutamicum, by which benzoate was oxidized to benzoate diol. Following this oxidation, the benzoate diol is decarboxylated by 2-hydro-1,2-dihydroxybenzoate dehydrogenase (putatively encoded by ncgl2323) (Fig. 1) to form catechol. These genes (ncgl2320–ncgl23260benABCDRKE) were clustered and were transcribed in opposite direction to the catechol 1,2-dioxygenase gene (catA0ncgl2319) (Fig. 2). This genetic organization in C. glutamicum is conserved in other Grampositive bacteria such as Rhodococcus opacus and different from that in the P. putida KT2440, of which the benABCDK and cat genetic clusters are distantly located (Jiménez et al. 2002). Hydroxyquinol is a central intermediate for many aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants, such as chlorophenol (Nordin et al. 2005), 2,4,6-trichlorophenol (Louie et al. 2002), dibenzo-p-dioxin (Armengaud et al. 1999), 4aminophenol (Takenaka et al. 2003), 4-nitrocatechol (Chauhan et al. 2000), and 4-nitrophenol (Kitagawa et al. 2004). C. glutamicum is able to assimilate resorcinol (1,3dihydroxybenzene), 2,4-dihydroxybenzoate, and 3,5-dihydroxytoluene for growth through the hydroxyquinol pathway (Shen et al. 2005a). Early studies with Gram-negative bacteria indicated that resorcinol was degraded via three different ringcleavage pathways, i.e., the pyrogallol 1,2-dioxygenase pathway in Azotobacter vinelandii (Groseclose and Ribbons 1981), and the 2,3,5-trihydroxytoluene 1,2-dioxygenase and hydroxyquinol 1,2-dioxygenase pathways in P. putida (Chapman and Ribbons 1976). By genome data mining, two genetic clusters, designated ncgl1110–ncgl1113 and ncgl2950–ncgl2953 (Table S1), were proposed to encode proteins involved in resorcinol catabolism. Genetic and biochemical studies demonstrated that both genetic clusters were involved, but only the ncgl1110–nclgl1113 were essential to hydroxyquinol pathway. Expression of ncgl1113 and ncgl2951 in E. coli revealed that both genes coded for hydroxyquinol 1,2-dioxygenases (Shen et al. 2005a; Huang et al. 2006). Researches have shown that Ncgl1111 represents a new type of hydroxylase involved in aromatic compound catabolism (Huang et al. 2006).

The β-ketoadipate pathway in C. glutamicum The β-ketoadipate pathway is the major pathway for ligninderived aromatic compounds assimilation that distributed widely in soil bacteria and fungi (Harwood and Parales 1996; Davis and Sello 2010). As in many other bacteria, the β-ketoadipate pathway in C. glutamicum consists of the

protocatechuate and catechol branches that converge at βketoadipate enol-lactone, and a central pathway of three additional steps (catalyzed by pcaDIJF gene products) leads to the Krebs cycle intermediates, acetyl-CoA and succinylCoA (Fig. 1). The genes involved in the catechol branch are organized in a single gene cluster (ncgl2317–ncgl2319) (Table S1). Very impressively, the genes involved in the protocatechuate branch of β-ketoadipate pathway in C. glutamicum are organized as a supraoperonic cluster, of which 10 genes organized in three independent transcriptional units, i.e., pcaHGBC, pcaIJ, and pcaRFDO. Only a few of these genes have been functionally identified, and the majority of the genes involved in the β-ketoadipate pathway were deduced from sequence identity searches (Shen and Liu 2005). The pca genes are generally more alike to their counterparts of Gram-positive bacteria such as Streptomyces species and R. opacus than to those in Gram-negative bacteria. But significant differences of gene structure and organization were also found between C. glutamicum and Streptomyces species and R. opacus. The genes of the catechol and protocatechuate branches, plus the genes of peripheral pathways leading to the β-ketoadipate central pathway, form a well-organized catabolic island (contribute to 1% of the entire genome) that have not been found in other Gram-positive or Gram-negative bacteria.

The phenylacetyl-CoA ring-cleavage pathway The aerobic phenylacetate catabolism was revealed in E. coli K12, and it represents an unorthodox aromatic ringcleavage strategy that differs significantly from the established chemistry for biodegradation of aromatic compounds (Teufel et al. 2010). The first step of the pathway was previously identified as the activation of phenylacetate into phenylacetyl-CoA by a phenylacetate-CoA ligase (Ferrández et al. 1998). All further intermediates likewise were processed as CoA thioesters, a typical feature of anaerobic rather than aerobic aromatic metabolism (Teufel et al. 2010). A cluster of paa genes involved in the phenylacetate pathway was identified in the genomes of C. glutamicum strain R and of the recently sequenced strain AS1.542 (data unpublished), but not of the ATCC13032 (Table S1). Genome data mining further revealed that the degradation of phenylethylamine proceeds through the phenylacetyl-CoA pathway in strain R (Fig. 1). Phenylethylamine is converted into phenylacetate via the catalysis of amine oxidase (MaoA) and phenylacetaldehyde dehydrogenase (PadA or FeaB) (Parrot et al. 1987; Hacisalihoglu et al. 1997; Díaz et al. 2001). Homologs to MaoA (CgR_0016 or NCgl0220) and PadA (CgR_0018 or Ncgl2698) have been detected in the genome of strain R (Table S1); their involvement and

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physiological functions in phenylethylamine degradation remain to be experimentally confirmed.

Transport of aromatic compounds in C. glutamicum The robust ability of C. glutamicum to grow on a variety of aromatic compounds partially relies on its multiple transporters for uptake of aromatic compounds. Although aromatic compounds can enter the cells by passive diffusion when present at high concentrations, active transport increases the efficiency and rate of substrate acquisition in natural environments where these compounds are present at low concentrations (Shen et al. 2005a). Among the various categories of transporters classified (Ren et al. 2004), most of the functionally identified aromatic compound transport systems belong to either the aromatic acid/H+ symporter (AAHS) family transporters within the major facilitator superfamily (MFS) or the ATP-binding cassette (ABC) superfamily. The following aromatic compound transporters have been identified in C. glutamicum, protocatechuate/4hydroxybenzoate transporter encoded by ncgl1031, vanillate transporter encoded by ncgl2302, gentisate transporter encoded by ncgl2922, two benzoate transporters encoded by ncgl2325 and ncgl2326 (Xu et al. 2006; Chaudhry et al. 2007; Wang et al. 2011), as well as two aromatic amino acids transporters, AroP (Wehrmann et al. 1995) and PheP (Zhao et al. 2011). A putative phenylacetate transporter (CgR_0643) was observed in the putative paa cluster of C. glutamicum strain R, but the function of this gene needs to be confirmed.

Regulation of aromatic compounds metabolism in C. glutamicum So far, regulation of aromatic degradation happens at transcriptional level (Díaz and Prieto 2000; Gerischer 2002; Tropel and van der Meer 2004). A global analysis of C. glutamicum genome revealed that the transcriptional regulation of aromatic compound degradation in C. glutamicum is mainly controlled by single regulatory protein sensing the presence of aromatic compounds, thus representing single input motifs within the transcriptional regulatory network (Brinkrolf et al. 2006). Several genes encoding transcriptional regulators have been identified, including the PcaO and PcaR that regulate the protocatechuate pathway (Brune et al. 2005; Zhao et al. 2010), the NagR that regulates the gentisate pathway (Shen et al. 2005b), the RolR that regulates the hydroxyquinol pathway (Huang et al. 2006; Li et al. 2011). The genes involved in the protocatechuate pathway are regulated in a hierarchical manner exerted by two regulatorencoding genes located in the pca genetic cluster, pcaR and

pcaO (Fig. 2a). The pcaR gene encodes a transcriptional regulator of the IclR family and pcaO codes for a transcriptional activator of the large ATP-binding LuxR (LAL)-type regulator family (Brune et al. 2005; Zhao et al. 2010). PcaR exerts its regulatory role by interacting with 15-bp operator sequences that are located in the pcaI–pcaR intergenic region and upstream of pcaH and pobA. PcaO is the first LAL-type regulator involved in aromatic compounds catabolism (Zhao et al. 2010), although the involvement of LAL-type regulators in regulation of different metabolic pathways has been intensively characterized (Panagiotidis et al. 1998; van Beilen et al. 2001; Evangelista-Martínez et al. 2006). In vitro EMSA results showed that ATP weakened the binding between PcaO and its target sequence but ADP strengthened this binding, while the effect of protocatechuate on PcaO binding was dependent on the protocatechuate concentration. These findings suggest that in the presence of protocatechuate, the transcription of pcaHG is probably controlled by the ratio of ATP to ADP in cells (Zhao et al. 2010). The PcaR–PcaO hierarchical regulatory system in C. glutamicum provides a flexible control that necessary for the degradation of various aromatic compounds that are channeled to the protocatechuate pathway. Several regulatory proteins of the LysR and AraC/XylS family regulators involved in the transcriptional control of benzoate degradation have been characterized (Collier et al. 1998; Cowles et al. 2000). A putative transcriptional regulator, BenR, of benzoate degradation in C. glutamicum was proposed, and the expression level of the benR was significantly upregulated along with other benzoate-degrading genes during growth on benzoate (Haussmann et al. 2009). Sequence analysis showed that BenR was probably a LuxRtype regulator. The LuxR-type regulators are generally functioning as transcriptional activators (Bateman et al. 2002; Hansmeier et al. 2006; Cramer et al. 2006). Thus, it is assumable that BenR activates the ben and/or cat genes in C. glutamicum. It was reported that the expression of catechol 1,2-dioxygenase, an iron-containing enzyme, was also controlled by the RipA repressor, which itself is negatively regulated by the iron sensing regulator DtxR (Wennerhold et al. 2005; Brune et al. 2006) (Fig. 2b). Transcriptional regulation of the vanillate degradation genes (vanABK gene tags0ncgl2300–2302) in C. glutamicum undergoes with a PadR family regulator (VanR) encoded by ncgl2299 (Brune et al. 2005) (Fig. 2c). Deletion of vanR resulted in enhanced transcription of the vanABK genes, demonstrating that VanR is a negative transcriptional regulator. While ferulate and vanillate can induce the transcription of the vanABK gene cluster, glucose and protocatechuate have no positive effect on vanABK expression. Similar to other PadRtype regulators (Barthelmebs et al. 2000; Gury et al. 2004), a region of short dyad symmetry represents a putative operator sequence of VanR was identified downstream of the mapped vanABK promoter in C. glutamicum (Brinkrolf et al. 2006).

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Unlike Ralstonia strain U2, in which the gentisate pathway is regulated by a LysR-type transcriptional activator (Jones et al. 2003), the gentisate pathway in C. glutamicum is regulated by NagR (Ncgl2921), a transcriptional activator that exhibits moderate identities to the transcriptional regulators of the IclR family (Fig. 2d). Similar to other IclR-type regulator genes, nagR (ncgl2921) locates upstream of its target gene cluster and transcribed in the opposite direction, and contains an HTH motif at its N terminus. Activation of gentisate pathway gene transcription by nagR required the existence of gentisate (or 3-hydroxybenzoate) (Shen et al. 2005b). The regulation of the hydroxyquinol pathway at transcriptional level is exerted by a TetR-type repressor, RolR (Resorcinol Regulator), of which is encoded by rolR (gene tag0ncgl1110) and located at the opposite DNA strand upstream of the hydroxyquinol hydroxylase gene (ncgl1111) (Fig. 2e). RolR negatively regulates the transcription of other rol genes. Deletion of the rolR gene resulted in elevated transcription levels of ncgl1111, ncgl1112, and ncgl1113 in C. glutamicum (Brinkrolf et al. 2006). Hyperexpression of rolR completely inhibited the transcription of its target genes, and the hydroxyquinol 1,2-dioxygenase activity in cells was no longer detectable (Huang et al. 2006). A 29-bp operator sequence essential for RolR binding was identified in the intergenic region of ncgl1110 and ncgl1111. The binding of RolR to the operator was affected by resorcinol and hydroxyquinol, two starting compounds of resorcinol catabolic pathway. The structure of resorcinol–RolR complex reveals that the hydrogen-bonded network mediated by the four-residue motif (Asp94-Arg145-Arg148-Asp149) with two water molecules and the hydrophobic interaction via five residues (Phe107, Leu111, Leu114, Leu142, and Phe172) are the key factors for the recognition and binding between the resorcinol and RolR molecules. RolR represents a new subfamily of TetR proteins that are involved in the regulation of microbial degradation of aromatics (Li et al. 2011). The quinate/shikimate utilization operon qsuABCD is regulated by QsuR in C. glutamicum, a LysR-type transcriptional regulator located immediately upstream and opposite to qsuA (Fig. 2f). The expression of the qsuABCD genes was inducible in the presence of quinate or shikimate. Induction of qsuABCD gene transcription by shikimate was also observed in the presence of glucose, and simultaneous consumption of glucose and shikimate was observed during growth (Teramoto et al. 2009). This property is different from that of other microorganisms in which the expression of quinate/shikimate utilization genes is subject to stringent carbon catabolite repression (Dal et al. 2002; Siehler et al. 2007; Hawkins et al. 1993). Deletion of qsuR resulted in the loss of qsuABCD transcription in the presence of shikimate, suggesting that QsuR acts as an activator of the qsuABCD genes (Teramoto et al. 2009).

A TetR family regulator PaaR (CgR_0649) located in the paa gene locus is a potential candidate involving in controlling the paa cluster gene expression in C. glutamicum strain R (Fig. 2g). Regulation of the paa gene cluster by a similar TetR family regulator has been reported in Thermus thermophilus HB8 recently (Sakamoto et al. 2011). In T. thermophilus HB8, this TetR family regulator negatively regulated the transcription of the paa gene cluster by binding pseudopalindromic sequences surrounding the promoters. Phenylacetyl-CoA is an effector of this TetR-type regulator for transcriptional derepression with a proposed binding stoichiometry of 1:1 protein monomer (Sakamoto et al. 2011). However, whether CgR_0649 in C. glutamicum plays a similar role in paa gene regulation remains to be functionally identified. Aromatic compound degradation in C. glutamicum is regulated mainly by pathway-specific regulators. No global regulator had been proposed until putative GlxR binding sites were observed in front of transcription units involved in aromatic compound degradation (Kohl et al. 2008). The GlxR is a DNA-binding transcription factor of the CRP family (Kim et al. 2004), and the CRP protein as a global regulator on multiple carbon metabolism via carbon catabolite repression has been extensively investigated in E. coli (Deutscher 2008). In C. glutamicum, GlxR has been reported to regulate more than 400 genes (Kohl et al. 2008; Kohl and Tauch 2009) covering diverse cellular functions including gluconate metabolism (Letek et al. 2006), acetate metabolism (Park et al. 2010), phosphate uptake (Panhorst et al. 2011), and anaerobic metabolism (Nishimura et al. 2011). However, whether or not the GlxR was involved in catabolite repression to aromatic compound metabolism in C. glutamicum still remains to be determined in the future. Carbon catabolite repression is an important global regulation which allows bacteria to adapt economically and quickly to preferred carbon and energy sources. So far, there are very few carbon catabolite repression responses that have been described for C. glutamicum, except the preferential assimilation of glucose over ethanol and glutamate (Arndt and Eikmanns 2007; Arndt et al. 2008). C. glutamicum simultaneously utilizes glucose with other sugars and organic acids, such as lactate, pyruvate, acetate, and propionate, as well as with aromatic compounds such as vanillate, gentisate, or shikimate (Merkens et al. 2005; Qi et al. 2007; Teramoto et al. 2009). C. glutamicum shows monophasic growth on these substrate mixtures (Cocaign et al. 1993; Domínguez et al. 1997; Wendisch et al. 2000; Claes et al. 2002). This property of C. glutamicum is in contrast to those of other microorganisms such as P. putida, of which the aromatic compound utilization genes (ben, cat, pca, and pobA) are subject to stringent catabolite regulation (Morales et al. 2004). Simultaneous utilization of various carbon

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sources by C. glutamicum is a hallmark of this bacterium setting it apart from yeasts, E. coli and Bacillus subtilis, which typically show sequential utilization of substrates present in blends.

Cross-talking during aromatic compound degradation and central carbon metabolisms in C. glutamicum The growth of C. glutamicum on aromatic compounds is dependent on the functioning of other cellular processes, e.g., gluconeogenesis. Comparative analysis on the proteomes of C. glutamicum from different aromatic compounds and glucose revealed that Fbp (Fructose-1,6bisphophatase) consistently increased its abundance by 2.0–2.7-fold on various aromatic compounds over glucose (Qi et al. 2007). Fbp catalyzes the conversion of fructose1,6-bisphosphate into fructose-6-phospate, and plays a key role in gluconeogenesis that supplies cellular building blocks such as hexose and intermediates of the pentose phosphate pathway for cell growth (Rittmann et al. 2003). Deletion of the fbp gene resulted in the loss of ability to grow on aromatic compounds, indicating that Fbp is essential for aromatic compounds assimilation in C. glutamicum. Notably, increased abundance of phosphoglycerate kinase was also observed when phenol, 4-cresol, resorcinol, and gentisate were used as sole carbon and energy sources during the process of gluconeogenesis (Qi et al. 2007), although phosphoglycerate kinase was reported to be also involved in glycolysis (Eikmanns 1992). This enzyme had also been reportedly increased in Pseudomonas alcaligenes strain NCIMB9867 (Zhao et al. 2005). All these data strongly suggest that the gluconeogenesis pathway, as a main cellular building blocks supplier, is essential for the growth of C. glutamicum when aromatic compounds are used as sole carbon sources. It is reasonable to expect that other enzymes essential for gluconeogenesis, such as pck (Riedel et al. 2001), may also be involved in aromatic degradation in C. glutamicum. The assimilation of aromatic compounds needs other physiological processes, such as central carbon and energy metabolisms, being adjusted accordingly in C. glutamicum (Qi et al. 2007; Haussmann et al. 2009). Comparative proteome analysis of cells grown on gentisate, benzoate, phenol, 4-cresol, and resorcinol indicated that the central carbon metabolism changed differently among various aromatic compounds. With phenol as carbon source, the abundance of isocitrate lyase was reduced 2.8-fold, indicating that carbon flow into the glyoxylate shunt was possibly decreased. When benzoate, 4-cresol, phenol, or resorcinol served as carbon source, the abundances of citrate synthase (GltA) and aconitase A (Acn) that catalyze the first two reactions of TCA cycle were increased, implicating that

the intermediates generated from these aromatic compounds were further metabolized through the TCA cycles (Qi et al. 2007). The gltA and acn are subject to transcriptional control by several regulators such as RamA, RamB, AcnR, and RipA (van Ooyen et al. 2011; Krug et al. 2005; Wennerhold et al. 2005; Emer et al. 2009). More importantly, similar to the transcriptional units involved in aromatic compound degradation, both gltA and acn have functional GlxR operator sites in their regulatory regions (van Ooyen et al. 2011; Han et al. 2008). Indeed, transcriptomics analyses with C. glutamicum grown on glucose or acetate have shown that there is a carbon-source-dependent expression of gltA (Muffler et al. 2002; Gerstmeir et al. 2003). These findings suggest that aromatic degradation genes and TCA cycle genes might be coordinately regulated by carbon catabolite repression. Common for all aromatic compounds examined, the pyruvate/quinone oxidoreductase and pyruvate kinase were newly synthesized, which probably indicated that the carbon flux via the phosphoenolpyruvate–pyruvate–oxaloacetate node (Eikmanns 2005) was increased.

Conclusions and perspectives As a result of extensive genomic analysis and experimental studies in recent years, knowledge on the aromatic compound metabolism in C. glutamicum is accumulating. To further understand the C. glutamicum workhorse for biodegradation and bioconversion, many questions about the regulation and tolerance of aromatic compound metabolism in C. glutamicum remain to be answered. Cross-talking among different aromatic catabolic pathways and other cellular physiological processes is largely an unexplored field that should be addressed in the future. Engineering of highly efficient C. glutamicum strains is also attractive since such strains are potentially useful for bioremediation/bioconversion in lignocellulosic feedstock usage by C. glutamicum. The multiple and advanced molecular and systems biology tools available for C. glutamicum will still be the great advantages for the future studies. It is well known that lignin-derived phenolic compounds (such as vanillin, ferulic acid, benzoate, 4-hydroxybenzoate, vanillate, and phenol) produced in the lignocellulosic hydrolysates are the main growth inhibitors that greatly reduce microbial fermentation into desired products (Klinke et al. 2004; Mills et al. 2009; Parawira and Tekere 2011). Indeed, C. glutamicum has been shown to withstand pretreatmentderived inhibitors of lignocellulosic feedstocks like furfural, hydroxymethyl furfural, and 4-hydroxybenzaldehyde under growth-arrested conditions (Sakai et al. 2007). Although the necessity in the use of cheap feedstock in biorefinery has long been appreciated, this is still a much overlooked area by researchers dealing with C. glutamicum, one of the most

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biotechnologically important workhorses. Thus, a deeper and systematic understanding of aromatic compound assimilation in C. glutamicum is needed. Efforts have been made recently to produce amino acids from aromatic feedstock. Most of the intermediates generated from aromatic compound assimilation are further metabolized through the TCA cycle in C. glutamicum (Shen et al. 2005a; Qi et al. 2007), and amino acids are coupled with TCA cycle in C. glutamicum (Bott 2007). The newly defined amino acid production processes through the utilization of aromatic compounds such as phenol and naphthalene in C. glutamicum provide an alternative way in bioremediation/bioconversion of aromatic pollutants. In phenol-grown cultures (8.5 mM), the production of glutamate and proline were 149.2 mM and 143.3 mM, 1.2 and 14.7 times higher, respectively, than the culture conditions without phenol, suggesting that the metabolic intermediates from phenol degradation fluxed into the central carbon metabolism and were used to produce glutamate and proline (Lee et al. 2010a). When cultured with 4.2 mM naphthalene, aspartate and glutamate production also increased to 15.2 mM and 100.4 mM, 1.5- and 1.3-fold, respectively, compared to control (without naphthalene) (Lee et al. 2010b). Much work is needed in the future to turn this into reality of the concept of using aromatic compounds as feedstock for bioproduction. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (30725001) and Ministry of Science and Technology (2012CB721104).

References Armengaud J, Timmis KN, Wittich RM (1999) A functional 4hydroxysalicylate/hydroxyquinol degradative pathway gene cluster is linked to the initial dibenzo-p-dioxin pathway genes in Sphingomonas sp. strain RW1. J Bacteriol 181:3452–3461 Arndt A, Eikmanns BJ (2007) The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression. J Bacteriol 189:7408–7416 Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ (2008) Ethanol catabolism in Corynebacterium glutamicum. J Mol Microbiol Biotechnol 15:222–233 Barthelmebs L, Lecomte B, Divies C, Cavin JF (2000) Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator. J Bacteriol 182:6724–6731 Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL (2002) The Pfam protein families database. Nucleic Acids Res 30:276–280 Becker J, Klopprogge C, Schroder H, Wittmann C (2009) Metabolic engineering of the tricarboxylic acid cycle for improved lysine production by Corynebacterium glutamicum. Appl Environ Microbiol 75:7866–7869 Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, Eikmanns BJ (2011) Corynebacterium glutamicum tailored

for efficient isobutanol production. Appl Environ Microbiol 77:3300–3310 Bott M (2007) Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends Microbiol 15:417–425 Brinkrolf K, Brune I, Tauch A (2006) Transcriptional regulation of catabolic pathways for aromatic compounds in Corynebacterium glutamicum. Genet Mol Res 5:773–789 Brune I, Brinkrolf K, Kalinowski J, Puhler A, Tauch A (2005) The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences. BMC Genomics 6:86 Brune I, Werner H, Hüser AT, Kalinowski J, Pühler A, Tauch A (2006) The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum. BMC Genomics 7:21 Cao B, Nagarajan K, Loh KC (2009) Biodegradation of aromatic compounds: current status and opportunities for biomolecular approaches. Appl Microbiol Biotechnol 85:207–228 Carmona M, Zamarro MT, Blázquez B, Durante-Rodríguez G, Juárez JF, Valderrama JA, Barragán MJ, García JL, Díaz E (2009) Anaerobic catabolism of aromatic compounds: a genetic and genomic view. Microbiol Mol Biol Rev 73:71–133 Chapman PJ, Ribbons DW (1976) Metabolism of resorcinylic compounds by bacteria: orcinol pathway in Pseudomonas putida. J Bacteriol 125:974–984 Chaudhry MT, Huang Y, Shen XH, Poetsch A, Jiang CY, Liu SJ (2007) Genome-wide investigation of aromatic acid transporters in Corynebacterium glutamicum. Microbiology 153:857–865 Chauhan A, Samanta SK, Jain RK (2000) Degradation of 4nitrocatechol by Burkholderia cepacia: a plasmid-encoded novel pathway. J Appl Microbiol 88:764–772 Claes WA, Pühler A, Kalinowski J (2002) Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle. J Bacteriol 184(10):2728–2739 Cocaign M, Monnet C, Lindley ND (1993) Batch kinetics of Corynebacterium glutamicum during growth on various carbon substrates: use of substrate mixtures to localise metabolic bottlenecks. Appl Microbiol Biotechnol 40:526–530 Collier LS, Gaines GL III, Neidle EL (1998) Regulation of benzoate degradation in Acinetobacter sp. strain ADP1 by BenM, a LysRtype transcriptional activator. J Bacteriol 180:2493–2501 Cowles CE, Nichols NN, Harwood CS (2000) BenR, a XylS homologue, regulates three different pathways of aromatic acid degradation in Pseudomonas putida. J Bacteriol 182:6339–6346 Cramer A, Gerstmeir R, Schaffer S, Bott M, Eikmanns BJ (2006) Identification of RamA, a novel LuxR-type transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J Bacteriol 188:2554–2567 Dal S, Steiner I, Gerischer U (2002) Multiple operons connected with catabolism of aromatic compounds in Acinetobacter sp. strain ADP1 are under carbon catabolite repression. J Mol Microbiol Biotechnol 4:389–404 Davis JR, Sello JK (2010) Regulation of genes in Streptomyces bacteria required for catabolism of lignin-derived aromatic compounds. Appl Microbiol Biotechnol 86:921–929 Deutscher J (2008) The mechanisms of carbon catabolite repression in bacteria. Curr Opin Microbiol 11:87–93 Díaz E (2004) Bacterial degradation of aromatic pollutants: a paradigm of metabolic versatility. Int Microbiol 7:173–180 Díaz E, Prieto MA (2000) Bacterial promoters triggering biodegradation of aromatic pollutants. Curr Opin Biotechnol 11(5):467– 475

Appl Microbiol Biotechnol Díaz E, Ferrández A, Prieto MA, García JL (2001) Biodegradation of aromatic compounds by Escherichia coli. Microbiol Mol Biol Rev 65:523–569 Domínguez H, Cocaign-Bousquet M, Lindley ND (1997) Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum. Appl Microbiol Biotechnol 47:600–603 Dosanjh M, Newton GL, Davies J (2008) Characterization of a mycothiol ligase mutant of Rhodococcus jostii RHA1. Res Microbiol 159:643–650 Eikmanns BJ (1992) Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase. J Bacteriol 174:6076–6086 Eikmanns BJ (2005) In: Eggeling L, Bott M (eds) Handbook of Corynebacterium glutamicum. Taylor and Francis, New York, pp 241–276 Elsemore DA, Ornston LN (1994) The pca–pob supraoperonic cluster of Acinetobacter calcoaceticus contains quiA, the structural gene for quinate-shikimate dehydrogenase. J Bacteriol 176:7659–7666 Emer D, Krug A, Eikmanns BJ, Bott M (2009) Complex expression control of the Corynebacterium glutamicum aconitase gene: identification of RamA as a third transcriptional regulator besides AcnR and RipA. J Biotechnol 140:92–98 Evangelista-Martínez Z, González-Cerón G, Servín-González L (2006) A conserved inverted repeat, the LipR box, mediates transcriptional activation of the Streptomyces exfoliatus lipase gene by LipR, a member of the STAND class of P-loop nucleoside triphosphatases. J Bacteriol 188:7082–7089 Feng J, Che Y, Milse J, Yin YJ, Liu L, Ruckert C, Shen XH, Qi SW, Kalinowski J, Liu SJ (2006) The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum. J Biol Chem 281:10778–10785 Ferrández A, Miñambres B, García B, Olivera ER, Luengo JM, García JL, Díaz E (1998) Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic hybrid pathway. J Biol Chem 273:25974–25986 Fuchs G, Boll M, Heider J (2011) Microbial degradation of aromatic compounds—from one strategy to four. Nat Rev Microbiol 9:803–816 Gerischer U (2002) Specific and global regulation of genes associated with the degradation of aromatic compounds in bacteria. J Mol Microbiol Biotechnol 4:111–121 Gerstmeir R, Wendisch VF, Schnicke S, Ruan H, Farwick M, Reinscheid D, Eikmanns BJ (2003) Acetate metabolism and its regulation in Corynebacterium glutamicum. J Biotechnol 104:99–122 Groseclose EE, Ribbons DW (1981) Metabolism of resorcinylic compounds by bacteria: new pathway for resorcinol catabolism in Azotobacter vinelandii. J Bacteriol 146:460–466 Gury J, Barthelmebs L, Tran NP, Divies C, Cavin JF (2004) Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum. Appl Environ Microbiol 70:2146–2153 Hacisalihoglu A, Jongejan JA, Duine JA (1997) Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca. Microbiology 143:505–512 Han SO, Inui M, Yukawa H (2008) Effect of carbon source availability and growth phase on expression of Corynebacterium glutamicum genes involved in the tricarboxylic acid cycle and glyoxylate bypass. Microbiology 154:3073–3083 Hansmeier N, Albersmeier A, Tauch A, Damberg T, Ros R, Anselmetti D, Pühler A, Kalinowski J (2006) The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a

LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032. Microbiology 152:923–935 Harwood CS, Parales RE (1996) The beta-ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50:553–590 Haussmann U, Qi SW, Wolters D, Rögner M, Liu SJ, Poetsch A (2009) Physiological adaptation of Corynebacterium glutamicum to benzoate as alternative carbon source—a membrane proteome-centric view. Proteomics 9:3635–3651 Hawkins AR, Lamb HK, Moore JD, Charles IG, Roberts CF (1993) The pre-chorismate (shikimate) and quinate pathways in filamentous fungi: theoretical and practical aspects. J Gen Microbiol 139:2891–2899 Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172 Huang Y, Zhao KX, Shen XH, Chaudhry MT, Jiang CY, Liu SJ (2006) Genetic characterization of the resorcinol catabolic pathway in Corynebacterium glutamicum. Appl Environ Microbiol 72:7238–7245 Huang Y, Zhao KX, Shen XH, Jiang CY, Liu SJ (2008) Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum. Appl Microbiol Biotechnol 78:75–83 Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109 Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H (2004) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254 Jiménez JI, Miñambres B, García JL, Díaz E (2002) Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environ Microbiol 4:824–841 Johannes J, Bluschke A, Jehmlich N, von Bergen M, Boll M (2008) Purification and characterization of active-site components of the putative p-cresol methylhydroxylase membrane complex from Geobacter metallireducens. J Bacteriol 190:6493–6500 Jones RM, Britt-Compton B, Williams PA (2003) The naphthalene catabolic (nag) genes of Ralstonia sp. strain U2 are an operon that is regulated by NagR, a LysR-type transcriptional regulator. J Bacteriol 185:5847–5853 Jothivasan VK, Hamilton CJ (2008) Mycothiol: synthesis, biosynthesis and biological functions of the major low molecular weight thiol in actinomycetes. Nat Prod Rep 25:1091–1117 Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, Dusch N, Eggeling L, Eikmanns BJ, Gaigalat L, Goesmann A, Hartmann M, Huthmacher K, Krämer R, Linke B, McHardy AC, Meyer F, Möckel B, Pfefflerle W, Pühler A, Rey DA, Ruckert C, Rupp O, Sahm H, Wendisch VF, Wiegräbe I, Tauch A (2003) The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartatederived amino acids and vitamins. J Biotechnol 104:5–25 Kawaguchi H, Vertès AA, Okino S, Inui M, Yukawa H (2006) Engineering of a xylose metabolic pathway in Corynebacterium glutamicum. Appl Environ Microbiol 72:3418–3428 Kawaguchi H, Sasaki M, Vertès AA, Inui M, Yukawa H (2007) Engineering of an L-arabinose metabolic pathway in Corynebacterium glutamicum. Appl Microbiol Biotechnol 77:1053–1062 Kim HJ, Kim TH, Kim Y, Lee HS (2004) Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J Bacteriol 186:3453–3460 Kinoshita S, Udaka S, Shimono M (2004) Studies on amino acid fermentation. Part I. Production of L-glutamic acid by various microorganisms. J Gen Appl Microbiol 50:331–343 Kitagawa W, Kimura N, Kamagata Y (2004) A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101. J Bacteriol 186:4894–4902

Appl Microbiol Biotechnol Klinke HB, Thomsen AB, Ahring BK (2004) Inhibition of ethanolproducing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66:10–26 Kohl TA, Tauch A (2009) The GlxR regulon of the amino acid producer Corynebacterium glutamicum: detection of the corynebacterial core regulon and integration into the transcriptional regulatory network model. J Biotechnol 143:239–246 Kohl TA, Baumbach J, Jungwirth B, Pühler A, Tauch A (2008) The GlxR regulon of the amino acid producer Corynebacterium glutamicum: in silico and in vitro detection of DNA binding sites of a global transcription regulator. J Biotechnol 135:340–350 Krug A, Wendisch VF, Bott M (2005) Identification of AcnR, a TetRtype repressor of the aconitase gene acn in Corynebacterium glutamicum. J Biol Chem 280:585–595 Lee SY, Kim YH, Min J (2010a) Conversion of phenol to glutamate and proline in Corynebacterium glutamicum is regulated by transcriptional regulator ArgR. Appl Microbiol Biotechnol 85:713–720 Lee SY, Le TH, Chang ST, Park JS, Kim YH, Min J (2010b) Utilization of phenol and naphthalene affects synthesis of various amino acids in Corynebacterium glutamicum. Curr Microbiol 61:596– 600 Letek M, Valbuena N, Ramos A, Ordóñez E, Gil JA, Mateos LM (2006) Characterization and use of catabolite-repressed promoters from gluconate genes in Corynebacterium glutamicum. J Bacteriol 188:409–423 Leuchtenberger W, Huthmacher K, Drauz K (2005) Biotechnological production of amino acids and derivates: current status and prospects. Appl Microbiol Biotechnol 69:1–8 Li DF, Zhang N, Hou YJ, Hu Y, Zhang Y, Liu SJ, Wang DC (2011) Crystal structures of the transcriptional repressor RolR reveals a novel recognition mechanism between inducer and regulator. PLoS One 6:e19529 Liu TT, Xu Y, Liu H, Luo S, Yin YJ, Liu SJ, Zhou NY (2011) Functional characterization of a gene cluster involved in gentisate catabolism in Rhodococcus sp. strain NCIMB 12038. Appl Microbiol Biotechnol 90:671–678 Louie TM, Webster CM, Xun L (2002) Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134. J Bacteriol 184:3492–3500 Lv Y, Wu Z, Han S, Lin Y, Zheng S (2011) Genome sequence of Corynebacterium glutamicum S9114, a strain for industrial production of glutamate. J Bacteriol 193:6096–6097 Lv Y, Liao J, Wu Z, Han S, Lin Y, Zheng S (2012) Genome sequence of Corynebacterium glutamicum ATCC 14067, which provides insight into amino acid biosynthesis in coryneform bacteria. J Bacteriol 194:742–743 Merkens H, Beckers G, Wirtz A, Burkovski A (2005) Vanillate metabolism in Corynebacterium glutamicum. Curr Microbiol 51:59– 65 Mills TY, Sandoval NR, Gill RT (2009) Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnol Biofuels 2:26 Mimitsuka T, Sawai H, Hatsu M, Yamada K (2007) Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation. Biosci Biotechnol Biochem 71:2130–2135 Mitra A, Kitamura Y, Gasson MJ, Narbad A, Parr AJ, Payne J, Rhodes MJ, Sewter C, Walton NJ (1999) 4-Hydroxycinnamoyl-CoA hydratase/lyase (HCHL)—an enzyme of phenylpropanoid chain cleavage from Pseudomonas. Arch Biochem Biophys 365:10–16 Morales G, Linares JF, Beloso A, Albar JP, Martínez JL, Rojo F (2004) The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds. J Bacteriol 186:1337–1344 Muffler A, Bettermann S, Haushalter M, Hörlein A, Neveling U, Schramm M, Sorgenfrei O (2002) Genome-wide transcription

profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose. J Biotechnol 98:255–268 Newton GL, Buchmeier N, Fahey RC (2008) Biosynthesis and functions of mycothiol, the unique protective thiol of Actinobacteria. Microbiol Mol Biol Rev 72:471–494 Nishimura T, Teramoto H, Toyoda K, Inui M, Yukawa H (2011) Regulation of nitrate reductase operon narKGHJI by cyclic AMP-dependent regulator GlxR in Corynebacterium glutamicum. Microbiology 157:21–28 Nordin K, Unell M, Jansson JK (2005) Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A6. Appl Environ Microbiol 71:6538–6544 Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H (2008a) An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain. Appl Microbiol Biotechnol 81:459–464 Okino S, Suda M, Fujikura K, Inui M, Yukawa H (2008b) Production of D-lactic acid by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 78:449–454 Overhage J, Priefert H, Steinbüchel A (1999) Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp. strain HR199. Appl Environ Microbiol 65:4837–4847 Panagiotidis CH, Boos W, Shuman HA (1998) The ATP-binding cassette subunit of the maltose transporter MalK antagonizes MalT, the activator of the Escherichia coli mal regulon. Mol Microbiol 30:535–546 Panhorst M, Sorger-Herrmann U, Wendisch VF (2011) The pstSCAB operon for phosphate uptake is regulated by the global regulator GlxR in Corynebacterium glutamicum. J Biotechnol 154:149– 155 Parawira W, Tekere M (2011) Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review. Crit Rev Biotechnol 31:20–31 Park SY, Moon MW, Subhadra B, Lee JK (2010) Functional characterization of the glxR deletion mutant of Corynebacterium glutamicum ATCC 13032: involvement of GlxR in acetate metabolism and carbon catabolite repression. FEMS Microbiol Lett 304:107– 115 Parrot S, Jones S, Cooper RA (1987) 2-Phenylethylamine catabolism by Escherichia coli K12. J Gen Microbiol 133:347–351 Peters F, Heintz D, Johannes J, van Dorsselaer A, Boll M (2007) Genes, enzymes, and regulation of para-cresol metabolism in Geobacter metallireducens. J Bacteriol 189:4729–4738 Priefert H, Rabenhorst J, Steinbüchel A (1997) Molecular characterization of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate. J Bacteriol 179:2595– 2607 Qi SW, Chaudhry MT, Zhang Y, Meng B, Huang Y, Zhao KX, Poetsch A, Jiang CY, Liu S, Liu SJ (2007) Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase. Proteomics 7:3775–3787 Rawat M, Av-Gay Y (2007) Mycothiol-dependent proteins in actinomycetes. FEMS Microbiol Rev 31:278–922 Ren Q, Kang KH, Paulsen IT (2004) TransportDB: a rational database of cellular membrane transport system. Nucleic Acids Res 32: D284–D288 Riedel C, Rittmann D, Dangel P, Möckel B, Petersen S, Sahm H, Eikmanns BJ (2001) Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production. J Mol Microbiol Biotechnol 3:573–583 Rittmann D, Schaffer S, Wendisch VF, Sahm H (2003) Fructose-1,6bisphosphatase from Corynebacterium glutamicum: expression

Appl Microbiol Biotechnol and deletion of the fbp gene and biochemical characterization of the enzyme. Arch Microbiol 180:285–292 Rittmann D, Lindner SN, Wendisch VF (2008) Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 74:6216–6222 Sakai S, Tsuchida Y, Nakamoto H, Okino S, Ichihashi O, Kawaguchi H, Watanabe T, Inui M, Yukawa H (2007) Effect of lignocellulose-derived inhibitors on growth of and ethanol production by growth-arrested Corynebacterium glutamicum R. Appl Environ Microbiol 73:2349–2353 Sakamoto K, Agari Y, Kuramitsu S, Shinkai A (2011) Phenylacetyl coenzyme A is an effector molecule of the TetR family transcriptional repressor PaaR from Thermus thermophilus HB8. J Bacteriol 193:4388–4395 Schneider J, Wendisch VF (2010) Putrescine production by engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 88:859– 868 Segura A, Bünz PV, D’Argenio DA, Ornston LN (1999) Genetic analysis of a chromosomal region containing vanA and vanB, genes required for conversion of either ferulate or vanillate to protocatechuate in Acinetobacter. J Bacteriol 181:3494–3504 Shen XH, Liu SJ (2005) Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum. Sci China C Life Sci 48:241–249 Shen XH, Liu ZP, Liu SJ (2004) Functional identification of the gene locus ncg12319 and characterization of catechol 1,2-dioxygenase in Corynebacterium glutamicum. Biotechnol Lett 26:575–580 Shen XH, Huang Y, Liu SJ (2005a) Genomic analysis and identification of catabolic pathways for aromatic compounds in Corynebacterium glutamicum. Microb Environ 20:160–167 Shen XH, Jiang CY, Huang Y, Liu ZP, Liu SJ (2005b) Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum. Appl Environ Microbiol 71:3442–3452 Siehler SY, Dal S, Fischer R, Patz P, Gerischer U (2007) Multiple level regulation of genes for protocatechuate degradation in Acinetobacter baylyi includes cross-regulation. Appl Environ Microbiol 73:232–242 Takenaka S, Okugawa S, Kadowaki M, Murakami S, Aoki K (2003) The metabolic pathway of 4-aminophenol in Burkholderia sp. strain AK-5 differs from that of aniline and aniline with C-4 substituents. Appl Environ Microbiol 69:5410–5413 Tateno T, Fukuda H, Kondo A (2007) Production of L-lysine from starch by Corynebacterium glutamicum displaying α-amylase on its cell surface. Appl Microbiol Biotechnol 74:1213–1220 Teramoto H, Inui M, Yukawa H (2009) Regulation of expression of genes involved in quinate and shikimate utilization in Corynebacterium glutamicum. Appl Environ Microbiol 75:3461–3468 Teufel R, Mascaraque V, Ismail W, Voss M, Perera J, Eisenreich W, Haehnel W, Fuchs G (2010) Bacterial phenylalanine and phenylacetate catabolic pathway revealed. Proc Natl Acad Sci USA 107:14390–14395 Tropel D, van der Meer JR (2004) Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500 Udaka S (1960) Screening method for microorganisms accumulating metabolites and its use in the isolation of Micrococcus glutamicus. J Bacteriol 79:754–755 van Beilen JB, Panke S, Lucchini S, Franchini AG, Röthlisberger M, Witholt B (2001) Analysis of Pseudomonas putida alkanedegradation gene clusters and flanking insertion sequences:

evolution and regulation of the alk genes. Microbiology 147:1621–1630 van Ooyen J, Emer D, Bussmann M, Bott M, Eikmanns BJ, Eggeling L (2011) Citrate synthase in Corynebacterium glutamicum is encoded by two gltA transcripts which are controlled by RamA, RamB, and GlxR. J Biotechnol 154:140–148 Venturi V, Zennaro F, Degrassi G, Okeke BC, Bruschi CV (1998) Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358. Microbiology 144:965–973 Wang R, Yin YJ, Wang F, Li M, Feng J, Zhang HM, Zhang JP, Liu SJ, Chang WR (2007) Crystal structures and site-directed mutagenesis of a mycothiol-dependent enzyme reveal a novel folding and molecular basis for mycothiol-mediated maleylpyruvate isomerization. J Biol Chem 282:16288–16294 Wang SH, Xu Y, Liu SJ, Zhou NY (2011) Conserved residues in the aromatic acid/H+ symporter family are important for benzoate uptake by NCgl2325 in Corynebacterium glutamicum. Int Biodeterior Biodegrad 65:527–532 Wehrmann A, Morakkabati S, Krämer R, Sahm H, Eggeling L (1995) Functional analysis of sequences adjacent to dapE of Corynebacterium glutamicum reveals the presence of aroP, which encodes the aromatic amino acid transporter. J Bacteriol 177:5991–5993 Wendisch VF, de Graaf AA, Sahm H, Eikmanns BJ (2000) Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J Bacteriol 182:3088–3096 Wennerhold J, Krug A, Bott M (2005) The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR. J Biol Chem 280:40500–40508 Wieschalka S, Blombach B, Eikmanns BJ (2012) Engineering Corynebacterium glutamicum for the production of pyruvate. Appl Microbiol Biotechnol 94:449–459 Xu Y, Yan DZ, Zhou NY (2006) Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032. Biochem Biophys Res Commun 346 (2):555–561 Yang YF, Zhang JJ, Wang SH, Zhou NY (2010) Purification and characterization of the ncgl2923-encoded 3-hydroxybenzoate 6hydroxylase from Corynebacterium glutamicum. J Basic Microbiol 50:599–604 Yukawa H, Omumasaba CA, Nonaka H, Kós P, Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vertès AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042– 1058 Zhao B, Yeo CC, Poh CL (2005) Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB9867. Proteomics 5:1868– 1876 Zhao KX, Huang Y, Chen X, Wang NX, Liu SJ (2010) PcaO positively regulates pcaHG of the beta-ketoadipate pathway in Corynebacterium glutamicum. J Bacteriol 192:1565–1572 Zhao Z, Ding JY, Li T, Zhou NY, Liu SJ (2011) The ncgl1108 (phePCg) gene encodes a new L-Phe transporter in Corynebacterium glutamicum. Appl Microbiol Biotechnol 90:2005–2013 Zhou NY, Fuenmayor SL, Williams PA (2001) nag genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding enzymes for gentisate catabolism. J Bacteriol 183:700–708