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Characterization of a Unique Pathway for 4-Cresol Catabolism Initiated by Phosphorylation in Corynebacterium glutamicum*

  • Lei Du
    Affiliations
    From the CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology and

    the University of the Chinese Academy of Sciences, Beijing 100049, China, and
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  • Li Ma
    Affiliations
    From the CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology and
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  • Feifei Qi
    Affiliations
    From the CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology and
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  • Xianliang Zheng
    Affiliations
    From the CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology and

    the University of the Chinese Academy of Sciences, Beijing 100049, China, and
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  • Chengying Jiang
    Affiliations
    the State Key Laboratory of Microbial Resources, and Environmental Microbiology and Biotechnology Research Center at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • Ailei Li
    Affiliations
    the CAS Key Laboratory of Bio-based Materials at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China,
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  • Xiaobo Wan
    Affiliations
    the CAS Key Laboratory of Bio-based Materials at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China,
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  • Shuang-Jiang Liu
    Correspondence
    To whom correspondence may be addressed. E-mail: .
    Affiliations
    the State Key Laboratory of Microbial Resources, and Environmental Microbiology and Biotechnology Research Center at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • Shengying Li
    Correspondence
    To whom correspondence may be addressed. E-mail: .
    Affiliations
    From the CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology and
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  • Author Footnotes
    * This work was supported by National Natural Science Foundation of China under Grant NSFC 31422002 and Shandong Provincial Natural Science Foundation Grant JQ201407, and the funding from Recruitment Program of Global Experts (to S. L.) and 973 Project from Ministry of Science and Technology Grant 2012CB7211-04 (to S.-J. L.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Tables S1 and S2 and Figs. S1–S25.
Open AccessPublished:January 27, 2016DOI:https://doi.org/10.1074/jbc.M115.695320
      4-Cresol is not only a significant synthetic intermediate for production of many aromatic chemicals, but also a priority environmental pollutant because of its toxicity to higher organisms. In our previous studies, a gene cluster implicated to be involved in 4-cresol catabolism, creCDEFGHIR, was identified in Corynebacterium glutamicum and partially characterized in vivo. In this work, we report on the discovery of a novel 4-cresol biodegradation pathway that employs phosphorylated intermediates. This unique pathway initiates with the phosphorylation of the hydroxyl group of 4-cresol, which is catalyzed by a novel 4-methylbenzyl phosphate synthase, CreHI. Next, a unique class I P450 system, CreJEF, specifically recognizes phosphorylated intermediates and successively oxidizes the aromatic methyl group into carboxylic acid functionality via alcohol and aldehyde intermediates. Moreover, CreD (phosphohydrolase), CreC (alcohol dehydrogenase), and CreG (aldehyde dehydrogenase) were also found to be required for efficient oxidative transformations in this pathway. Steady-state kinetic parameters (Km and kcat) for each catabolic step were determined, and these results suggest that kinetic controls serve a key role in directing the metabolic flux to the most energy effective route.

      Introduction

      The aromatic compound 4-cresol (i.e. p-cresol or p-methylphenol) is an important synthetic precursor for manufacturing a great variety of chemical products including synthetic resins, disinfectants, antioxidants, preservatives, fumigants, explosives, and others (
      • Fiege H.
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      ). This compound is mainly derived from diverse industrial processes such as coal gasification and fractionation of coal tar. In nature, 4-cresol is generated by anaerobic bacteria as a byproduct during the metabolism of phenylalanine and tyrosine (
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      ).
      Like other aromatic compounds, 4-cresol in polluted environments is degraded by various aerobic and anaerobic microorganisms. Thus, studies on biodegradation mechanisms of 4-cresol hold significant potential for industrial application in environmental protection. In the past decades, a growing number of microorganisms capable of degrading 4-cresol have been discovered, and great efforts have been made to elucidate their biodegradation pathways (
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      ).
      Currently known 4-cresol catabolic pathways can be classified into one of three categories based on the initial step (Fig. 1). The microorganisms falling into the first category (Fig. 1A) begin their degradation of 4-cresol from a methyl hydroxylation step. For instance, in Gram-negative Pseudomonas species, 4-cresol is initially oxidized to 4-hydroxybenzyl alcohol, and then to 4-hydroxybenzyl aldehyde by the same enzyme 4-cresol methylhydroxylase (PCMH)
      The abbreviations used are: PCMH, 4-cresol methylhydroxylase; P450, cytochrome P450 enzyme; HRMS, high resolution mass spectrometry; PEP, phosphoenolpyruvate.
      (
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      ). Subsequently, 4-hydroxybenzyl aldehyde undergoes variant modifications to form protocatechuatic acid (i.e. 3,4-dihydroxybenzoate) or benzoyl-CoA, both of which can be diverted into the central metabolism (i.e. TCA) (
      • Peters F.
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      Genes, enzymes, and regulation of para-cresol metabolism in Geobacter metallireducens.
      ,
      • Londry K.L.
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      Cresol metabolism by the sulfate-reducing bacterium Desulfotomaculum sp. strain Groll.
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      p-Cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol.
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      Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida.
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      Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1.
      ).
      Figure thumbnail gr1
      FIGURE 1Known pathways for 4-cresol catabolism by variant microorganisms. Degradation of 4-cresol begins from methyl hydroxylation (A), linking fumarate to the methyl group (B), and direct aromatic ring hydroxylation (C).
      In the second category, the obligate anaerobe Desulfobacterium cetonicum (
      • Müller J.A.
      • Galushko A.S.
      • Kappler A.
      • Schink B.
      Initiation of anaerobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in Desulfobacterium cetonicum.
      ) initiates the degradation of 4-cresol by linking a fumarate moiety to the methyl group to generate 4-hydroxybenzyl succinate, which is further degraded to 4-hydroxybenzoyl-CoA via β-oxidation (Fig. 1B). Subsequent reduction to benzoyl-CoA leads the metabolic flux to the central metabolism.
      The fungus Aspergillus fumigates likely adopts the third type of degradation pathway to assimilate 4-cresol (
      • Jones K.H.
      • Trudgill P.W.
      • Hopper D.J.
      Metabolism of p-cresol by the fungus Aspergillus fumigatus.
      ). To form protocatechuatic acid, the hydroxylation of 4-cresol by an NADPH-dependent hydroxylase first occurs on aromatic ring to produce 4-methylcatecol, followed by a series of methyl oxidations (Fig. 1C). The intermediates and the enzyme activities for the proposed steps have been identified using cell free extracts (
      • Jones K.H.
      • Trudgill P.W.
      • Hopper D.J.
      Metabolism of p-cresol by the fungus Aspergillus fumigatus.
      ). In yeast Trichosporon cutaneum (
      • Powlowski J.B.
      • Dagley S.
      β-Ketoadipate pathway in Trichosporon cutaneum modified for methyl-substituted metabolites.
      ), an ortho-fission enzyme directly transforms 4-methylcatecol into 3-methyl-cis,cis-muconic acid, which can readily enter the central metabolism via the β-ketoadipate pathway (
      • Jõesaar M.
      • Heinaru E.
      • Viggor S.
      • Vedler E.
      • Heinaru A.
      Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens.
      ) (Fig. 1C).
      More recently, focus has shifted toward understanding the capacity of the Gram-positive bacterium Corynebacterium glutamicum to metabolize diverse aromatic compounds including 4-cresol (
      • Li T.
      • Chen X.
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      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
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      Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium?.
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      PcaO positively regulates pcaHG of the β-ketoadipate pathway in Corynebacterium glutamicum.
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      Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.
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      • Qi S.W.
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      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.
      ,
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      Genome-wide investigation of aromatic acid transporters in Corynebacterium glutamicum.
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      Genetic characterization of the resorcinol catabolic pathway in Corynebacterium glutamicum.
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      ). This high GC content bacterium is not only an important industrial microorganism for production of amino acids and vitamins (
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      ,
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      ,
      • Peters-Wendisch P.
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      • Stäbler N.
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      ), but is also a useful model system to understand the genetics, biochemistry, and mechanisms for biodegradation, especially for assimilation of aromatic compounds (
      • Shen X.H.
      • Zhou N.Y.
      • Liu S.J.
      Degradation and assimilation of aromatic compounds by Corynebacterium glutamicum: another potential for applications for this bacterium?.
      ).
      In our previous studies, we employed proteomic analysis and genome mining to identify a gene cluster in C. glutamicum, creABCDEFGHIJR, which is involved in 4-cresol catabolism (
      • Li T.
      • Chen X.
      • Chaudhry M.T.
      • Zhang B.
      • Jiang C.Y.
      • Liu S.J.
      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
      ,
      • Qi S.W.
      • Chaudhry M.T.
      • Zhang Y.
      • Meng B.
      • Huang Y.
      • Zhao K.X.
      • Poetsch A.
      • Jiang C.Y.
      • Liu S.
      • Liu S.J.
      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.
      ). Specifically, six (creD, creE, creG, creH, creI, and creJ) of eleven cre genes were successfully knocked out from the genome of C. glutamicum RES167. Each knock-out strain lost the ability of the wild-type strain to grow in the minimal medium in which 4-cresol was the sole carbon source. For each mutant, genetic complementation with the corresponding gene restored the wild-type phenotype (
      • Li T.
      • Chen X.
      • Chaudhry M.T.
      • Zhang B.
      • Jiang C.Y.
      • Liu S.J.
      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
      ). These results clearly demonstrated that at least these six genes were required for 4-cresol biodegradation in C. glutamicum. Furthermore, subjecting the six mutants to medium containing diverse aromatic compounds as the sole carbon source, such as 4-hydroxybenzyl alcohol, 4-hydroxybenzyl aldehyde, and 4-hydroxybenzoate, suggested a catabolic pathway responsible for the degradation of 4-cresol (
      • Li T.
      • Chen X.
      • Chaudhry M.T.
      • Zhang B.
      • Jiang C.Y.
      • Liu S.J.
      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
      ). However, details of this putative catabolic pathway could not be rationalized from our previous experiments.
      Although the cre pathway was genetically identified in part, the catalytic functions of the majority of enzymes were not determined. Of the remaining five genes in the pathway, creR is a putative regulatory gene and is therefore not predicted to function as a catabolic catalyst. Based on bioinformatics analysis, creA and creB are believed to reside outside of the cre gene cluster boundary. Interestingly, both Corynebacterium efficiens YS-314 and Arthrobacter sp. FB24 possess the identical cre genetic organization to that of C. glutamicum with the noted absence of homologues to creA and creB. Previously, we generated the creA and creB deletion mutant in C. glutamicum and observed that each resulting strain grew similarly to wild type on the medium supplemented with 4-cresol (
      • Li T.
      • Chen X.
      • Chaudhry M.T.
      • Zhang B.
      • Jiang C.Y.
      • Liu S.J.
      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
      ). Thus, neither creA nor creB are predicted to be involved in 4-cresol biodegradation. Finally, numerous attempts to generate creC and creF deletion mutants were unsuccessful. Therefore, the role of these gene products in 4-cresol degradation could not be anticipated.
      In the current work, all biodegradation enzymes encoded by the cre gene cluster of C. glutamicum were cloned, expressed, and functionally characterized using in vitro enzymatic assays. Through these efforts, an unprecedented 4-cresol catabolic pathway was unveiled that features a novel activating mechanism and a group of enzymes possessing remarkable substrate flexibility. Of particular interest, a unique phosphorylation reaction mediated by CreHI was identified as a novel initiating step of 4-cresol biodegradation. Further, a class I cytochrome P450 (P450) system (
      • Hannemann F.
      • Bichet A.
      • Ewen K.M.
      • Bernhardt R.
      Cytochrome P450 systems: biological variations of electron transport chains.
      ) consisting of CreJ (P450 enzyme), CreF (ferredoxin), and CreE (ferredoxin reductase) was elucidated during this investigation. In vitro characterization of this system demonstrated that it accepts multiple phosphorylated aromatic compounds as substrates and plays a central role in assimilation of 4-cresol.

      Discussion

      Two unusual enzymatic systems represent the most important discoveries of the current study: 1) CreHI initiates the pathway by phosphorylating 4-cresol to give 4-methylbenzyl phosphate, and 2) CreJEF catalyzes the three-step sequential oxidation of 4-methylbenzyl phosphate to benzylalcohol-4-phosphate, benzylaldehyde-4-phosphate, and benzoate-4-phosphate.
      Conserved domain searches revealed that CreH has a PEP-utilizing enzyme mobile domain, whereas CreI has a PEP/pyruvate binding domain (
      • Herzberg O.
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      • Kapadia G.
      • McGuire M.
      • Carroll L.J.
      • Noh S.J.
      • Dunaway-Mariano D.
      Swiveling-domain mechanism for enzymatic phosphotransfer between remote reaction sites.
      ). Interestingly, both CreH and CreI possess respective conserved domains with strong similarity to different parts of the PEP synthase of E. coli (supplemental Fig. S9), suggesting a possible evolutionary relationship between CreH, CreI, and PEP synthase. The Orf1 and Orf2 subunits from the anaerobic T. aromatica, which show 45 and 46% amino acid identity to CreH and CreI, respectively, are responsible for converting phenol into phenylphosphate (
      • Schmeling S.
      • Narmandakh A.
      • Schmitt O.
      • Gad'on N.
      • Schühle K.
      • Fuchs G.
      Phenylphosphate synthase: a new phosphotransferase catalyzing the first step in anaerobic phenol metabolism in Thauera aromatica.
      ,
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      Genes involved in anaerobic metabolism of phenol in the bacterium Thauera aromatica.
      ,
      • Schühle K.
      • Fuchs G.
      Phenylphosphate carboxylase: a new C-C lyase involved in anaerobic in phenol metabolism in Thauera aromatica.
      ). The creHI homologous genes are also found in many other microbial genomes such as C. efficiens, Cladosporium halotolerans, Brevibacterium flavum, Arthrobacter species, Kocuria palustris, Bradyrhizobium species, and Runella slithyformis. Taken together, these gene products may form a new enzyme family capable of phosphorylating phenolic compounds. Moreover, the genomes of C. efficiens, C. halotolerans, B. flavum, Arthrobacter species, and K. palustris contain gene clusters with strong homology to the cre gene cluster of C. glutamicum, suggesting that these bacteria are likely able to degrade 4-cresol using the same mechanism employed by C. glutamicum.
      CreJEF is able to catalyze the successive oxidations of phosphorylated intermediates. It is possible that the phosphate group of substrates could act as an anchoring group that delivers the substrate to the correct position within the active site of CreJ, as done by the deoxysugar that is attached to substrates of the PikC P450 enzyme (
      • Li S.
      • Chaulagain M.R.
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      Selective oxidation of carbolide C-H bonds by an engineered macrolide P450 mono-oxygenase.
      ,
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      ). To the best of our knowledge, this is the first reported example of a P450 enzyme that recognizes a spectrum of substrates via a phosphate group. This discovery may have great biotechnological potential for selective oxidation of unactivated C–H bonds.
      Some known P450 enzymes are capable of catalyzing the complete oxidation of a C–H bond into a carboxylic group. One important example is the multifunctional ent-kaurene oxidase encoded by the P450–4 gene in the gibberellin biosynthetic pathway of Gibberella fujikuroi. This P450 enzyme catalyzes three successive oxidation steps between ent-kaurene and ent-kaurenoic acid (
      • Tudzynski B.
      • Hedden P.
      • Carrera E.
      • Gaskin P.
      The P450–4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthesis pathway.
      ). Another example is CYP71AV1 from Artemisia annua, which performs a three-step oxidation of amorpha-4,11-diene to form artemisinic acid (
      • Ro D.K.
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      ). To complete this challenging six-electron oxidation, CYP71AV1 recruits an artemisinic aldehyde dehydrogenase and an alcohol dehydrogenase to achieve the optimal transformation (
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      ). This is similar to what we have observed with CreJEF, CreC, and CreG. Likewise, in the tirandamycin biosynthetic pathway, TamI P450 enzyme employs the FAD-containing TamL oxidase to overcome the conversion from an alcohol to a keto group (
      • Carlson J.C.
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      ). These examples suggest that the successive oxidations are likely a heavy burden for P450 enzymes to accomplish on their own; therefore, assistance is required from other types of oxidases.
      It is obvious that the initial oxidation of 4-cresol requiring a prephosphorylation step in C. glutamicum is more complicated than the direct oxidation catalyzed by a 4-cresol methylhydroxylase (PCMH). Kinetically, the latter flavocytochrome is much more efficient. For example, the PCMH from a denitrifying Achromobacter sp. exhibited Km and kcat values for 4-cresol of 21 μm and 112 s−1, respectively (
      • Hopper D.J.
      • Bossert I.D.
      • Rhodes-Roberts M.E.
      p-Cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol.
      ). The kcat/Km value of another PCMH from P. putida NCIMB 9869 was calculated to be 2.9 × 105 mm−1 min−1 (
      • Efimov I.
      • Cronin C.N.
      • Bergmann D.J.
      • Kuusk V.
      • McIntire W.S.
      Insight into covalent flavinylation and catalysis from redox, spectral, and kinetic analyses of the R474K mutant of the flavoprotein subunit of p-cresol methylhydroxylase.
      ). Both enzymes are >2000 times more efficient than CreJEF using 4-methylbenzyl phosphate as substrate (Km = 0.34 mm, kcat = 44.5 min−1; Table 1). This is not surprising because the kcat values of many P450 enzymes are within 1–300 min−1, which can mainly be attributed to the slow electron transfer step (
      • Bernhardt R.
      • Urlacher V.B.
      Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations.
      ). Considering that the additional enzymes in the pathway (except for CreC against benzylaldehyde-4-phosphate) also displayed relatively high Km and low kcat values (Table 1), C. glutamicum appears not to be a good 4-cresol assimilating microorganism. However, without a PCMH gene on its genome (data not shown), it is intriguing to speculate that C. glutamicum was forced to evolve a novel, albeit less efficient, pathway for 4-cresol degradation.
      Based on the kinetic analyses and competition experiments, we propose that the primary degradation pathway for 4-cresol in C. glutamicum is the phosphorylated route (Fig. 6): 4-cresol is first activated by CreHI via conversion into 4-methylbenzyl phosphate, and subsequent oxidations of phosphorylated intermediates are co-mediated by CreJEF, CreG, and CreC. This proposed pathway sequence is supported by the fact that the conversion from benzoate-4-phosphate to 4-hydroxybenzoate is a unidirectional reaction (Fig. 6), because CreHI does not take 4-hydroxybenoate as substrate.
      However, it remains yet premature to suggest that the primary route consisting of steps 1 → 3 → 4 → 10 → 14 represents the physiological pathway for 4-cresol degradation in C. glutamicum because of details lacking with respect to the cellular concentration and localization of enzymes, availability of cofactors, and other intracellular environmental factors. It is noteworthy that there is both agreement as well as inconsistency between the proposed pathway in this study and the previous in vivo results (
      • Li T.
      • Chen X.
      • Chaudhry M.T.
      • Zhang B.
      • Jiang C.Y.
      • Liu S.J.
      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
      ). For example, according to the pathways shown in Fig. 6, the inactivation of CreJEF, CreHI, or CreD would block the major catabolic pathway, thus abolishing the ability of knock-out strains to grow on 4-cresol as the sole carbon source. This observation is consistent with the previous observations (
      • Li T.
      • Chen X.
      • Chaudhry M.T.
      • Zhang B.
      • Jiang C.Y.
      • Liu S.J.
      Genetic characterization of 4-cresol catabolism in Corynebacterium glutamicum.
      ). In contrast, based on the results obtained from the one-pot reactions (Fig. 7B), CreG and CreC appear to be unnecessary in the major route from 4-cresol to 4-hydroxybenzoate. This observation, however, contradicts our earlier finding that the creG knock-out strain was unable to grow using 4-cresol as the sole carbon source. We reason that this inconsistency might be derived from a yet unknown function of CreG or the result of the complexity within the in vivo environment such as balances of energy and cofactors or potential toxicity of produced intermediates that could adversely affect cell growth.
      Another interesting aspect of the 4-cresol catabolic pathway is the differential usage of cofactors by CreJEF (NADPH), CreG (NAD+), and CreC (NADP+). This may be of physiological importance, because these enzymes may rely on a cofactor balancing system during degradation of 4-cresol. For instance, CreJEF and CreC may form an NADPH/NADP+ recycling system in vivo to avoid too much loss of reducing power during multiple P450 oxidations.
      It is intriguing to speculate that the substrate flexibility of the catabolic enzymes involved in 4-cresol degradation may have physiological significance because this pathway could also assimilate other phenolic compounds. To further assess this possibility, we are currently pursuing more detailed biochemical studies in our laboratories. Enzyme function redundancy may be useful when microorganisms face harsh polluted environments because multiple enzymes with a common functionality could maximally release the metabolic potential by better taking advantage of substrates, cofactors, and energy stored in different forms.

      Author Contributions

      L. D., X. W., S.-J. L., and S. L. conceived this study, analyzed the results, and wrote the manuscript. L. D., L. M., F. Q., X. Z., C. J., and A. L. conducted experiments. All authors read and approved the manuscript.

      Acknowledgments

      We thank Dr. Shaohua Huang, Dr. Ying Yang, and Fali Bai (Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences) for assistance during NMR and LC-MS data collections. We are grateful to Dr. Jeffrey D. Kittendorf (Alluvium Biosciences Inc.) for proofreading the manuscript.

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