Degradation and assimilation of aromatic compounds by ...

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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).

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