Production of Aromatic Compounds by Metabolically EngineeredEscherichia coli with an Expanded Shikimate Pathway

Daisuke Koma, Hayato Yamanaka, Kunihiko Moriyoshi, Takashi Ohmoto, and Kiyofumi Sakai

Osaka Municipal Technical Research Institute, Osaka, Japan

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing sixkinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid,2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agro-chemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid,the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine andtyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and thephenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and ty-rosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC andthe alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hy-droxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter.While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehydeand 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endoge-nous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of eachgene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxy-phenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols.During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid,accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the con-structed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six con-structed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aro-matic compounds are able to be produced from renewable resources without supplementing with expensive precursors.

Aromatic compounds are an important class of chemicals thatare used as organic solvents, dyes, and precursors in the pro-

cessing of foods, pharmaceuticals, polymers, etc. However, theseare currently manufactured from petroleum which is recognizedas a limited resource and as a cause of global warming. Thus,renewable sources such as biomass feedstock are considered to bean alternative and sustainable source for manufacturing aromaticcompounds.

Many bacteria are natural producers of aromatic compoundsby virtue of a pathway that synthesizes aromatic amino acidsknown as the shikimate pathway (7, 13, 36). In this pathway, phos-phoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) con-verted from glucose through the central metabolic pathway areinitially combined to form 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), which is then converted to chorismate. Fromchorismate, the pathway branches to form a variety of aromaticend products, including phenylalanine (Phe), tyrosine (Tyr),tryptophan (Trp), folic acid, etc. To date, the shikimate pathwayhas been exploited in the manufacture and investigation of severalaromatic compounds, some of which are currently commerciallyavailable. Three aromatic amino acids, Phe, Tyr, and Trp, are pro-duced by fermentation and used in a range of 150 to 12,000 tons/year (7). These amino acids are mainly used as food and feedadditives, pharmaceutical intermediates, sweetener precursors,etc. Shikimate, an intermediate in the shikimate pathway, is a cru-cial starting material for the synthesis of neuramidase inhibitor

GS4104 (Tamiflu), administered as a precaution against influenzainfection (18).

To enhance the productivity of aromatic compounds, tech-niques for genetic modification of strains are frequently applied.For example, in Phe production, the most important steps are thefirst and last steps of the shikimate pathway. However, enzymesincluding these steps, DAHP synthase (encoded by aroG) andchorismate mutase/prephenate dehydratase (encoded by pheA),are strongly inhibited by Phe (20). Therefore, feedback-resistantmutants (fbr) have been studied and exploited for Phe production(16, 25). In addition, the levels of expression of both aroG andpheA are controlled by the transcriptional repressor TyrR (29) sothat deletion of tyrR is also efficient for Phe production (5). Recentworks address modifications of the central metabolic pathway toenhance the availability of the DAHP precursors PEP and E4P (7,13, 36). Such modifications include overexpression of transketol-ase (tktA) and PEP synthase (pps) genes (28), deletion of PEPcarboxylase gene (ppc) (23), deletion or overexpression of carbonstorage regulator genes (csrA or csrB) (38, 44), and glucose trans-

Received 11 April 2012 Accepted 23 June 2012

Published ahead of print 29 June 2012

Address correspondence to Daisuke Koma, koma@omtri.or.jp.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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port system exchange from PEP-dependent sugar phosphotrans-ferase system (PTS) to either galactose permease (GalP)-glucoki-nase (Glk) system (3, 45) or Zymomonas mobilis glucose facilitator(Glf)-Glk system (28). These modifications have been used insuitable combinations to enhance the production of aromaticcompounds.

Expansion of the shikimate pathway by introducing homolo-gous/heterologous genes into engineered Escherichia coli strainshas been applied to a diversity of aromatic compounds. Produc-tion of p-hydroxybenzoic acid has been achieved by homologousoverexpression of both ubiC-encoded chorismate lyase and aroF-encoded DAHP synthase in E. coli (4) and Pseudomonas putida(22, 40). In this strategy, PEP and E4P synthesized from glucoseenter the shikimate pathway to form chorismic acid, which is thenconverted to p-hydroxybenzoic acid with the release of pyruvicacid. Phenol production from glucose has been studied using sol-vent-tolerant Pseudomonas putida S12 (43). In this study, a Tyr-overproducing strain of P. putida S12 was modified by introduc-tion of Tyr phenol lyase gene (encoded by tpl from Pantoeaagglomerans) to generate a phenol producer. Cinnamic acid (CA)(26) and coumaric acid (4HCA) (27) producers have also beenconstructed from aromatic amino acid-overproducing strains ofP. putida S12 by introducing the pal gene encoding the bifunc-tional enzyme, Phe-ammonia lyase/Tyr-ammonia lyase fromRhodosporidium toruloides. The constructed strains were able todeaminate Phe or Tyr converted from glucose to produce copiousamounts of CA or 4HCA. Introduction of pdc-encoding 4HCAdecarboxylase from Lactobacillus plantarum into the 4HCA over-producer enabled p-hydroxystyrene (4HSTY) production fromglucose (41). Production of 4HCA and 4HSTY from glucose alsoproved successful when a Tyr-overproducing strain of E. coli wasused as the host strain (31, 33, 39). Recently, production of bio-based styrene (STY) was achieved by simultaneous overexpressionof pal2 from Arabidopsis thaliana and fdc1 from Saccharomycescerevisiae in a Phe-overproducing strain of E. coli (21).

We previously presented tyramine (TYM) and phenethyl-amine (PEA) production from glucose by expanding the shiki-mate pathway in E. coli (17). The TYM producer was constructedby overexpression of the tyramine decarboxylase gene from Lac-tobacillus brevis in a Tyr overproducer, whereas the PEA producerwas constructed by overexpression of the aromatic amino aciddecarboxylase gene from Pseudomonas putida in a Phe overpro-ducer. In this report, we investigate the possibility of bioproduc-tion of an additional six aromatic compounds, phenyllactic acid(PLA), 4-hydroxyphenyllactic acid (4HPLA), 2-phenylethanol(PE), 2-(4-hydroxyphenyl)ethanol (4HPE) (also known as ty-rosol), phenylacetic acid (PAA), and 4-hydroxyphenylacetic acid(4HPAA) by metabolically engineered E. coli. The shikimate path-way was expanded by introducing T7 promoter-controlled ho-mologous/heterologous genes into the chromosomes of Phe andTyr overproducers to generate strains capable of producing therespective aromatic compounds from glucose. Target aromaticcompounds have been widely used in some industries, and someof these compounds have the potential of being a biopolymer unit.PE is used for flavor and fragrance compound with a rose-likeodor. PAA, 4HPAA, and 4HPE are used as building blocks for anantibiotic (penicillin G), pharmaceuticals, agrochemicals, etc.PLA is known as a broad-spectrum antimicrobial agent. In addi-tion, PLA and 4HPLA have the possibility of being building blocksof aromatic polymers, e.g., blending with polylactate. To the best

of our knowledge, this is the first assessment of the capability ofmetabolically modified E. coli to produce PLA, 4HPLA, PAA, and4HPAA from glucose.

MATERIALS AND METHODSCloning of genes. Phusion Hot Start DNA polymerase (Novagen) wasused to amplify the relevant genes. The lactate dehydrogenase gene (ldhA)was amplified from genomic DNA from Cupriavidus necator JCM20644(synonym Ralstonia eutropha) using the primer pair ReADH2-F-Nde (5=-CCAACCATATGCCTGCTCCCCAGATCCTCC-3= [the NdeI site is un-derlined]) and ReADH2-R-Xho (5=-CACTCGAGTTACAGCACTGGCGTCAGCAC-3= [the XhoI site is underlined]). The NdeI-XhoI-digestedamplicon was introduced into the corresponding site of pET21a-FRT (17)to construct pARO133. The alcohol dehydrogenase gene (adhC) was am-plified from genomic DNA from Lactobacillus brevis JCM1170 using theprimer pair LbADH-F-Nde (5=-CCAACCATATGATGCAAATCAAAACAGCTTTTTC-3=) and LbADH-R-Xho (5=-CACTCGAGTTAGAATGTGATTACGGGC-3=). The NdeI-XhoI-digested amplicon was introducedinto the corresponding site of pET21a-FRT to construct pARO131. Thephenylacetaldehyde (PAAL) dehydrogenase gene (feaB) was amplifiedfrom genomic DNA from Escherichia coli BW25113. Two segments wereinitially amplified by PCR using primer pair EcPAALDH-Nde (5=-CCAACCATATGACAGAGCCGCATGTAGC-3=) and EcPAALDH-RM1-Nco(5=-CGGAAAGTTCCACGGCACAATTCCCGCC-3=) and primer pairEcPAALDH-FM1-Nco (5=-GGCGGGAATTGTGCCGTGGAACTTTCCG-3=) and EcPAALDH-Xho (5=-CACTCGAGTTAATACCGTACACACACCGAC-3=); the segments were then combined by overlap extension PCR(32) using the primer pair EcPAALDH-Nde and EcPAALDH-Xho.Aldehyde reductase genes (yqhD, yjgB, and yahK) were also amplifiedfrom genomic DNA from Escherichia coli BW25113. Primer pair yqhD-Nde (5=-CCAACCATATGAACAACTTTAATCTGCAC-3=) and yqhD-Xho (5=-CACTCGAGTTAGCGGGCGGCTTCG-3=), primer pairyjgB-Nde (5=-CCAACCATATGTCGATGATAAAAAGCTATG-3=) andyjgB-Xho (5=-CACTCGAGTCAAAAATCGGCTTTCAACACC-3=), andprimer pair yahK-Nde (5=-CCAACCATATGAAGATCAAAGCTGTTGGTG-3=) and yahK-Xho (5=-CACTCGAGTCAGTCTGTTAGTGTGCG-3=) were used to amplify the respective genes. The NdeI-XhoI-digestedamplicons of feaB, yqhD, yjgB, and yahK were introduced into the corre-sponding site of pET21a-FRT to generate pARO132, pARO167,pARO168, and pARO176, respectively. The phenylpyruvate (PP) decar-boxylase gene (ipdC) was synthesized with optimizing codon usage for E.coli using OptimumGene algorithm (GenScript USA Inc.), and insertedinto the NdeI-XhoI site of pET21a-FRT to construct pARO136.

Construction of strains. Escherichia coli BW25113(DE3) was genet-ically modified by the previously developed method (17) to generatearomatic compound producers. The method consisted of Red-medi-ated recombination, FLP (flippase)/FRT (FLP recognition target) re-combination, and P1 transduction. All of the first strains harboring asingle desired gene were constructed by Red-mediated recombination.With adhC for example, the Km cassette flanking two FRT sites and adhC(FRT-Km-FRT-adhC) was amplified by PCR using pARO131 as the tem-plate DNA and then introduced into the acs locus on the chromosome togenerate strain AR-G37 (acs::FRT-Km-FRT-T7p-adhC [T7p-adhC indi-cates that the adhC gene is controlled by the T7 promoter]). Similarly,strains AR-G38 (acs::FRT-Km-FRT-T7p-feaB [pARO132 used as the tem-plate]), AR-G39 (acs::FRT-Km-FRT-T7p-ldhA [pARO133 used as thetemplate]), AR-G84 (mtlA::FRT-Km-FRT-T7p-ipdC [pARO136 used asthe template]), AR-G85 (mtlA::FRT-Km-FRT-T7p-ldhA [pARO133 usedas the template]), AR-G92 (acs::FRT-Km-T7p-FRT-yqhD [pARO167used as the template]), AR-G93 (acs::FRT-Km-FRT-T7p-yjgB [pARO168used as the template]), and AR-G94 (acs::FRT-Km-FRT-T7p-yahK[pARO176 used as the template]) were constructed. Next, genetic traitswere assembled by P1 transduction, and a kanamycin resistance markerwas excised by FLP/FRT recombination to generate aromatic compoundproducers. The genetic trait of strain AR-G39 (acs::FRT-Km-FRT-T7p-

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ldhA) was incorporated into strains AR-G91 (tyrR::T7p-aroFfbr-pheAfbr;Phe overproducer) and AR-G2 (tyrR::T7p-aroFfbr-tyrAfbr; Tyr overpro-ducer) (17), which were derived from E. coli strain BW25113(DE3), togenerate PLA producer PAR-57 and 4HPLA producer PAR-3, respec-tively. Strain PAR-57 was further modified by assembling the genetic traitof strain AR-G85 (mtlA::FRT-Km-FRT-T7p-ldhA) to generate PLA pro-ducer PAR-58 harboring two copies of the chromosomal T7p-ldhA gene.The genetic trait of strain AR-G84 (mtlA::FRT-Km-FRT-T7p-ipdC) wasincorporated into strains AR-G91 and AR-G2 to generate strains PAR-60and PAR-47, respectively. Both strains were further modified by assem-bling the genetic trait of AR-G38 (acs::FRT-Km-FRT-T7p-feaB) to gener-ate PAA producer PAR-61 and 4HPAA producer PAR-51. Similarly, PE

producer PAR-62 and 4HPE producer PAR-53 were constructed fromstrains PAR-60 and PAR-47, respectively, by assembling the genetic traitof AR-G37 (acs::FRT-Km-FRT-T7p-adhC). Instead of the genetic trait ofstrain AR-G37, the genetic traits of strains AR-G92 (acs::FRT-Km-T7p-yqhD), AR-G93 (acs::FRT-Km-FRT-T7p-yjgB), and AR-G94 (acs::FRT-Km-FRT-T7p-yahK) were incorporated into strains PAR-60 and PAR-47to generate PE producer PAR-66, PAR-67, and PAR-68 and 4HPE pro-ducers PAR-63, PAR-64, and PAR-65. The strains constructed to producearomatic compounds are summarized in Table 1.

Gene deletion. The feaB, paaK, yahK, tyrA, and pheA genes were in-dividually deleted from the chromosome of E. coli BW25113(DE3) aspreviously reported (2, 9), yielding strains AR-G49 (feaB::FRT-Km-FRT),

TABLE 1 E. coli strains used in this study

Straina Relevant genotype Relevant characteristic or phenotype

BW25113(DE3) lacI rrnBT14 �lacZWJ16 hsdR514 �araBADAH33 �rhaBADLD78 dcm(DE3) Host strain in this study (9, 17)AR-G2 tyrR::T7p-aroFfbr-tyrAfbr Tyr overproducer (17)AR-G37 acs::FRT-Km-FRT-T7p-adhC Donor of T7p-adhCAR-G38 acs::FRT-Km-FRT-T7p-feaB Donor of T7p-feaBAR-G39 acs::FRT-Km-FRT-T7p-ldhA Donor of T7p-ldhAAR-G49 feaB::FRT-Km-FRT feaB deficientAR-G51 paaK::FRT-Km-FRT paaK deficientAR-G78 tyrA::FRT-Km-FRT tyrA deficientAR-G79 pheA::FRT-Km-FRT pheA deficientAR-G84 mtlA::FRT-Km-FRT-T7p-ipdC Donor of T7p-ipdCAR-G85 mtlA::FRT-Km-FRT-T7p-ldhA Donor of T7p-ldhAAR-G91 tyrR::T7p-aroFfbr-pheAfbr Phe overproducer (17)AR-G92 acs::FRT-Km-FRT-T7p-yqhD Donor of T7p-yqhDAR-G93 acs::FRT-Km-FRT-T7p-yjgB Donor of T7p-yjgBAR-G94 acs::FRT-Km-FRT-T7p-yahK Donor of T7p-yahKAR-G98 yahK::FRT-Km-FRT yahK deficientJW2978 yqhD::FRT-Km-FRT yqhD deficient (2)JW5761 yjgB::FRT-Km-FRT yjgB deficient (2)PAR-3 tyrR::T7p-aroFfbr-tyrAfbr acs::T7p-ldhA 4HPLA producerPAR-47 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC 4HPAAL synthesisPAR-51 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-feaB 4HPAA producerPAR-53 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-adhC 4HPE producerPAR-57 tyrR::T7p-aroFfbr-pheAfbr acs::T7p-ldhA PLA producerPAR-58 tyrR::T7p-aroFfbr-pheAfbr acs::T7p-ldhA mtlA::T7p-ldhA PLA producerPAR-60 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC PAAL synthesisPAR-61 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-feaB PAA producerPAR-62 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-adhC PE producerPAR-63 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-yqhD 4HPE producerPAR-64 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-yjgB 4HPE producerPAR-65 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-yahK 4HPE producerPAR-66 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-yqhD PE producerPAR-67 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-yjgB PE producerPAR-68 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-yahK PE producerPAR-75 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yqhD 4HPAAL synthesisPAR-77 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yjgB 4HPAAL synthesisPAR-79 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yahK 4HPAAL synthesisPAR-82 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-feaB paaK PAA producerPAR-83 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-yahK feaB 4HPE producerPAR-84 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-yahK feaB PE producerPAR-89 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yqhD yjgB 4HPAAL synthesisPAR-90 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yqhD yahK 4HPAAL synthesisPAR-91 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yjgB yahK 4HPAAL synthesisPAR-92 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC yqhD yjgB yahK 4HPAAL synthesisPAR-97 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-feaB yahK 4HPAA producerPAR-100 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-feaB tyrA PAA producerPAR-102 tyrR::T7p-aroFfbr-tyrAfb mtlA::T7p-ipdC acs::T7p-feaB yahK pheA 4HPAA producerPAR-104 tyrR::T7p-aroFfbr-tyrAfbr mtlA::T7p-ipdC acs::T7p-yahK feaB pheA 4HPE producerPAR-105 tyrR::T7p-aroFfbr-pheAfbr mtlA::T7p-ipdC acs::T7p-yahK feaB tyrA PE producera Strains BW25113(DE3), JW2978, and JW5761 were derived from E. coli BW25113, whereas the other strains were derived from E. coli BW25113(DE3).

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AR-G51 (paaK::FRT-Km-FRT), AR-G98 (yahK::FRT-Km-FRT), AR-G78(tyrA::FRT-Km-FRT), and AR-G79 (pheA::FRT-Km-FRT). Two deriva-tives of strain BW25113, strains JW2978 (yqhD::FRT-Km-FRT) andJW5761 (�yjgB::FRT-Km-FRT), which are members of a single-geneknockout mutant library known as the Keio collection (2), were obtainedfrom the National BioResource Project (NIG, Japan) on E. coli strains. Thetraits of gene-deficient strains were transferred to aromatic compoundproducers by P1 transduction, and then the kanamycin resistance markerwas excised by FLP/FRT recombination (17) to generate strains PAR-75,PAR-77, PAR-79, PAR-82, PAR-83, PAR-84, PAR-89, PAR-90, PAR-91,PAR-92, PAR-97, PAR-100, PAR-102, PAR-104, and PAR-105, which arelisted in Table 1.

Production of aromatic compounds. Each strain was precultivatedovernight in 5 ml LB-G medium (10 g polypeptone, 5 g dried yeast extractD-3, 10 g NaCl per liter [pH 7.0]) at 37°C. Fifty microliters of the precul-ture was inoculated into 5 ml M9M medium (10 g glucose, 6 g Na2HPO4,3 g KH2PO4, 0.5 g NaCl, 2 g NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 50 �MFeSO4, 1 �M ZnSO4, 1 �M CoCl2, and 0.001% thiamine per liter), M9Mmedium plus 1 mM Phe, or M9M medium plus 1.5 mM Tyr. The timingto induce gene expression by the addition of isopropyl-�-D-thiogalacto-pyranoside (IPTG) was varied depending on the strains because thegrowth of some constructed strains was depressed by IPTG addition andglucose consumption became poor. As a standard, the strains were culti-vated at 37°C with shaking at 250 rpm/min (BR-23FH·MR; Taitec Co.Ltd.) in M9M medium until an optical density at 660 nm (OD660) of 0.3was attained (17). Strains PAR-47, PAR-51, PAR-53, PAR-63, PAR-64,PAR-65, PAR-75, PAR-77, PAR-79, PAR-83, PAR-89, PAR-90, PAR-91,PAR-92, and PAR-97, which were AR-G2 derivatives harboring T7p-ipdCat the mtlA locus of the chromosome, were cultivated in M9M medium at37°C (250 rpm/min) until an OD660 of 4.0 was attained. Phe auxotrophstrains PAR-102 and PAR-104 and Tyr auxotroph strains PAR-100 andPAR-105 were cultivated at 37°C (250 rpm/min) in M9M medium plus 1mM Phe and M9M medium plus 1.5 mM Tyr, respectively, until an OD660

of 2.0 was attained. IPTG was added to the culture at a final concentrationof 1 mM to induce gene expression, and the strains were continuouslycultivated at 27°C with shaking at 250 rpm/min (TC-300; Takasaki Scien-tific Instruments Corp.). The total cultivation time at 37°C and 27°C was48 h. If necessary, cultivation time was prolonged for up to 72 h. In orderto determine volumetric and specific productivity parameters, an aliquotof the culture (0.2 ml) was periodically sampled.

Following cultivation, 1/20 volume of 2 M HCl and 1/5 volume ofmethanol were mixed with 1/10 diluted culture. The mixture was centri-fuged at 10,000 � g for 5 min, and the obtained supernatant was filtered byCosmonice filter W (0.45 �l) (Millipore). The filtrate was preserved for ahigh-performance liquid chromatography (HPLC) assay.

Enzyme assay. E. coli BW25113(DE3) harboring pARO131 (adhC),pARO133 (ldhA), pARO136 (ipdC), pARO167 (yqhD), pARO168 (yjgB),or pARO176 (yahK) was cultivated in LB-G medium containing ampicil-lin until an OD660 of 0.5 was attained. IPTG was added to the culture at afinal concentration of 1 mM to induce gene expression, and the strain wascontinuously cultivated at 27°C for 16 h. After the strain was cultivated at27°C for 16 h, cells were harvested from 1 ml of culture by centrifugationat 10,000 � g for 5 min, washed once with 1 ml of 100 mM phosphatebuffer (PB) (pH 7.2), and then dissolved with 100 �l PB. The cells weredisrupted by sonication in ice-chilled water and centrifuged at 10,000 � gfor 15 min. The supernatant was used as cell extract (CFE [named CFE forcell-free extract]) in the enzyme assay. The protein concentration of theCFE was measured using Coomassie protein assay reagent (Pierce) withbovine serum albumin as a standard. For assay of PLA or 4HPLA dehy-drogenase activity, the reaction mixture contained 2 mM PP or 4-hy-droxyphenylpyruvate (4HPP), 2 mM NADH, and each CFE in PB. Forassay of PAAL reductase activity, the reaction mixture contained 2 mMPAAL, 2 mM NADH, 0.2 mM ZnSO4, and each CFE in PB. The reactionswere started by the addition of 10 �l of each CFE. For assay of 4-hydroxy-phenylacetaldehyde (4HPAAL) reductase activity, the reaction mixture

contained 2 mM 4HPP, 2 mM NADH, 2 mM thiamine pyrophosphate(TPP), 0.2 mM ZnSO4, 0.2 mM MgSO4, 100 �l of CFE (4 mg/ml) from E.coli BW25113(DE3)/pARO136 (IpdC solution), and each CFE. The reac-tion was started by the addition of 100 �l of IpdC solution and 10 �l ofeach CFE. The reactions were performed at 30°C for 10 min and thenstopped by adding 50 �l of 2 M HCl. After 0.2 ml acetonitrile was mixedinto the reaction mixture, the amount of each product was determined byHPLC.

Analytical method. An HPLC system fitted with a photodiode array(SPD-M10AVP; Shimadzu Corp.) and an octadecyl silica column (Cos-mosil 5C18-MS-II column [3.0 by 150 mm] from Nacalai Tesque Inc.) wasused to measure the concentrations of aromatic compounds. The com-pounds were eluted at a flow rate of 0.4 ml/min of mobile phase contain-ing methanol and 0.1% trifluoroacetic acid. The methanol concentrationwas increased from 20% to 80% for 5 min, and this concentration wasthen maintained for 10 min. Under such conditions, Tyr (3.8 min),4HPLA (5.1 min), Phe (5.2 min), 4HPE (5.4 min), 4HPAA (5.7 min), PLA(6.8 min), PAA (7.2 min), and PE (7.3 min) were eluted with acceptableseparation. The wavelength used to detect Phe, PLA, PAA, and PE was 206nm, whereas the wavelength used to detect Tyr, 4HPLA, 4HPE, and4HPAA was 222 nm. The compounds were quantified using standardcurves of the respective commercial chemicals (except for PAA, which isdifficult to purchase). For PAA quantification, PE was used as the stan-dard. To determine enzyme activity (see “Enzyme assay” above), acetoni-trile was used for the mobile phase instead of methanol. Under such con-ditions, PLA, 4HPLA, PE, and 4HPE were eluted at 5.2, 3.2, 5.9, and 3.9min, respectively.

To analyze the chirality of produced PLA and 4HPLA, an HPLC sys-tem fitted with a Sumichiral OA-5000 column (4.6 by 150 mm; SumikaChemical Analysis Service Ltd.) was used. The compounds were eluted ata flow rate of 1.0 ml/min from the mobile phase containing 15% isopro-panol and 2 mM CuSO4. Under such conditions, the standard chemicalcompounds L-4HPLA, D-4HPLA, L-PLA, and D-PLA were eluted at 19, 25,72, and 94 min, respectively, and detected at 254 nm.

Optical density for cell growth was measured at 660 nm (UV-160A;Shimadzu Corp). Cell weight (dry weight) (CDW) values were calculatedfrom OD660 values using the formula CDW (g liter�1) � OD660 � 0.5.The glucose concentration was colorimetrically determined using Glu-cose CII Test Wako (Wako Pure Chemical Industries, Ltd.).

RESULTSOverview of the production of aromatic compounds by expand-ing the shikimate pathway. E. coli synthesizes Tyr and Phe fromglucose through the central metabolic pathway and the shikimatepathway (Fig. 1). Glucose is converted to PEP and E4P in thecentral metabolic pathway, and then DAHP was synthesized byDAHP synthases encoded by aroF, aroG, and/or aroH. Seven stepsinto the shikimate pathway, prephenic acid, precursor to both PPand 4HPP, is synthesized. Here, pheA- or tyrA-encoded bifunc-tional enzyme acts on prephenic acid to form PP or 4HPP, respec-tively, which is converted to Phe or Tyr, respectively, by tyrB-encoded aminotransferase.

Expansion of the shikimate pathway by introduction and over-expression of homologous/heterologous genes may allow tailor-ing the production of aromatic compounds from glucose (Fig. 1).For instance, PLA can be produced from PP by introducing adehydrogenase gene that facilitates reduction of the PP carbonylgroup. PP can also be converted to PAAL by introducing a decar-boxylase gene. PAAL is further converted to PE or PAA by intro-duction and overexpression of an aldehyde reductase gene or analdehyde dehydrogenase gene, respectively. Phe synthesized fromPP by transamination can be converted to PEA or CA by intro-duction of a decarboxylase gene or an ammonia lyase gene. CA can

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be further converted to STY by introducing a decarboxylase gene.These compounds are derived by expansion of the Phe syntheticroute; a similar pathway may be constructed by expanding the Tyrsynthetic route. In such cases, compounds possessing hydroxylgroups on their aromatic ring, namely, 4HPLA, 4HPE, 4HPAA,TYM, 4HCA, and 4HSTY, are obtainable from glucose. A PAAdehydrogenase gene (feaB) is known to reside in the genome of E.coli, enabling the potential conversion of PAAL and 4HPAAL toPAA and 4HPAA in this organism. However, E. coli lacks a decar-boxylase gene for conversion of PP or 4HPP to PAAL or 4HPAAL.So far, production of CA, 4HCA, STY, and 4HSTY from glucosehas been well studied by using metabolically engineered E. coliand/or Pseudomonas strains (21, 26, 27, 31, 33, 41). Production ofPEA and TYM from glucose by metabolically engineered E. colihas been demonstrated in our previous study (17). In the presentstudy, we address the issue of whether PLA, 4HPLA, PE, 4HPE,PAA, and 4HPAA can be produced via a similar mechanism(shaded products in Fig. 1).

Genes for expansion of the shikimate pathway. To achievethe production of six aromatic compounds, PLA, 4HPLA, PE,4HPE, PAA, and 4HPAA, the desired genes were surveyed us-

ing the BRENDA database (http://www.brenda-enzymes.org/)and some biological tools (e.g., BLAST search) and informa-tion from the National Center for Biotechnology Information(NCBI) (http://www.ncbi.nlm.nih.gov/). Because E. coli is usedas a host strain in the present study, bacteria (rather than fungi,yeasts, or archaea) were targeted as natural sources of the genes.Finally, selected genes were cloned from three members of thephylum Proteobacteria (including E. coli) and one member ofthe phylum Firmicutes (Lactobacillus brevis). The cloned genesand the organisms from which they were derived are listed inTable 2.

A reductase capable of acting on PP and 4HPP is required forPLA and 4HPLA production. Suitable known reductases are (R)-4HPLA dehydrogenase (EC 1.1.1.222) and 4HPP reductase (EC1.1.1.237). There are some eukaryotic enzymes, such as 4HPP re-ductase from Solenostemon scutellarioides (GenBank protein iden-tification [ID] CAD47810.2), known to belong to these categories,so the corresponding genes were surveyed in bacteria by BLASTsearch using the amino acid sequence of S. scutellarioides 4HPPreductase as a query sequence. The amino acid sequence of ldhA-encoded dehydrogenase from C. necator showed 45% identity,

FIG 1 Overview of production of aromatic compounds by expanding the shikimate pathway. Solid lines indicate inherent pathways in E. coli, whereas brokenlines indicate pathways that may be constructed by introducing heterologous genes. The genes in brackets are the introduced and overexpressed genes to producethe respective aromatic compounds. The shaded (gray shading) compounds are the target aromatic compounds in the present study.

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and its gene was cloned for our purpose. Although E. coli harborsldhA gene encoding lactate dehydrogenase in the genome, thesimilarity of the amino acid sequence against 4HPP reductasefrom S. scutellarioides and LdhA from C. necator was low (�24%).

PP decarboxylase (EC 4.1.1.43) is widely distributed in bacte-ria. The enzyme encoded by the ipdC gene derived from Azospi-rillum brasilense has been extensively investigated, and its three-dimensional structure has been published (35, 42). The enzymeacts not only on PP but also on 4HPP to produce the correspond-ing aldehydes. Although the native ipdC gene is expressed in activeform in E. coli, codon modification (without changing the aminoacid) was undertaken to reduce the GC content, rendering it suit-able for amplification by PCR.

PAAL dehydrogenase, which is encoded by feaB and involvedin PEA and TYM degradation pathways in E. coli, has been well-studied. The feaB gene was cloned for conversion of PAAL and4HPAAL to PAA and 4HPAA, respectively.

Reduction of PAAL and 4HPAAL yields PE and 4HPE, but noPAAL or 4HPAAL reductase gene has yet been found in E. coli.Instead, the heterologous adhC gene, which encodes Zn-depen-dent alcohol dehydrogenase, was cloned from L. brevis. The adhCgene exhibited similarity (�50%) with aryl-alcohol dehydroge-nase (EC 1.1.1.90) derived from a variety of bacteria. However,our data (described below) suggest that reductase genes capable ofacting on PAAL and 4HPAAL do exist in E. coli. Therefore, we

surveyed such genes in E. coli by BLAST search using query aminoacid sequences of some heterologous aromatic reductases, phen-ylacetaldehyde reductase of Rhodococcus sp. strain ST-10(GenBank protein ID BAD51480), cinnamyl alcohol dehydroge-nase of Helicobacter pylori (AAD08150.1), and benzyl alcohol de-hydrogenases of Acinetobacter calcoaceticus (AAC32671.1) and ofPseudomonas putida (BAJ06499 and BAJ06503). Twelve genes,adhE, adhP, eutG, ydjJ, ydjL, frmA, gatD, yiaY, yjgB, yjiN, yphC,and yqhD, exhibited similarities to the query sequences suffi-ciently and were introduced into the multicloning site (MCS) ofpET21a-FRT or pET21d-FRT as 4HPAAL reductase candidates.4HPAAL-producing strain PAR-47 (Table 1) was transformedwith the respective recombinant plasmids, and 4HPE and 4HPAAproduction in M9M medium by the transformants was prelimi-narily examined (data not shown). In conclusion, three aldehydereductase gene candidates seemed to increase 4HPE production,so these genes were used to generate 4HPE-producing strains.

Enzyme assays. Since two exogenous genes, ldhA and adhC,and two endogenous genes, yjgB and yahK, encode the enzymeswithout experimental data on the function, the genes were ex-pressed in E. coli, and their enzymatic activities were evaluated(Table 3). Although two kinds of enzymatic reactions, PP to PLAand 4HPP to 4HPLA, were carried out by using CFE preparedfrom the E. coli BW25113(DE3) host strain as a control, suchreductase activities were not detected. In contrast, the reductase

TABLE 2 Genes for production of aromatic compounds

Gene GenBank protein ID Enzyme Species of origin

ldhA CAJ91827 Lactate dehydrogenase or related dehydrogenase Cupriavidus necator JCM20644ipdC CAA67899 Indol-3-pyruvate/phenylpyruvate decarboxylase Azospirillum brasilense NBRC102289feaB AAC74467 Phenylacetaldehyde dehydrogenase Escherichia coli BW25113adhC ABJ64046 Zn-dependent alcohol dehydrogenase Lactobacillus brevis JCM1170yqhD AAC76047.1 Aldehyde reductase, NADPH dependent Escherichia coli BW25113yjgB AAC77226.2 Predicted alcohol dehydrogenase, Zn dependent and NAD(P) binding Escherichia coli BW25113yahK AAC73428.1 Predicted oxidoreductase, Zn dependent and NAD(P) binding Escherichia coli BW25113

TABLE 3 Evaluation of enzyme activities for putative lactate dehydrogenase and aldehyde reductases

Reactiona Supplemented cofactor(s) and enzymeb StrainOverexpressedgene

Sp act(U/mg)c

PP ¡ PLA NADH BW25113(DE3) (control) NDBW25113(DE3)/pARO133 ldhA 1.60 0.01

4HPP ¡ 4HPLA NADH BW25113(DE3) (control) NDBW25113(DE3)/pARO133 ldhA 0.58 0.02

PAAL ¡ PE NADH ZnSO4 BW25113(DE3) (control) 0.02 0.00BW25113(DE3)/pARO131 adhC 0.06 0.01BW25113(DE3)/pARO167 yqhD 0.40 0.00BW25113(DE3)/pARO168 yjgB 2.80 0.04BW25113(DE3)/pARO176 yahK 2.24 0.09

4HPP ¡ 4HPAAL ¡ 4HPE NADH ZnSO4 TPP MgCl2 IpdC BW25113(DE3) (control) NDBW25113(DE3)/pARO131 adhC NDBW25113(DE3)/pARO167 yqhD 0.08 0.00BW25113(DE3)/pARO168 yjgB 0.44 0.01BW25113(DE3)/pARO176 yahK 0.84 0.02

a The assay for conversion of 4HPP to 4HPE was performed by a coupling reaction of IpdC [CFE from BW25113(DE3)/pARO136] and each overexpressed reductase.b Thiamine pyrophosphate (TPP) and MgCl2 were cofactors for IpdC.c The values are means standard deviations of 3 independent enzyme assays. One unit of activity was defined as the amount of enzyme that produced 1 �mol of product perminute at 30°C. ND, not detected.

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activities of PP to 4HPLA and 4HPP to 4HPLA were distinctlydetected in CFE prepared from an ldhA-expressing strain. On theother hand, the reductase activity of PAAL to PE was detected instrains overexpressing adhC, yjgB, or yahK as well as in a strainwith the yqhD gene, which is known to encode a broad-substratealcohol dehydrogenase, and their reductase activities were consid-erably higher than those of host strain BW25113(DE3).

An enzymatic assay for reduction of 4HPAAL to 4HPE was notperformed because sufficient amounts of 4HPAAL could not beobtained. Instead, an assay for conversion of 4HPP to 4HPE wasperformed by a coupling reaction of phenylpyruvate decarboxyl-ase (IpdC) and each putative reductase. Although 4HPP (2 mM)was almost completely converted to 4HPAAL (1.9 mM) in thecontrol reaction [using CFE from E. coli BW25113(DE3)], 4HPEwas not detected. In contrast, the conversion of 4HPP to 4HPEwas advanced by using CFEs prepared from yqhD-, yjgB-, andyahK-overexpressing strains, indicating that yqhD-, yjgB-, andyahK-encoded reductases have 4HPAAL reductase activity. Theseresults suggest that the cloned putative genes are candidates forconstruction of PLA, 4HPLA, PE, and 4HPE producers.

Construction and evaluation of PLA and 4HPLA producers.To generate PLA- and 4HPLA-producing strains, T7 promoter-controlled ldhA (T7p-ldhA) was introduced into the chromo-somes of E. coli strains AR-G91 (Phe overproducer) and AR-G2(Tyr overproducer). The resulting strains, PAR-57 and PAR-3,produced PLA and 4HPLA, respectively (Table 4). During PLAproduction, accumulation of a small amount of Phe was observed.The accumulated Phe was reduced by introducing additional T7p-ldhA into the PAR-57 chromosome, yielding strain PAR-58. Un-der the same cultivation conditions, the level of accumulated PLAin strain PAR-58 (6.0 0.3 mM) was identical to the level ofaccumulated Phe in strain AR-G91 (7.6 0.3 mM). Surprisingly,although strain AR-G2 produced only 4.7 0.5 mM Tyr, strainPAR-3 produced a considerably higher concentration of 4HPLA(8.1 0.8 mM). By HPLC chirality analysis, the PLA and 4HPLAproduced by strains PAR-58 and PAR-3, respectively, were iden-tified with standard chemicals of D-forms, and the optical puritieswere above 99%ee (enantiomeric excess). These results indicate

that we had successfully constructed strains that produced D-PLAand D-4HPLA from glucose.

Construction and evaluation of PE and 4HPE producers. Togenerate strains PAR-60 and PAR-47, strains capable of synthesiz-ing PAAL and 4HPAAL, respectively, T7 promoter-controlledipdC (T7p-ipdC) was incorporated into the chromosomes of therespective strains, strains AR-G91 (Phe overproducer) and AR-G2(Tyr overproducer). T7 promoter-controlled adhC (T7p-adhC)was then incorporated into the chromosomes of strains PAR-60and PAR-47 to generate PAR-62 (PE producer) and PAR-53(4HPE producer), respectively (Table 1).

The PAR-60 and PAR-47 strains and their derivatives, whichharbored T7p-ipdC, did not accumulate Phe and Tyr when cul-tured. In addition, accumulation of aldehydes (PAAL and4HPAAL) was not observed in cultures of strains PAR-60 andPAR-47. Instead, PE, 4HPE, PAA, and 4HPAA accumulated, sug-gesting that PAAL and 4HPAAL produced from glucose werereadily reduced and oxidized to their corresponding alcohols andacids. The titers and yields of PE and 4HPE by the constructedstrains are summarized in Table 5. Overexpression of both T7p-ipdC and T7p-adhC genes in strain PAR-62 increased the PE pro-duction of that strain relative to that of PAR-60. However, a sim-ilar effect was lacking in strain PAR-53, which was constructed asa 4HPE producer. The reductase encoded by adhC appeared to acton PAAL in vivo but not on 4HPAAL. During the production ofPE by strain PAR-62, a significant amount of 4HPE by-product,which was synthesized from the Tyr synthetic route (describedbelow), accumulated in the culture.

As strains PAR-60 and PAR-47 accumulated PE and 4HPE, it isthought that endogenous aldehyde reductases are able to targetPAAL and 4HPAAL in E. coli. Simultaneous overexpression of theT7p-ipdC gene and T7 promoter-controlled candidate genes(T7p-yqhD, T7p-yjgB, or T7p-yahK) increased PE production bystrains PAR-66, PAR-67, and PAR-68 and 4HPE production bystrains PAR-63, PAR-64, and PAR-65 (Table 5). This was espe-cially true in strain PAR-65, in which the yahK gene was overex-pressed and the accumulation of 4HPAA by-product was consid-erably decreased.

TABLE 4 Production of PLA and 4HPLA by the genetically modified strainsa

Strainb

Strain description and relevantinserted genec

Amt (mM)of glucoseconsumedd,e

OD660d,f Amt (mM) of major product producedg

Yieldh (%,mol/mol)

Ind. Fin. PLA 4HPLA Phe Tyr Yp/s Ybp/s

Strains constructed to produce PLAAR-G91 Phe overproducer 54.1 0.3 7.2 ND ND 7.6 0.5 ND NC 13.7PAR-57 Phe overproducer ldhA 53.3 0.3 6.4 5.5 0.2 ND 2.4 0.2 ND 9.9 4.3PAR-58 Phe overproducer ldhA ldhA 54.0 0.3 6.4 6.0 0.3 ND 1.3 0.1 ND 10.8 2.3

Strains constructed to produce 4HPLAAR-G2 Tyr overproducer 55.5 0.3 7.9 ND ND ND 4.7 0.2 NC 8.5PAR-3 Tyr overproducer ldhA 54.0 0.3 8.6 ND 8.1 0.8 ND ND 14.6 NC

a The strains were cultivated in M9M medium at 37°C (before induction of gene expression) and 27°C (after induction of gene expression) for 48 h.b Strains AR-G91 and AR-G2 were phenylalanine and tyrosine overproducers, respectively, and used as control strains.c before a gene indicates that the gene was inserted. Expression of the inserted genes was controlled by the T7 promoter.d Values were obtained from three independent cultures, and the maximum coefficient of variation was below 10%.e The initial glucose concentration was 55.6 mM (10 g/liter).f Ind., OD660 when the induction of gene overexpression was started; Fin., final OD660.g The values are means standard deviations obtained from 3 independent cultures. ND, not detected.h Yp/s, yield in accumulated target product (mM) per initial glucose (55.6 mM) � 100%; Ybp/s, yield in accumulated by-product (mM) per initial glucose (55.6 mM) � 100%. NC,not calculable.

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To identify which reductases contribute to 4HPE production,three reductases encoded by yqhD, yjgB, and yahK were individu-ally or simultaneously deleted from the chromosome of strainPAR-47. Since 4HPE and 4HPAA were produced from 4HPAALby endogenous aldehyde reductase and feaB-encoded aldehydedehydrogenase, respectively, in strain PAR-47, deletion of the rel-evant aldehyde reductase would cause a decrease in 4HPE produc-tion, coinciding with an increase in 4HPAA production. The ratioof 4HPE/4HPAA accumulation by strain PAR-47 was 1.5, whereasthe ratios by strain PAR-75 (yqhD deficient), PAR-77(yjgB defi-cient), and PAR-79 (yahK deficient) were 0.7, 1.0, and 1.0, respec-tively (Table 6). Furthermore, the ratio was reduced to 0.5 bystrain PAR-90 lacking both yqhD and yahK. However, 4HPE titerswere not significantly reduced in all deletion variants, suggestingthat another endogenous reductase acts on the production of4HPE.

The pathways for PE and 4HPE syntheses include somebranching points. The first branching point is the locus of pre-

phenic acid, the second branching point is the locus of PP or4HPP, and the third one is the locus of PAAL or 4HPAAL (Fig. 2).Because undesirable metabolic flow would reduce the yield of eachtarget compound and increase the amount of by-products, unde-sirable pathways should be blocked. Indeed, throughout PE and4HPE syntheses, undesired PAA and 4HPAA syntheses occurred,due to the presence of endogenous feaB-encoded PAAL dehydro-genase (Table 5). Endogenous feaB was therefore deleted fromPAR-68 (PE producer) and PAR-65 (4HPE producer) to block thePAA and 4HPAA synthetic routes, generating strains PAR-84 andPAR-83, respectively (modification of the third branching pointin Fig. 2). The amounts of PE and 4HPE produced by strainsPAR-84 and PAR-83, respectively, were nearly equal to those bystrains PAR-68 and PAR-65 (Table 5). PAA and 4HPAA by-prod-ucts were not detected after deletion of endogenous feaB, as shownby strains PAR-84 and PAR-83.

4HPE by-product was accumulated in PE production by strainPAR-84, whereas the accumulation of PE by-product was in-

TABLE 5 Production of PE and 4HPE by the genetically modified strainsa

Strainb

Strain description and relevantinserted and deleted gene(s)c Mediumd

Amt (mM)of glucoseconsumede,f

OD660e,g

Amt (mM) of major product producedh

(mM)Yieldi (%,mol/mol)

Ind. Fin. PE 4HPE PAA 4HPAA Yp/s Ybp/s

Strains constructedto produce PE

AR-G91 Phe overproducer M9M 54.1 0.3 6.5 ND ND ND ND NC NCPAR-60 Phe overproducer ipdC M9M 48.4 0.3 4.5 4.6 0.5 2.4 0.1 1.7 0.2 0.9 0.1 8.3 9.0PAR-62 Phe overproducer ipdC

adhCM9M 53.4 0.3 4.2 5.7 0.4 2.0 0.3 0.2 0.0 0.2 0.0 10.3 4.3

PAR-66 Phe overproducer ipdC yqhD

M9M 54.0 0.3 5.5 5.1 0.4 2.0 0.2 �0.2 �0.2 9.2 �4.3

PAR-67 Phe overproducer ipdC yjgB M9M 54.1 0.3 5.4 5.8 0.4 2.3 0.2 �0.2 �0.2 10.4 �4.9PAR-68 Phe overproducer ipdC yahK M9M 53.9 0.3 5.3 6.5 0.1 2.7 0.2 �0.2 �0.2 11.7 �5.6PAR-84 Phe overproducer ipdC yahK

� feaBM9M 51.7 0.3 4.9 7.7 0.2 3.4 0.1 ND ND 13.8 6.1

PAR-105 Phe overproducer ipdC yahK� feaB � tyrA

M9M Tyr 54.1 2.0 3.2 6.9 0.2 1.3 0.1 ND ND 12.4 2.3

Strains constructedto produce4HPE

AR-G2 Tyr overproducer M9M 55.5 0.3 7.9 ND ND ND ND NC NCPAR-47 Tyr overproducer ipdC M9M 50.2 4.0 5.1 0.3 0.0 2.6 0.1 �0.2 1.7 0.0 4.7 �4.0PAR-53 Tyr overproducer ipdC adhC M9M 52.4 4.0 5.2 0.3 0.0 2.4 0.6 �0.2 1.6 0.1 4.3 �3.8PAR-63 Tyr overproducer ipdC yqhD M9M 55.4 4.0 5.0 0.3 0.0 3.0 0.1 �0.2 1.6 0.0 5.4 �3.8PAR-64 Tyr overproducer ipdC yjgB M9M 54.3 4.0 5.1 0.4 0.0 3.9 0.7 �0.2 1.5 0.5 7.0 �3.8PAR-65 Tyr overproducer ipdC yahK M9M 54.1 4.0 4.8 0.5 0.0 4.1 0.2 �0.2 0.5 0.1 7.4 �2.2PAR-83 Tyr overproducer ipdC yahK

� feaBM9M 53.5 4.0 5.4 2.4 0.7 3.8 0.3 ND ND 6.8 4.3

PAR-104 Tyr overproducer ipdC yahK� feaB � pheA

M9M Phe 51.7 2.0 3.2 1.0 0.1 8.3 0.2 ND ND 14.9 1.8

a The strains were cultivated at 37°C (before induction of gene expression) and 27°C (after induction of gene expression) for 48 h.b Strains AR-G91 and AR-G2 were phenylalanine and tyrosine overproducers, respectively, and used as control strains.c before a gene indicates that the gene was inserted, whereas � before a gene indicates that the gene was deleted. Expression of the inserted genes was controlled by the T7promoter.d The concentrations of supplemented Phe and Tyr were 1.0 and 1.5 mM, respectively.e Values were obtained from 3 independent cultures, and the maximum coefficient of variation was below 10%.f The initial glucose concentration was 55.6 mM (10 g/liter).g Ind., OD660 when the induction of gene overexpression was started; Fin., final OD660.h The values are means standard deviations obtained from 3 independent cultures. ND, not detected.i Yp/s, yield in accumulated target product (mM) per initial glucose (55.6 mM) � 100%; Ybp/s, yield in accumulated by-product (mM) per initial glucose (55.6 mM) � 100%. NC,not calculable.

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creased in 4HPE production by strain PAR-83 (Table 5). Becausethe host strain BW25113 accumulated PE and PAA but not 4HPEand 4HPAA when cultivated in M9M medium supplemented with1 mM PAAL, we inferred that 4HPE by-product had accumulatedthrough leakage into the Tyr synthetic route in strain PAR-84 (thefirst branching point in Fig. 2). Similarly, it was thought that ac-cumulation of PE by-product by strain PAR-83 was due to leakageinto the Phe synthetic route. To avoid such leakage, the tyrA geneof PE producer PAR-84 and the pheA gene of 4HPE producerPAR-83 were deleted to generate strains PAR-105 and PAR-104,respectively (modification of the first branching point in Fig. 2).Since these strains became Tyr or Phe auxotrophic, each aromaticamino acid was added to the medium: the addition of aromaticamino acids contributes to cell growth rather than production oftarget product. As a result, accumulation of 4HPE by-productduring PE production by strain PAR-105 was appreciably de-

creased (Table 5). Similarly, PE by-product during 4HPE produc-tion by strain PAR-104 was also decreased. In addition, theamount of 4HPE produced by strain PAR-104 considerably in-creased more than that by strain PAR-83.

Blockage of degradation pathways of the target compoundsproduced is one of the considerable issues that must be resolved(Fig. 2). As the host strain BW25113(DE3) is a K-12 derivative, itcan degrade PAA through the PAA degradation pathway, compo-nents of which are encoded by the paa operon, but it cannot de-grade 4HPAA because it lacks the hpa operon (10). On the otherhand, degradation pathways of PE and 4HPE are unclear. Thestrain could not degrade 4HPE in M9M medium supplementedwith 1 mM 4HPE, nor could 4HPE be used as the sole carbon andenergy source in glucose-free M9M medium, suggesting that4HPE is the final product. In contrast, PE was slightly degraded bythe strain (less than 10%) when the strain was cultivated at 27°Cfor 48 h in M9M medium supplemented with 1 mM PE. Althoughthe degradation pathway of PE is unclear, it is thought that PE wasoxidized to PAA through PAAL formation and then degraded viathe PAA degradation pathway. In fact, PE was not degraded by afeaB-deficient strain (AR-G47) when the strain was cultivated at27°C for 48 h in M9M medium supplemented with 1 mM PE.Therefore, we think that deletion of feaB, shown as strains PAR-84and PAR-105, might contribute to prevent PE degradation.

Construction and evaluation of PAA and 4HPAA producers.The PAR-60 and PAR-47 strains were further modified by incor-poration of the T7 promoter-controlled feaB (T7p-feaB) gene togenerate PAR-61 (PAA producer) and PAR-51 (4HPAA pro-ducer), respectively (Table 1). Overexpression of both T7p-ipdCand T7p-feaB in strains PAR-61 and PAR-51 resulted in accumu-lation of PAA and 4HPAA (Table 7). However, a small amount ofaccumulated PAA by-product was detected in 4HPAA production

TABLE 6 Effect of gene deletion on production of 4HPE and 4HPAAa

Strain Deleted gene(s)

Amt (mM) of productformedb Ratio of

4HPE/4HPAAformed4HPE 4HPAA

PAR-47 2.6 0.1 1.7 0.0 1.5PAR-75 yqhD 3.4 0.1 4.8 0.3 0.7PAR-77 yjgB 4.3 0.3 4.5 0.4 1.0PAR-79 yahK 2.7 0.3 2.9 0.4 0.9PAR-89 yqhD yjgB 2.0 0.7 2.7 0.8 0.7PAR-90 yqhD yahK 2.2 0.4 4.3 0.2 0.5PAR-91 yjgB yahK 2.5 0.4 2.4 0.3 1.0PAR-92 yqhD yjgB yahK 3.0 0.1 2.2 0.1 1.4a The strains were cultivated at 37°C (before induction of gene expression) and 27°C(after induction of gene expression) for 48 h.b The values are means standard deviations obtained from 3 independent cultures.

FIG 2 PE, 4HPE, PAA, and 4HPAA synthesis and degradation pathways in E. coli BW25113(DE3). Three metabolic branching points are surrounded by brokenlines, whereas degradation pathways are emphasized by gray shading. Host strain BW25113(DE3), which is a derivative of E. coli strain K-12, lacks the hpa operonwhich encodes enzymes involved in 4HPAA degradation. TCA, tricarboxylic acid.

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by strain PAR-51. Similarly, PAR-61 acquired small quantities of4HPAA by-product during PAA production.

As strain PAR-61 can operate the PAA degradation pathway(10), we modified the strain by deleting paaK, which encodes phe-nylacetyl-coenzyme A (CoA) ligase (catalyzing the first step of thePAA degradation pathway), to prevent PAA degradation. How-ever, the accumulated PAA by the resulting strain, PAR-82, wasconsiderably decreased (Table 7); the reason is unclear. On theother hand, endogenous yahK of strain PAR-51 was deleted toblock the 4HPE synthetic route (the third branching point in Fig.2), but it was not meaningful with respect to 4HPAA production,as shown by strain PAR-97. To prevent the production of 4HPAAby-product via leakage into the Tyr synthetic route (the firstbranching point in Fig. 2) in strain PAR-61, the tyrA gene wasdeleted to generate strain PAR-100. Similarly, the pheA gene of4HPAA producer PAR-97 was deleted to generate strain PAR-102.The deletion of tyrA or pheA allowed strain PAR-100 or PAR-102to produce a larger amount of PAA or 4HPAA and a smalleramount of by-product.

Productivities of aromatic compounds by the constructedstrains. Production of aromatic compounds by the respectiveconstructed strains, which contained PLA by strain PAR-58,4HPLA by strain PAR-3, PE by strain PAR-105, 4HPE by strainPAR-104, PAA by strain PAR-100, and 4HPAA by strain PAR-102, was periodically analyzed (Fig. 3). For comparison, time

course data of the production of Phe (by strain AR-G91) and Tyr(by strain AR-G2) were also shown in Fig. 3. Gene overexpressionwas induced by the addition of IPTG at an OD660 of 0.3 whenstrains AR-G91, AR-G2, PAR-3 and PAR-58 were cultivated. Onthe other hand, since the cell growth of strains PAR-105, PAR-104,PAR-100, and PAR-102 were depressed by IPTG addition, geneoverexpression was induced at an OD660 of 2.0. Under such con-ditions, the final OD660 of the former strains ranged from 5.8 to8.8, whereas that of the latter strains ranged from 2.4 to 3.4.

Time course experiments revealed the occurrence of by-prod-ucts during the production of the respective aromatic com-pounds. In 4HPLA production by strain PAR-3, Tyr by-productwas detected in the early stage (within 24 h) of cultivation (Fig. 3).However, the by-product disappeared later (after 24 h). In con-trast, Phe by-product was gradually increased during PLA pro-duction by strain PAR-58. On the other hand, by-products of PE,4HPE, PAA, and 4HPAA production arose in the early stage(within 10 h) and their accumulated levels were maintained untilthe end of cultivation, suggesting that these by-products were de-rived from supplemented Phe or Tyr but not from glucose.

Maximum volumetric and specific productivities of PLA,4HPLA, PE, 4HPE, PAA, and 4HPAA by the respective con-structed strains were determined from the time course data andsummarized in Table 8. Due to the by-product produced, themaximum specific productivity (qp, max) of PLA by strain AR-G58

TABLE 7 Production of PAA and 4HPAA by the genetically modified strainsa

Strainb

Strain description and relevantinserted and deleted gene(s)c Mediumd

Amt (mM) ofglucoseconsumede,f

OD660e,g Amt (mM) of major product producedh

Yieldi (%,mol/mol)

Ind. Fin. PE 4HPE PAA 4HPAA Yp/s Ybp/s

Strains constructedfor PAAproduction

AR-G91 Phe overproducer M9M 54.1 0.3 6.5 ND ND ND ND NC NCPAR-60 Phe overproducer ipdC M9M 48.4 0.3 4.5 4.6 0.5 2.4 0.1 1.7 0.2 0.9 0.1 3.1 12.9PAR-61 Phe overproducer ipdC feaB M9M 48.4 0.3 4.5 ND ND 5.2 0.0 1.8 0.0 9.4 3.2PAR-82 Phe overproducer ipdC feaB

� paaKM9M 46.2 0.3 5.1 ND ND 2.9 0.0 2.3 0.0 5.2 4.1

PAR-100 Phe overproducer ipdC feaB� tyrA

M9M Tyr 47.0 2.0 3.1 ND ND 8.8 0.1 1.3 0.0 15.8 2.3

Strains constructedfor 4HPAAproduction

AR-G2 Tyr overproducer M9M 55.5 0.3 7.9 ND ND ND ND NC NCPAR-47 Tyr overproducer ipdC M9M 50.2 4.0 5.1 0.3 0.0 2.6 0.1 �0.2 1.7 0.0 3.1 �5.6PAR-51 Tyr overproducer ipdC feaB M9M 50.2 4.0 5.1 ND ND 0.5 0.0 5.3 1.0 9.5 0.9PAR-97 Tyr overproducer ipdC feaB

� yahKM9M 48.4 4.0 4.6 ND ND 0.6 0.0 4.9 0.5 8.8 1.1

PAR-102 Tyr overproducer ipdC feaB� yahK � pheA

M9M Phe 44.5 2.0 2.4 ND ND 0.8 0.0 6.1 0.4 13.2 1.4

a The strains were cultivated at 37°C (before induction of gene expression) and 27°C (after induction of gene expression) for 48 h.b Strains AR-G91 and AR-G2 were phenylalanine and tyrosine overproducers, respectively, and used as control strains.c before a gene indicates that the gene was inserted, whereas � before a gene indicates that the gene was deleted. Expression of the inserted genes was controlled by the T7promoter.d The concentrations of supplemented Phe and Tyr were 1.0 and 1.5 mM, respectively.e Values were obtained from three independent cultures, and the maximum coefficient of variation was below 10%.f The initial glucose concentration was 55.6 mM (10 g/liter).g Ind., OD660 when the induction of gene overexpression was started; Fin., final OD660.h The values are means standard deviations obtained from 3 independent cultures. ND, not detected.i Yp/s, yield in accumulated target product (mM) per initial glucose (55.6 mM) � 100%; Ybp/s, yield in accumulated by-product (mM) per initial glucose (55.6 mM) � 100%. NC,not calculable.

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was slightly lower (2.6 �mol g�1 CDW min�1) than that of Phe bystrain AR-G91 (3.3 �mol g�1 CDW min�1). In contrast, the max-imum specific productivity of 4HLA by strain PAR-3 (2.5 �molg�1 CDW min�1) was similar to that of Tyr by strain AR-G2 (2.2�mol g�1 CDW min�1). On the other hand, the maximum spe-cific productivities of PE by strain PAR-105 (4.2 �mol g�1 CDWmin�1), 4HPE by strain PAR-104 (4.5 �mol g�1 CDW min�1),PAA by strain PAR-100 (5.8 �mol g�1 CDW min�1), and 4HPAAby strain PAR-102 (4.6 �mol g�1 CDW min�1) were obviously

higher than those of Phe by strain AR-G91 and Tyr by strainAR-G2.

DISCUSSION

E. coli has become a promising host organism for the microbialproduction of a variety of valuable chemicals, including biofuel(8), lactic acid (19), succinic acid (6), etc., from renewable re-sources. The aim of this study was to generate E. coli strains able toproduce a range of aromatic compounds from glucose. Our strat-

FIG 3 Time course analyses of the production of the respective aromatic compounds by the constructed strains on a shaking test tube platform. E. coli strainsAR-G91 (Phe overproducer), AR-G2 (Tyr overproducer), PAR-58 (PLA producer), and PAR-3 (4HPLA producer) were cultivated in M9M medium. StrainsPAR-105 (PE producer) and PAR-100 (PAA producer) were cultivated in M9M medium plus 1.5 mM Tyr, whereas strains PAR-104 (4HPE producer) andPAR-102 were cultivated in M9M medium plus 1 mM Phe. The black arrows indicate the time of the addition of IPTG for gene overexpression. The strains werecultivated at 37°C (before IPTG addition) and 27°C (after IPTG addition). The data are averages of triplicate cultures, and the maximum variation was less than10%. The respective symbols represent as follows: , glucose concentration; �, OD660; open circle, Phe concentration; open triangle, Tyr concentration; opensquare, PLA concentration; open diamond, 4HPLA concentration; closed circle, PE concentration; closed triangle, 4HPE concentration; closed square, PAAconcentration; closed diamond, 4HPAA concentration. The broken lines represent by-products. Abbreviations are summarized in Materials and Methods.

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egy— expanding the shikimate pathway—is exceedingly simple.Homologous/heterologous genes were introduced into the chro-mosomal DNA of aromatic amino acid overproducers. To accom-plish this, the heterologous genes were surveyed from bacteria tobe expressed in soluble and active form in E. coli. Conclusively, wesuccessfully constructed metabolically engineered E. coli strainscapable of producing six kinds of aromatic compounds, namely,PLA, 4HPLA, PE, 4HPE, PAA, and 4HPAA. To the best of ourknowledge, this is the first report of the bioproduction of PLA,4HPLA, PAA, and 4HPAA from glucose by metabolically engi-neered E. coli.

PLA is known as a broad-spectrum antimicrobial agent whichis naturally produced by some classes of microorganisms, espe-cially lactic acid bacteria. An enzyme responsible for convertingPP to PLA is required in PLA production. Batch and fed-batchfermentation by Lactobacillus, optimized by PP feeding and pHcontrol, has been proposed as one mechanism of PLA production(24). Whole cells of Bacillus coagulans SDM have also been shownto effectively convert PP to PLA (46). In both cases, PP was used asa substrate for PLA production; however, PP is too unstable andexpensive for industrial and/or commercial use. In our study, glu-cose could be used as a substrate, because the dehydrogenase gene(ldhA), responsible for PP reduction activity, was introduced intothe chromosomal DNA of Phe-overproducing E. coli. Applyingthis strategy to the Tyr overproducer, we found that 4HPLA couldbe similarly produced from a glucose substrate.

PE is an important flavor and fragrance compound with a rose-like odor. Little is currently known about whether bacteria cansynthesize PE, but Microbacterium sp. and Brevibacterium linensare known to do so (15). By comparison, many fungal and yeastspecies are PE producers. Indeed, yeasts are the most prominentnatural PE-synthesizing microorganisms, and PE production byyeast has been extensively studied (11). Yeasts produce PEthrough the Ehrlich pathway which involves transamination tothe corresponding alpha-keto acids, followed by decarboxylationto an aldehyde, and final reduction to an alcohol. Current yeastproduction processes require Phe as a substrate. On the otherhand, we successfully produced PE by metabolically engineeringE. coli, using glucose as a substrate instead of Phe. In our study, the

shikimate pathway of a Phe overproducer was expanded by intro-ducing two bacterial heterologous genes (T7p-ipdC and T7p-adhC) whose effects mimic the Ehrlich pathway. Our attempt wassuccessful, but an unexpected phenomenon arose. Introducingthe PP decarboxylase gene (T7p-ipdC) induced automatic reduc-tion of PAAL in vivo, supposedly by endogenous aldehyde reduc-tases present in E. coli, leading to PE accumulation. By surveyingsuch reductase genes, three candidate genes, yqhD, yjgB, and yahK,were identified. Of these, yqhD-encoded reductase alone has beenexperimentally investigated. This reductase is a broad-substratealcohol dehydrogenase (14), and as such, has been used to gener-ate strains capable of producing 1,3-propandiol (37) and isobuta-nol (1). We propose here that other genes, yjgB and yahK, could beused for PE production from glucose. The Ehrlich pathway wasalso simulated in a Tyr overproducer to generate a 4HPE-produc-ing strain. In this case, the T7p-adhC gene product did not suffi-ciently function as a 4HPAAL reductase (Table 5). However, threeof the genes encoding aldehyde reductases (yqhD, yjgB, and yahK)were implicated in successful 4HPE production. Introducing T7p-yahK resulted in lower accumulated levels of 4HPAA by-product.In recent work, it was shown that an E. coli strain overexpressingboth the tyramine oxidase gene from Micrococcus luteus and theTyr decarboxylase gene from Papaver somniferum, without anyexogenous 4HPAAL reductases, produced 4HPE from glucose,suggesting the presence of a reductase capable of acting on4HPAAL (34). The enzymatic data in the present study indicatedthat yqhD-, yjgB-, and yahK-encoded reductases can act as4HPAAL reductases (Table 3). However, the results of the genedeletion experiment suggest that other reductases can also pro-mote 4HPE production (Table 6).

Oxidation of PAAL to PAA and 4HPAAL to 4HPAA are well-known processes. In our study, oxidation was carried out usingfeaB-encoded PAAL dehydrogenase, which is involved in E. coliPEA and TYM degradation pathways (10, 30). Introduction ofT7p-ipdC alone thus resulted in PAA and/or 4HPAA productionvia decarboxylation of Phe and Tyr by ipdC-encoded decarboxyl-ase, followed by oxidation by the endogenous feaB-encoded dehy-drogenase in strains PAR-60 and PAR47 (Table 7). PAA and4HPAA production was enhanced when feaB was overexpressedtogether with ipdC in strains PAR-61 and PAR-51.

We encountered several obstacles in the production of the re-spective aromatic compounds, which we attempted to resolve.During 4HPE production by strain PAR-65, small amounts ofPAA, 4HPAA, and PE by-products were accumulated (Table 5). Inthis case, we thought that deletion of the endogenous feaB geneencoding aldehyde dehydrogenase might prevent PAA and4HPAA production (10, 12). In fact, the constructed strain PAR-83, a PAR-65 derivative lacking feaB, did not produce such by-products. The same effect was observed in PE producer PAR-84lacking the feaB gene (Table 5).

The most serious problem was that hydroxyphenyl com-pounds, 4HPE and 4HPAA, accumulated as by-products in theproduction of PE and PAA. That is, a certain amount of 4HPE wasaccumulated during PE production by strain PAR-68 (Table 5),while the PAA produced by strain PAR-61 was contaminated with4HPAA (Table 7). For reasons that are not entirely clear, 4HPEand 4HPAA leakages may have occurred through Tyr synthesis. Insuch cases, we thought that blocking the Tyr synthetic route mightreduce accumulation of unwanted by-products. In addition,blockage of the Tyr synthetic route might contribute to the en-

TABLE 8 Volumetric and specific productivitiesa

StrainTargetproduct Mediumb

rp, max

(�mol l�1

min�1)

qp, maxc

(�molg�1 CDWmin�1)

AR-G91 Phe M9M 10.0 0.6 3.3 0.1AR-G2 Tyr M9M 4.8 0.6 2.2 0.1PAR-58 PLA M9M 6.4 0.2 2.6 0.1PAR-3 4HPLA M9M 7.6 0.3 2.5 0.2PAR-105 PE M9M Tyr 6.2 0.1 4.2 0.2PAR-104 4HPE M9M Phe 7.9 0.3 4.5 0.5PAR-100 PAA M9M Tyr 8.2 0.2 5.8 0.1PAR-102 4HPAA M9M Phe 5.8 0.2 4.6 0.6a The strains were cultivated at 37°C (before induction of gene expression) and 27°C(after induction of gene expression) for 72 h. An aliquot of the culture was periodicallysampled to determine volumetric and specific productivity parameters.b Phe and Tyr were supplied at 1.0 and 1.5 mM, respectively.c Maximum specific productivity was calculated by the formula qp � rp/Mx (27), whererp is the volumetric productivity (�mol liter�1 min�1) and Mx is the cell dry weight(CDW) (g l�1).

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hanced yield of target product by depressing carbon flux into thecell. Indeed, in a previous study of Phe production, deletion of thetyrA gene in E. coli strains overexpressing both aroF and pheA wasshown to markedly enhance Phe production (36). Blockage of theTyr synthetic route by deletion of tyrA in PE- and PAA-producingstrains did indeed repress accumulation of 4HPE (strain PAR-105in Table 5) and 4HPAA (strain PAR-100 in Table 7). Furthermore,the titers and yields of 4HPE and 4HPAA were increased. How-ever, deletion of the tyrA gene rendered strains PAR-105 and PAR-100 Tyr auxotrophic, so they required supplemental Tyr in theM9M medium for growth. The observed lower levels of 4HPE(PAR-105) and 4HPAA (PAR-100) by-products accumulatedwere thought to mean that supplemental Tyr for cell growth wasconverted to 4HPE and 4HPAA through the Ehrlich pathway.Although PP decarboxylase encoded by the ipdC gene from A.brasilense, which acts on both PP and 4HPP, was used in the pre-sent study, use of a PP-specific enzyme might encourage PE andPAA production without amassing 4HPE and 4HPAA as un-wanted by-products.

Maximum theoretical yield coefficients of produced Phe fromglucose have been calculated from the known stoichiometry ofPhe biosynthesis from glucose in different metabolic scenarios(28). Although PEP is converted to pyruvate during glucose trans-port by PTS without recycling of pyruvate to PEP in wild-type E.coli, the calculated maximum theoretical yield is 0.275 g/g, whichcorresponds to 30% (mol/mol). On the other hand, by PTS inac-tivation or pyruvate recycling in an engineered strain, the maxi-mum theoretical yield increased 2-fold (0.55 g/g), which corre-sponds to 60% (mol/mol); these modifications have already beenachieved in Phe biosynthesis (7, 13, 36). These scenarios are alsoapplicable to the yields of Tyr and our target aromatic com-pounds. Since Phe overproducer AR-G91 and Tyr overproducerAR-G2, which are used as host strains for aromatic compoundproducers, take up glucose through PTS and do not recycle pyru-vate, the maximum theoretical yields of Phe and Tyr by thesestrains are 30% (mol/mol). Similarly, maximum theoretical yieldsof PLA, 4HPLA, PE, 4HPE, PAA, and 4HPAA by the constructedstrains derived from strains AR-G91 and AR-G2 are also 30%(mol/mol). Therefore, the constructed strains in this study shouldbe further genetically modified to increase the maximum theoret-ical yield up to 60% (mol/mol) as a future issue. Such modifica-tions would also enhance the volumetric and specific productivi-ties of the respective aromatic compounds.

The replacement of petroleum with renewable sources in in-dustrial chemical processing is of urgent concern, and here weaddress this rapidly expanding field of research. As an initial steptoward our goal, we verified that six kinds of aromatic compoundscan be produced from glucose, a primary component of renew-able resources such as starch and cellulose. The productivities ofsix aromatic compounds by the constructed strains were the sameor higher in comparison to those of Phe (by strain AR-G91) andTyr (by strain AR-2) (Table 8), so that the possibility of the pro-duction of aromatic compounds from renewable resources wasshown here. We further found that yjgB- and yahK-encoded en-zymes probably act as aldehyde reductases on PAAL and 4HPAAL.Although these reductases require enzymological data, novelfunctions of some putative genes could be revealed through met-abolic engineering studies such as that presented here.

ACKNOWLEDGMENT

This work was partially supported by a Grant-in-Aid for Young Scientists(B) (19780082 and 21780105).

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