Production of 4-Hydroxybenzoic Acid by an Aerobic Growth-Arrested Bioprocess Using Metabolically EngineeredCorynebacterium glutamicum

Yukihiro Kitade,a,b Ryoma Hashimoto,b Masako Suda,a,b Kazumi Hiraga,a,b Masayuki Inuia,b,c

aMolecular Microbiology and Biotechnology Group, Research Institute of Innovative Technology for the Earth(RITE), Kizugawa, Kyoto, Japan

bGreen Phenol Development Co., Ltd., Kizugawa, Kyoto, JapancGraduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan

ABSTRACT Corynebacterium glutamicum was metabolically engineered to produce4-hydroxybenzoic acid (4-HBA), a valuable aromatic compound used as a raw mate-rial for the production of liquid crystal polymers and paraben. C. glutamicum wasfound to have a higher tolerance to 4-HBA toxicity than previously reported hostsused for the production of genetically engineered 4-HBA. To obtain higher titers of4-HBA, we employed a stepwise overexpression of all seven target genes in the shi-kimate pathway in C. glutamicum. Specifically, multiple chromosomal integrations of amutated aroG gene from Escherichia coli, encoding a 3-deoxy-D-arabinoheptulosonic acid7-phosphate (DAHP) synthase, and wild-type aroCKB from C. glutamicum, encodingchorismate synthase, shikimate kinase, and 3-dehydroquinate synthase, were effec-tive in increasing product titers. The last step of the 4-HBA biosynthesis pathwaywas recreated in C. glutamicum by expressing a highly 4-HBA-resistant chorismatepyruvate-lyase (UbiC) from the intestinal bacterium Providencia rustigianii. To en-hance the yield of 4-HBA, we reduced the formation of by-products, such as 1,3-dihydroxyacetone and pyruvate, by deleting hdpA, a gene coding for a haloacid de-halogenase superfamily phosphatase, and pyk, a gene coding for a pyruvate kinase,from the bacterial chromosome. The maximum concentration of 4-HBA produced bythe resultant strain was 36.6 g/liter, with a yield of 41% (mol/mol) glucose after in-cubation for 24 h in minimal medium in an aerobic growth-arrested bioprocess us-ing a jar fermentor. To our knowledge, this is the highest concentration of 4-HBAproduced by a metabolically engineered microorganism ever reported.

IMPORTANCE Since aromatic compound 4-HBA has been chemically producedfrom petroleum-derived phenol for a long time, eco-friendly bioproduction of 4-HBAfrom biomass resources is desired in order to address environmental issues. In mi-crobial chemical production, product toxicity often causes problems, but we con-firmed that wild-type C. glutamicum has high tolerance to the target 4-HBA. Agrowth-arrested bioprocess using this microorganism has been successfully used forthe production of various compounds, such as biofuels, organic acids, and amino ac-ids. However, no production method has been applied for aromatic compounds todate. In this study, we screened for a novel final reaction enzyme possessing char-acteristics superior to those in previously employed microbial 4-HBA production. Wedemonstrated that the use of the highly 4-HBA-resistant UbiC from the intestinalbacterium P. rustigianii is very effective in increasing 4-HBA production.

KEYWORDS aromatic compound, bioprocess, Corynebacterium glutamicum,4-hydroxybenzoate, shikimate pathway, UbiC

Received 20 November 2017 Accepted 14December 2017

Accepted manuscript posted online 5January 2018

Citation Kitade Y, Hashimoto R, Suda M, HiragaK, Inui M. 2018. Production of 4-hydroxybenzoic acid by an aerobic growth-arrested bioprocess using metabolicallyengineered Corynebacterium glutamicum. ApplEnviron Microbiol 84:e02587-17. https://doi.org/10.1128/AEM.02587-17.

Editor Maia Kivisaar, University of Tartu

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Masayuki Inui,mmg-lab@rite.or.jp.

Y.K. and R.H. contributed equally to this work.

BIOTECHNOLOGY

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The aromatic compound 4-hydroxybenzoic acid (4-HBA) is industrially important andis useful as a building block for liquid crystals and as a raw material for paraben

production. 4-HBA and its derivatives are also used for various purposes, including asplasticizers of nylon resin, sensitizers for thermal paper, and raw materials for dyes andpigments. Recently, 4-HBA has attracted attention as a precursor in the bioproductionof the bulk chemical phenol, because using biophenol has a high potential to reduceCO2 emissions. In most cases, 4-HBA is produced from petroleum-derived phenol usingthe Kolbe-Schmitt reaction under high temperature and pressure (1). A limitation of theKolbe-Schmitt process is the production of dead-end products and the generation ofsolid waste products, such as tar residues (2). In contrast to a chemical process, abioprocess is sustainable and can be performed with a low environment impact underambient temperature and pressure. Aromatic compounds, including 4-HBA, are bio-synthesized via the shikimate pathway, which starts with the condensation of phos-phoenolpyruvate (PEP) from glycolysis and erythrose-4-phosphate (E4P) from the pen-tose phosphate pathway to yield 3-deoxy-D-arabinoheptulosonic acid 7-phosphate(DAHP). This reaction is catalyzed by the enzyme DAHP synthase, whose activity isregulated by feedback inhibition. After six more reactions, the 4-HBA precursor cho-rismate is synthesized. In the final reaction of 4-HBA synthesis, chorismate is con-verted into 4-HBA by chorismate pyruvate-lyase (UbiC) (Fig. 1).

Several 4-HBA production systems in Klebsiella pneumoniae, Escherichia coli, and thesolvent-tolerant bacterium Pseudomonas putida have been developed (3–6). The re-combinant E. coli strain has been successfully used to produce 12 g/liter 4-HBA fromglucose with a yield of 13% (mol/mol) after 72 h of cultivation in a jar fermentor. 4-HBAproduction from glycerol has been reported using P. putida, with a titer of 1.8 g/literand a yield of 8.5% (carbon mole of 4-HBA per carbon mole of sugar [C mol/C mol])after approximately 72 h of cultivation in a jar fermentor (5). Also, using P. putida, the4-HBA yield was improved using a mixed substrate (i.e., glycerol and xylose), reachinga value of up to 16.3% (C mol/C mol) (6). However, to date, bioproduction of 4-HBA hasnot been applied on a commercial basis because of low titers and/or yields.

Corynebacterium glutamicum is a Gram-positive member of the Actinobacteria,which represents one of the largest taxonomic units among the 18 major lineagescurrently recognized within the Bacteria domain. C. glutamicum is used in industry forthe production of L-glutamic acid (7). Additionally, metabolically engineered C. glutami-cum can efficiently produce commodity chemicals, such as organic acids and biofuels(8–10). During the last decade, improvements in bioprocesses based on the concept ofuncoupling the biocatalyst production phase and the product production phase havebeen achieved using this microorganism (11). The growth-arrested bioprocess per-formed under oxygen-deprived conditions has been successfully used for the produc-tion of valuable compounds, such as xylitol, alanine, L-valine, isobutanol, and ethanol(12–16). On the other hand, further efforts to expand the product portfolio that can bemanufactured by this organism are required. In some cases, an oxygen supply isneeded to establish the bioprocess in order to contribute more effectively to theindustrialization of the production of various bio-based chemicals, including aromaticcompounds. An alternate growth-arrested bioprocess under aerobic conditions isconducted in biotin-free minimal salts medium with aeration during the productproduction phase. This process has been applied to produce several amino acids andthe hydroaromatic compound shikimate (11, 17–20).

In this study, we constructed 4-HBA-overproducing C. glutamicum strains, usingrational metabolic engineering approaches. The production of 4-HBA from glucose bythe engineered strains was evaluated using a jar fermentor and minimal medium.

RESULTSEvaluation of 4-HBA tolerance. To obtain high product titers in microbial chemical

production, it is advantageous to have high tolerance to the target chemicals. Thus, weassayed the strains’ tolerance to 4-HBA. Growth tests in the presence of variousconcentrations of 4-HBA revealed that C. glutamicum was able to grow in medium

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containing 300 mM 4-HBA (Fig. 2a). P. putida was able to grow in the presence of 100mM 4-HBA, but the growth was strongly inhibited in the presence of 200 mM 4-HBA(Fig. 2b). E. coli did not grow at all in the presence of 200 mM 4-HBA (Fig. 2c). Thegrowth of Rhodobacter sphaeroides was inhibited in the medium containing 100 mM4-HBA (Fig. 2d). These results showed that C. glutamicum had higher tolerance for4-HBA than the other three bacterial species used for genetically engineered 4-HBAproduction, as reported earlier. In wild-type C. glutamicum, the pathway for thebiosynthesis of aromatic amino acids via chorismate is similar to that in other bacteria,but, unlike intestinal bacteria, such as E. coli, it lacks UbiC, which is the required enzymein the last step of the 4-HBA biosynthesis pathway. We therefore metabolically engi-neered C. glutamicum to increase carbon flow into the shikimate pathway and carriedout the heterologous expression of UbiC in this bacterial species.

Screening of UbiC enzymes. To construct a 4-HBA-overproducing C. glutamicumstrain, 21 ubiC genes were screened. The ubiC genes from various sources wereexpressed in C. glutamicum, and their activities and sensitivities to 4-hydroxybenzoicacid were examined in crude cell extracts. The measured activities and calculated 50%inhibitory concentrations (IC50s) of these enzymes are listed in Table 1. As shown inTable 1, enzymes of the genus Providencia exhibited significantly higher tolerance tohigh concentrations of 4-HBA than those of other genera tested, while UbiC enzymesfrom Cronobacter and Pantoea spp. exhibited activities about 3-fold higher than that of

FIG 1 Schematic representation of the biosynthesis pathway for the production of 4-HBA from glucose.The boxes indicate the relevant genes, which were chromosomally integrated. The double-line boxindicates that the gene is not originally present in the genome of C. glutamicum. A chorismatepyruvate-lyase gene, ubiC, from P. rustigianii was overexpressed via an expression plasmid. The genesldhA, qsuB, qsuD, pyk, and hdpA were deleted (crossed bars) from the chromosomal DNA of C. glutamicumto obtain a higher yield of 4-HBA. Enzymes are encoded by the following genes: haloacid dehalogenasesuperfamily phosphatase is encoded by hdpA, transketolase is encoded by tkt, lactate dehydrogenase isencoded by ldhA, transaldolase is encoded by tal, pyruvate kinase is encoded by pyk, DAHP synthaseis encoded by aroG, shikimate dehydrogenase is encoded by qsuD, 3-dehydroquinate synthase isencoded by aroB, dehydroshikimate dehydratase is encoded by qsuB, 3-dehydroquinate dehydratase isencoded by aroD, shikimate dehydrogenase is encoded by aroE, shikimate kinase is encoded by aroK,5-enolpyruvylshikimate 3-phosphate synthase is encoded by aroA, and chorismate synthase is encodedby aroC. PEP, phosphoenolpyruvate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; GAP, glycer-aldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; DHA, 1,3-dihydroxyacetone; TCA, tricarbox-ylic acid; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptu-lose-7-phosphate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonic acid 7-phosphate;DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; PCA, protocatechuate; 4-HBA, 4-hydroxybenzoic acid.

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UbiC from E. coli. To demonstrate the influence of different ubiC genes on 4-HBAproduction, test tube experiments were performed. As shown in Table 2, the highest4-HBA concentration (2.40 � 0.13 mM) was achieved by the C. glutamicum strainexpressing the ubiC gene of Providencia rustigianii. From these results, the UbiC of P.rustigianii was selected for 4-HBA production using C. glutamicum.

Metabolic engineering of 4-HBA-producing C. glutamicum strains. The geno-type of the basal strain (CRZ23) for the production of 4-HBA is shown in Table 3. Toprevent the generation of lactate and protocatechuate, we deleted ldhA, which en-codes a lactate dehydrogenase, and qsuB, which encodes a dehydroshikimate dehy-dratase, respectively (21, 22). To avoid the accumulation of 3-dehydroshikimate andquinate, qsuD, which encodes a shikimate dehydrogenase, was deleted (23). To achieve4-HBA production, the 4-HBA degradation pathway was blocked by disrupting pobA,which encodes a 4-hydroxybenzoate hydroxylase (24). To increase the supply of E4Pand DAHP, the native gene cluster tkt-tal, which encodes transketolase and transaldo-lase, and aroG(S180F) from E. coli [aroGEC(S180F)], which encodes a high-activity mutantDAHP synthase resistant to feedback inhibition, were integrated into the genome (25).The basal strain CRZ23 produced small amounts of shikimate (0.4 mM) but did notproduce any 4-HBA (Fig. 3). The P. rustigianii UbiC was heterologously expressed asdescribed above. As expected, the production of 4-HBA was achieved by the overex-pression of ubiC (PHE261 in Fig. 3). The production levels of 4-HBA and shikimate byPHE262, which was derived from PHE261 by introducing one additional aroGEC(S180F),were 21.2- and 13.7-fold higher than that by PHE261, respectively (Fig. 3). The produc-tion of 4-HBA and shikimate by PHE263, which was derived from PHE262 by introducingthe native shikimate pathway genes aroCKB, which encode chorismate synthase,shikimate kinase, and 3-dehydroquinate synthase, increased 78.9- and 8.5-fold, respec-

FIG 2 Growth curves in the presence of varied concentrations of 4-HBA. (a) C. glutamicum was grown inA-medium supplemented with 2% glucose at 33°C. (b) P. putida was grown in LB medium at 30°C. (c) E.coli was grown in LB medium at 37°C. (d) R. sphaeroides was grown in LB medium at 30°C. All bacteriawere cultured aerobically in microplates. Cell growth was monitored automatically every 60 min usingan incubation reader. 4-HBA was added to the medium at the indicated concentrations: 0 mM (opencircles), 100 mM (open diamonds), 200 mM (open squares), 250 mM (open triangles), and 300 mM (closedcircles). Data are presented as the mean and standard deviation (n � 5).

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tively, compared with the corresponding values in PHE261 (Fig. 3). This possibly meansthat the simultaneous transcription of these genes contributed effectively to increasecarbon flow to the shikimate pathway without any hindrance because of the use ofa native gene cluster within the C. glutamicum genome. The production of 4-HBAtogether with shikimate, in total, by PHE264, which was derived from PHE263 byintroducing the C. glutamicum native shikimate pathway genes aroD, which encodesa 3-dehydroquinate dehydratase, aroA, which encodes a 5-enolpyruvylshikimate3-phosphate synthase, and aroE, which encodes a shikimate dehydrogenase, exceededthat by PHE263. Specifically, 4-HBA production by PHE264 decreased 7% from that byPHE263, while shikimate production by PHE264 was 2.4-fold higher than that byPHE263 (Fig. 3). The accumulation of shikimate by PHE264 suggests that the overex-pression of genes downstream from shikimate in the shikimate pathway is especiallyneeded. The production of 4-HBA by PHE156, which was derived from PHE264 byintroducing two additional copies of aroCKB, was 1.4-fold higher than that by PHE264,while shikimate production by PHE156 decreased 82% compared to that by PHE264(Fig. 3). This means that the overexpression of these genes contributed to both anincrease in target 4-HBA production and a decrease in the levels of the by-productshikimate. However, 4-HBA production by PHE340 and PHE283, which were obtainedfrom PHE156 by introducing Cronobacter sakazakii and E. coli ubiC, respectively, both ofwhich were not resistant to 4-HBA, decreased 24% and 40% from that by PHE156,respectively (Fig. 3 and Tables 1 and 3). This suggests that the selection of a highly4-HBA-resistant form of UbiC is very important for 4-HBA production. The constructed

TABLE 1 Enzyme activities of chorismate pyruvate-lyase in C. glutamicum strains

Source of ubiC genea

Mean � SD

Initial activity (nmol · mg�1 · min�1)b 4-HBA IC50 (�M)

Providencia rustigianii 159 � 20 370 � 18Providencia stuartii 86 � 7 284 � 47Providencia sneebia 85 � 8 277 � 65Providencia rettgeri 42 � 9 291 � 8Providencia alcalifaciens 18 � 3 296 � 31Providencia burhodogranariea 8 � 7 305 � 48Cronobacter sakazakii 374 � 6 56 � 13Pantoea ananatis 339 � 9 35 � 7Pantoea agglomerans 262 � 55 51 � 1Citrobacter youngae 143 � 11 63 � 16Citrobacter koseri 92 � 22 75 � 20Pseudomonas putida 142 � 12 56 � 38Enterobacter cloacae 139 � 16 63 � 19Enterobacter aerogenes 132 � 9 57 � 14Pseudoalteromonas piscicida 116 � 21 60 � 15Pseudoalteromonas haloplanktis 112 � 2 61 � 24Morganella morganii 105 � 3 75 � 18Escherichia coli 103 � 18 74 � 20Azotobacter vinelandii 69 � 6 43 � 14Xenorhabdus nematophila 60 � 9 54 � 12Xenorhabdus bovienii 20 � 3 37 � 8aWild-type C. glutamicum was used as a host.bn � 5.

TABLE 2 4-HBA production of C. glutamicum strains after 20 h of test tube experiments

Source of ubiCgenea

4-HBA concn(mean � SD) (mM)b

Providencia rustigianii 2.40 � 0.13Providencia stuartii 1.48 � 0.11Cronobacter sakazakii 1.35 � 0.04Pantoea ananatis 1.01 � 0.05Escherichia coli 0.79 � 0.06aWild-type C. glutamicum was used as a host.bn � 5.

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strain PHE156 was used for subsequent strain improvement experiments and 4-HBAproduction using a jar fermentor.

Production of 4-HBA from glucose by an aerobic growth-arrested bioprocess.Using PHE156, 4-HBA was produced, with glucose as the sole carbon source. In a furtherattempt to improve 4-HBA yield, we constructed three strains shown in Table 3(PHE240, PHE242, and PHE243) and compared the yields of these strains to that ofPHE156. The deletion of pyk, which encodes a pyruvate kinase, was carried out tominimize excessive consumption of PEP, which does not contribute to 4-HBA produc-tion (Fig. 1). The 4-HBA yield by PHE240 (Δpyk mutant) was improved by 1% comparedto that by PHE156 (Table 4). The deletion of hdpA, which encodes a haloacid dehydro-genase (HAD) superfamily phosphatase, was carried out to prevent the formation of the

TABLE 3 Bacterial strains and plasmids used in this study

Strain or plasmid Relevant characteristic(s)a Source or reference

StrainsC. glutamicum

R Wild-type strain 43, JCM18229ACX-araE R with markerless integration of xylA, xylB, bglF317A-bglA, araBAD, and araE 44CRZ23 ACX-araE with ldhA::markerless, qsuB::markerless, qsuD::markerless, pobA::markerless, poxF::markerless,

markerless integration of C. glutamicum R tkt-tal and E. coli aroG(S180F)This study

PHE261 Kmr; CRZ23 harboring Pphe292 This studyPHE262 Kmr; PHE261 with markerless integration of one more aroG(S180F) (E. coli) This studyPHE263 Kmr; PHE262 with markerless integration of C. glutamicum R aroCKB This studyPHE264 Kmr; PHE263 with markerless integration of C. glutamicum R aroD, aroA, and aroE This studyPHE156 Kmr; PHE264 with markerless integration of two more sets of C. glutamicum R aroCKB This studyPHE340 Kmr; PHE156 harboring Pphe302 instead of Pphe292 This studyPHE283 Kmr; PHE156 harboring Pphe17 instead of Pphe292 This studyPHE240 Kmr; PHE156 with markerless deletion of pyk This studyPHE242 Kmr; PHE156 with markerless deletion of hdpA This studyPHE243 Kmr; PHE156 with markerless deletion of pyk and hdpA This study

PlasmidspCRB22 Kmr; E. coli-C. glutamicum shuttle vector derived from pHSG298 and pCASE1; 4.1 kb 13Pphe292 Kmr; pCRB22 with a PgapA-ubiC (P. rustigianii)-term gene; 5.6 kb This studyPphe302 Kmr; pCRB22 with a PgapA-ubiC (C. sakazakii)-term gene; 5.7 kb This studyPphe17 Kmr; pCRB22 with a PgapA-ubiC (E. coli)-term gene; 5.6 kb This study

aThe number in parentheses indicates the position of the altered amino acid residue. PgapA, a promoter of the gapA gene. Kmr, kanamycin resistance.

FIG 3 Accumulation of 4-HBA and shikimate in a 24-h batch culture of metabolically engineered C.glutamicum strains, using a baffled flask. CRZ23, the basal strain for the production of 4-HBA withoutoverexpression of the gene ubiC (genotype is shown in Table 3); PHE261, the strain overexpressing ubiCfrom P. rustigianii; PHE262, the strain overexpressing one additional aroGEC(S180F); PHE263, the strainoverexpressing aroCKB; PHE264, the strain overexpressing aroD, aroA, and aroE; PHE156, the strainoverexpressing two additional aroCKB genes; PHE340, the strain that shares the same genotype withPHE156 except for the overexpression of ubiC from C. sakazakii; and PHE283, the strain that shares thesame genotype with PHE156 except for the overexpression of ubiC from E. coli. The average values of theresults from triplicate experiments are shown. Error bars indicate standard deviations.

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by-product 1,3-dihydroxyacetone (DHA) (26) (Fig. 1). The 4-HBA yield by PHE242 (ΔhdpAmutant) was 2% higher than that by PHE156. The highest 4-HBA yield, with a value of41.6% (mol/mol) glucose, was achieved by PHE243 (Δpyk ΔhdpA mutant), although theyields by PHE243 and PHE242 were almost the same (Table 4). The amount of 4-HBAproduced by PHE243 reached 265.4 mM (36.6 g/liter) after 24 h of production reaction(Fig. 4). As expected, DHA formation was prevented in PHE243, while the formation ofother main by-products (pyruvate, shikimate, and phenylalanine) by PHE243 decreasedmore than that by PHE156 (Table 4).

DISCUSSION

Bioproduction of bulk chemicals typically requires high product titers, but this oftenleads to product toxicity. Product toxicity is one of the main bottlenecks in achievingoptimal production. Although various approaches toward improving tolerance tobiofuels have been reported (27, 28), our understanding of the molecular basis oftolerance to aromatic compounds, including 4-HBA, is quite poor. In a recent study onpara-aminobenzoate (PABA) production, C. glutamicum exhibited better PABA toler-ance than other microorganisms (29). According to Verhoef et al., E. coli was signifi-cantly less tolerant to 4-HBA than P. putida S12, which is known as a solvent-tolerantbacterium (5). Our data were consistent with these findings (Fig. 2b and c). Moreover,in our findings, C. glutamicum was much more tolerant to 4-HBA than P. putida S12 (Fig.2a and b). These results suggest that C. glutamicum has a high potential as a 4-HBAproduction host.

In a previous study on the microbial production of 4-HBA from glucose, where the

TABLE 4 4-HBA production, glucose consumption, and by-product formation ofmetabolically engineered C. glutamicum strainsa

Strain Time (h)4-HBAconcn (mM)b

Glucoseconsumption(mM)

Yield(% [mol/mol])c

Concn (mM)d

Shi Phe Pyr DHA

PHE156 10 111.8 289.8 39.1 � 0.5 1.5 0.5 2.6 ND24 255.7 649.1 39.4 � 1.4 4.2 2.4 18.3 10.6

PHE240 10 110.3 273.4 40.4 � 1.3 2.4 0.2 2.3 1.024 253.6 634.4 40.0 � 0.5 5.6 1.8 14.9 4.8

PHE242 10 112.3 273.0 41.1 � 0.7 1.0 0.1 2.1 ND24 263.4 639.4 41.2 � 0.6 3.2 1.4 12.6 0.1

PHE243 10 113.1 275.7 41.0 � 1.5 1.1 0.1 2.0 ND24 265.4 638.5 41.6 � 0.5 3.4 1.4 13.7 ND

aData are the means of the results from three experiments, unless otherwise indicated.b4-HBA, 4-hydroxybenzoic acid.cYield was calculated as mol 4-HBA produced per mole glucose consumed. Data are means � standarddeviations (SD) (n � 3).

dShi, shikimate; Phe, phenylalanine; Pyr, pyruvate; DHA, 1,3-dihydroxyacetone; ND, not detected.

FIG 4 4-HBA production by C. glutamicum PHE243 in a growth-arrested cell reaction. The average valuesof the results from triplicate experiments are shown. Error bars indicate standard deviations.

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currently highest titer of 12 g/liter and yield of 13% (mol/mol) were achieved, overex-pression of four chromosomally integrated target genes (aroB, aroL [encoding shiki-mate kinase II], aroA, and aroC) and the plasmid-based overexpression of four othertarget genes (tktA, aroF [encoding DAHP synthase], ubiC from E. coli, and serA forcomplementation of the gene disrupted by chromosomal integration of the genecassette PtacaroAaroLaroCaroB) in the auxotrophic E. coli strain D2704 (Phe�, Tyr�,Trp�) were performed (4). We succeeded in generating C. glutamicum strains thatshowed both higher titers and yields of 4-HBA using rational metabolic engineeringapproaches. Specifically, we employed stepwise overexpression and chromosomalintegration of all seven target genes, including aroD and aroE, in the shikimate pathwayand two target genes (tkt and tal) in the pentose phosphate pathway using a marker-less gene integration system. Moreover, aroGEC(S180F) and aroCKB were integrated intothe chromosome with a gapA promoter (Table 3). The last step of the 4-HBA biosyn-thesis pathway was recreated by the heterologous expression of ubiC from the intes-tinal bacterium P. rustigianii. It was demonstrated that the use of a selected highly4-HBA-resistant UbiC was very effective in increasing 4-HBA production (PHE156,PHE340, and PHE283 in Fig. 3). In this study, we used a genetically defined strain as ahost cell to construct 4-HBA-producing strains that have simple and clear geneticbackgrounds (Table 3). This was done because the commonly used shortcut strategy foraromatics production, which uses an existing overproducing strain that appeared inrandom screening approaches, such as chemical mutagenesis, etc., cannot eliminatemasking effects caused by unknown genetic factors within the host genome. Addition-ally, since we did not use an auxotrophic mutant for aromatic amino acids as a host cell,the generated 4-HBA-producing strains do not require expensive supplements (e.g.,Trp, Tyr, and Phe). Moreover, since the native gapA promoter was used to express alltarget proteins in this study, the 4-HBA-producing strains do not require expensivechemicals (e.g., isopropyl-�-D-thiogalactopyranoside [IPTG]) to induce gene expression.In PABA production, a considerable amount of by-product formation resulted from anonenzymatic reaction between a target product and the carbon source glucose (29).Since 4-HBA does not possess a reactive amino group within its molecular structure, the4-HBA-producing strains do not require an extra acid treatment to salvage the targetproduct from the by-product N-glucosyl.

According to a pathway model for DAHP production in E. coli, the theoretical yieldsof the phosphotransferase system (PTS) and non-PTS have been shown to be 43%(mol/mol) glucose and 86% (mol/mol) glucose, respectively (30, 31). In fact, yieldimprovement has been demonstrated for shikimic acid production (i.e., 21% [mol/mol]in PTS and 27% [mol/mol] in non-PTS [32]) and L-phenylalanine production (i.e., 0.21g/g [i.e., grams of product per gram of consumed glucose] in PTS and 0.33 g/g innon-PTS [33]), while it has not been demonstrated for anthranilate production (i.e., 0.20g/g in PTS and 0.11 g/g in non-PTS [34]). In C. glutamicum, non-PTS routes have beenindependently reported by two research groups in recent years (35, 36). These studiesassumed that a non-PTS route for glucose uptake provides a high potential for furtheryield improvement in 4-HBA production by metabolically engineered C. glutamicum.Optimization of the metabolic flows will be needed for an improvement of titer andyield (e.g., PEP and E4P balance of supply).

We set up experiments on an aerobic growth-arrested bioprocess with a final cellconcentration of 5% (wt/vol; wet weight) in this study. A higher cell density has beenused for high volumetric productivity in the case of alternate bioprocess of C. glutami-cum under oxygen deprivation (i.e., 20% [12, 14, 37], 25% [38], and 30% [16]). It wasfound that a higher cell density does not always provide a higher yield in an aerobicgrowth-arrested bioprocess because dissolved oxygen (DO), which is required for4-HBA production, tends to be short when the cell density is high. In the future, furtherimprovements in the bioconversion process, such as the introduction of a continuousculture system and chemical engineering technology, cost reduction for the precedingprocesses, such as the procurement of low-cost sugar, and countermeasures againstpollution in postprocesses, such as the development of a wastewater reuse system, will

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be required for the industrialization of 4-HBA production with low environmentalimpact.

MATERIALS AND METHODSBacterial strains and culture conditions. The C. glutamicum strains used in this study are listed in

Table 3. All strains were derived from C. glutamicum strain R. E. coli strains HST02 (TaKaRa Bio, Inc., Japan)and SCS110 (Toyobo, Japan) were used for genetic manipulations. E. coli strain K-12 MG1655 (ATCC47076), P. putida strain S12 (ATCC 700801), and R. sphaeroides (NBRC12203) were used for toleranceassays. C. glutamicum strains were routinely grown at 33°C in nutrient-rich medium (A-medium) orminimal medium (BT-medium [A-medium without yeast extract and Casamino Acids]) supplementedwith 4% glucose (39). E. coli strains were grown at 37°C in LB medium. Where appropriate, the mediawere supplemented with 50 �g/ml kanamycin.

Tolerance assay. C. glutamicum was grown in A-medium supplemented with 2% glucose at 33°C. E.coli was grown in LB medium at 37°C. P. putida and R. sphaeroides were grown in LB medium at 30°C.All bacteria were cultured in 150 �l of the medium in 96-well flat-bottom microplates. 4-HBA was addedto the medium at various concentrations (0, 100, 200, 250, or 300 mM). Cell growth was monitored every60 min with an automated incubation reader (mixing speed, 8; Bio microplate reader HiTS-S2; Scinics Co.,Tokyo, Japan), as described previously (29).

Construction of plasmids and strains. The plasmids used in this study are listed in Table 3, andinformation on the primers used to generate the plasmid constructs is provided in Table 5. In UbiCscreening experiments, the encoding genes were amplified from chromosomal DNA by PCR. The PCRproducts were cloned into the NdeI site of the expression vector pCRB22. The constructed plasmids weretransformed into wild-type C. glutamicum, and the resulting strains were assayed for enzyme activity.Chromosomal gene deletion and integration were achieved via a markerless system (13, 21). The plasmidpCRA728 (21) was used for markerless ldhA gene disruption. Site-directed mutagenesis of aroG(S180F)was performed by inverse PCR after cloning the wild-type aroG from E. coli, using primers 5 and 6 (Table5), and the mutated gene was named aroGEC(S180F). C. glutamicum strains were transformed byelectroporation, as described previously (40). E. coli strains were transformed using the CaCl2 procedure(41).

Enzyme assay. Recombinant C. glutamicum cells harboring ubiC genes from different sources wereharvested by centrifugation (5,000 � g, 4°C, 5 min), washed once with extraction buffer (100 mM Tris-HCl[pH 7.5], 1 mM MgCl2, 2 mM dithiothreitol [DTT]), and disrupted using a multibeads shocker [MB601U(S),Yasui Kikai, Osaka, Japan]. Cell debris was removed by centrifugation (20,000 � g, 4°C, 10 min), and celllysates were used as crude extracts for enzyme assays. Protein concentrations were measured using aprotein assay kit (Bio-Rad, USA). UbiC activity was measured at 33°C by the coupled assay protocoldescribed by Holden et al. (42). The IC50, defined as the concentration at which UbiC activity wasinhibited by 50% under the assay conditions, was estimated at various concentrations of 4-HBA (0, 20,40, 60, 80, 100, 200, 300, 400, and 500 �M) in the presence of 500 �M chorismate.

Conditions for 4-HBA production. For UbiC screening experiments, C. glutamicum strains wereaerobically grown in 10 ml of A-medium at 33°C for 24 h. The cultures were diluted in 10 ml of A-mediumsupplemented with 2% CaCO3 until the optical density at 610 nm (OD610) reached 0.1. The resultingmixture was incubated at 33°C for 20 h with agitation (200 rpm) in a test tube.

For batch-culture experiments, C. glutamicum strains were aerobically grown in 10 ml of A-mediumat 33°C for 24 h. The cultures were diluted in 100 ml of A-medium until an OD610 of 0.1 was reached. Theresulting mixture was incubated at 33°C for 24 h with agitation (180 rpm) in a 500-ml baffled flask.

For growth-arrested cell reaction experiments, C. glutamicum strains were precultured in 10 ml ofmodified A-medium, the recipe for which is essentially the same as that for A-medium, except that thebiotin concentration was reduced to 5 �g/liter, in duplicate. The cells were incubated with agitation (200rpm) at 33°C for 18 h. Twenty milliliters of precultured bacteria was inoculated into 500 ml of modifiedA-medium without urea. The main cultures were incubated in a 1-liter jar fermentor (Able Corporation,Tokyo, Japan) for 22 h (33°C, 900 rpm, 1 vol/vol/min). The pH was maintained at 7.0 via the automaticaddition of 5 N NaOH. Adekanol LG-126 (0.2% [vol/vol]; Adeka Corporation, Tokyo, Japan) was added tothe medium as an antifoamer. Exponentially growing cells in the main culture medium were harvestedby centrifugation, as described previously (18). The harvested cells were washed once with modifiedBT-medium, the recipe for which is essentially the same as that for BT-medium, except that the additionof urea, biotin, metals (i.e., FeSO4 and MnSO4), and thiamine was omitted. The cells were thenresuspended in 220 ml of modified BT-medium containing 400 mM glucose to a final cell concentrationof 5% (wt/vol; wet weight). The production reaction was performed in a 1-liter jar fermentor for 24 h(33°C, 900 rpm, 1 vol/vol/min). The pH was maintained at 7.3 via the automatic addition of 5 N NaOH.When necessary, glucose in the reaction solution was replenished before it was depleted. Since thereaction volume increased to some extent (�10%) because of the addition of NaOH solution, thecalculation of concentrations of 4-HBA, glucose, and by-products was corrected for the increased volumeand consequent dilution.

Analytical methods. Glucose concentration was measured by an enzyme electrode glucose sensor(BF-5; Oji Scientific Instruments, Hyogo, Japan). Concentrations of aromatic and related compounds,including 4-HBA and shikimate, were determined by high-performance liquid chromatography (HPLC;Prominence; Shimadzu Corporation, Kyoto, Japan) equipped with a Cosmosil 5C18-AR-II column (NacalaiTesque, Kyoto, Japan). This system was operated at 40°C with a mobile phase consisting of 20% methanoland 0.07% perchloric acid at a flow rate of 1.0 ml/min. Organic acid concentrations were determined byHPLC (Prominence; Shimadzu Corporation) equipped with a TSKgel OApak-A column (Tosoh Corporation,

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TABLE 5 Oligonucleotides used in this study

Primer Target gene Sequence (5= to 3=)a Restriction site

1 tkt-tal CTCTCATATGACGCTGTCACCTGAAC NdeI2 tkt-tal CTCTCATATGCTACTTCAGGCGAGCTTC NdeI3 aroG (Escherichia coli) CTCTGATATCATGAATTATCAGAACGACGATTTACGC EcoRV4 aroG (E. coli) CTCTGATATCGACTTATCAGGCCTGTGGTG EcoRV5 aroG(S180F) (E. coli) TTTGTCCGGTCGGCTTCAAAAATG6 aroG(S180F) (E. coli) AAAGCCCTGATGCCAGTTC7 aroCKB CTCTCATATGCTAGGCATGCTTCGATG NdeI8 aroCKB CTCTCATATGTTAGTGGCTGATTGCCTCATAAG NdeI9 aroA CTCTCCATGGTCTTTGTGTCTGATTCGTC NcoI10 aroA CTCTCCATGGCTAGCCAACCATCTCCTC NcoI11 aroD CTCTCATATGCTTGGAAAAATTCTCCTCCTC NdeI12 aroD CTCTCATATGCTACTTTTTGAGATTTGCCAGGATATC NdeI13 aroE CTCTCATATGGGTTCTCACATCACTCAC NdeI14 aroE CTCTCATATGCTAGTGTTCTTCCGAGATGC NdeI15 qsuB CTCTGTCGACCTCAGATTGGTTTCGCAGTC SalI16 qsuB CTGATTGCGCACCAAACCAAGAACGTATCCAAGCAGGTTC17 qsuB TTGGTTTGGTGCGCAATCAG18 qsuB CTCTGTCGACTCAACGGTAGGAAGCTCAG SalI19 qsuD CTCTGTCGACGTTCTTCGAAGTGGTGGAAC SalI20 qsuD GTGAGGCAGCTGACATCAAACGTTGAAGCCAAGGTAGAG21 qsuD TTTGATGTCAGCTGCCTCAC22 qsuD CTCTGTCGACTGATCACCTTAAAGGGCGAC SalI23 pobA CTCTTCTAGAGAAACGATCAAGTGCACCAG XbaI24 pobA GACACGAGCGTTTATACCTCTAATTGCCACTGGTACGTGG25 pobA GAGGTATAAACGCTCGTGTC26 pobA CTCTGAGCTCGAGAACACGAACCATACGAG SacI27 poxF CTCTTCTAGATACGTCCTAAACACCCGAC XbaI28 poxF GACCAACCATTGCTGACTTGCGTATCCATAGTCAGGCTTC29 poxF CAAGTCAGCAATGGTTGGTC30 poxF CTCTTCTAGATGATCAGTACCAAGGGTGAG XbaI31 pyk ATGCATGCTTGCTCTCTACGTAGCTGGT SphI32 pyk ATGCATGCCTCTCTTGGGTATCGAAGAG SphI33 pyk ATACTAGTTCGCTGAAACCGACGGTCGC SpeI34 pyk ATACTAGTGAAATATCCATACCTGGCAG SpeI35 hdpA CTCTCTGCAGTTGTGGTAGACCTTGGGTG PstI36 hdpA AACACCATTGTCCCTGTTTTGG37 hdpA TCGCCCAAAACAGGGACAATGGTGTTTATTCTGTAGGTCATGGCATTTGC38 hdpA CTCTTCTAGAATTGCAACACCTGCGATGC XbaI39 ubiC (Providencia rustigianii) CTCTCATATGCATGAAACAATTTTTACCCATCATCC NdeI40 ubiC (P. rustigianii) CTCTCATATGGATTATGTTAGATAGTTATCTATATGCAGGTG NdeI41 ubiC (E. coli) CTCTCATATGTCACACCCCGCGTTAA NdeI42 ubiC (E. coli) CTCTCATATGTTAGTACAACGGTGACGCC NdeI43 ubiC (Providencia stuartii) CTCTCATATGGATGAAACGCTTTTTATCTCTCAC NdeI44 ubiC (P. stuartii) CTCTCATATGTCCCTCCATTTGTTGTGCTC NdeI45 ubiC (Providencia sneebia) CTCTCATATGGATGATACGCTTTTTACCTCTC NdeI46 ubiC (P. sneebia) CTCTCATATGCTTCCCTTCACTTGTCATGC NdeI47 ubiC (Providencia rettgeri) CTCTCATATGGATGAAACGCTTTTTACTTCTCAG NdeI48 ubiC (P. rettgeri) CTCTCATATGTTAACGATATGCAGGTGATTCAGG NdeI49 ubiC (Providencia alcalifaciens) CTCTCATATGCATGAAACGATTTTTACCTCTCATC NdeI50 ubiC (P. alcalifaciens) CTCTCATATGGTTATCTATATGCAGGTGATTCAGG NdeI51 ubiC (Providencia burhodogranariea) CTCTCATATGGATGAAACGCTTTTTACCTCTC NdeI52 ubiC (P. burhodogranariea) CTCTCATATGATACTTCCCTCCACTTGTCG NdeI53 ubiC (Cronobacter sakazakii) CTCTCATATGTCCCATCCCGCGCTGAG NdeI54 ubiC (C. sakazakii) CTCTCATATGTATTCTGCGTCAGGCTCCAC NdeI55 ubiC (Pantoea ananatis) CTCTCATATGACGCAAGACCCGCT NdeI56 ubiC (P. ananatis) CTCTCATATGTTAACCTTGATCACGATAGAGCG NdeI57 ubiC (Pantoea agglomerans) CTCTCATATGAACTATCCTGCCGAGC NdeI58 ubiC (P. agglomerans) CTCTCATATGTTAAATAAAGTCAAAACGCGCAGTAAAG NdeI59 ubiC (Citrobacter youngae) CTCTCATATGCCACACCCTGCGTTAA NdeI60 ubiC (C. youngae) CTCTCATATGTCAGTACAACGGCGATGCA NdeI61 ubiC (Citrobacter koseri) CTCTCATATGTCACACCCTGCGTTAAC NdeI62 ubiC (C. koseri) CTCTCATATGTTAATACAACGGTGATGCGGG NdeI63 ubiC (Enterobacter cloacae) CTCTCATATGTCACACCCTGCGCTAA NdeI64 ubiC (E. cloacae) CTCTCATATGTCAGTACAACGGCGATGC NdeI65 ubiC (Enterobacter aerogenes) CTCTCATATGCCACATCCTGCGCTTAC NdeI

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Tokyo, Japan). This system was operated at 40°C with a 0.75 mM H2SO4 mobile phase at a flow rate of1.0 ml/min. Cell growth was monitored by measuring optical density using a spectrophotometer(Novaspec II; Amersham Pharmacia Biotech, USA).

ACKNOWLEDGMENTSWe thank Ryoji Noburyu (RITE) for his technical advice with the jar fermentor.This work was financially supported in part by the New Energy and Industrial

Technology Development Organization (NEDO), Japan.

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TABLE 5 (Continued)

Primer Target gene Sequence (5= to 3=)a Restriction site

66 ubiC (E. aerogenes) CTCTCATATGTTAATACAATGGCGATGCAGGC NdeI67 ubiC (Pseudomonas putida) CTCTCATATGTCGTACGAATCCCCG NdeI68 ubiC (P. putida) CTCTCATATGTCAGCGGTTTTCCTCCTTG NdeI69 ubiC (Pseudoalteromonas piscicida) CTCTCATATGCCTTTGCAATTACCCTTAGAG NdeI70 ubiC (P. piscicida) CTCTCATATGAAGCCTGCCATTTCTGGTGG NdeI71 ubiC (Pseudoalteromonas haloplanktis) CTCTCATATGATTACTTTCCCTGTTTCATTATCTGC NdeI72 ubiC (P. haloplanktis) CTCTCATATGTCATGAGTACAAATACGCTCCTG NdeI73 ubiC (Morganella morganii) CTCTCATATGACACAAACAGTGATAACACCC NdeI74 ubiC (M. morganii) CTCTCATATGCCACGTTATTCTTCTCCGAG NdeI75 ubiC (Azotobacter vinelandii) CTCTCATATGACCGCTGCTCCCG NdeI76 ubiC (A. vinelandii) CTCTCATATGTTATAGGGTGTCCGGGTC NdeI77 ubiC (Xenorhabdus nematophila) CTCTCATATGCCTATCCGCTGGTTTTC NdeI78 ubiC (X. nematophila) CTCTCATATGTCTGCGTCATACTGACCTCC NdeI79 ubiC (Xenorhabdus bovienii) CTCTCATATGGCAGATGACACAATATTAACTCC NdeI80 ubiC (X. bovienii) CTCTCATATGTTCTGCGTCATACTGGCCTC NdeIaThe restriction sites used in the cloning procedure are underlined.

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