Metabolic Engineering of Escherichia coli for Efficient Production of2‑Pyrone-4,6-dicarboxylic Acid from GlucoseZi Wei Luo,†,‡ Won Jun Kim,†,‡ and Sang Yup Lee*,†,‡,§

†Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering(BK21 Plus Program), Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon34141, Republic of Korea‡Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, KAIST, Daejeon 34141,Republic of Korea§BioProcess Engineering Research Center and BioInformatics Research Center, KAIST, Daejeon 34141, Republic of Korea

*S Supporting Information

ABSTRACT: 2-Pyrone-4,6-dicarboxylic acid (PDC) is a pseudoaromatic dicarboxylicacid and is a promising biobased building block chemical that can be used to makediverse polyesters with novel functionalities. In this study, Escherichia coli wasmetabolically engineered to produce PDC from glucose. First, an efficient biosyntheticpathway for PDC production from glucose was suggested by in silico metabolic fluxsimulation. This best pathway employs a single-step biosynthetic route toprotocatechuic acid (PCA), a metabolic precursor for PDC biosynthesis. On thebasis of the selected PDC biosynthetic pathway, a shikimate dehydrogenase (encodedby aroE)-deficient E. coli strain was engineered by introducing heterologous genes ofdifferent microbial origin encoding enzymes responsible for converting 3-dehydroshikimate (DHS) to PDC, which allowed de novo biosynthesis of PDC fromglucose. Next, production of PDC was further improved by applying stepwise rationalmetabolic engineering strategies. These include elimination of feedback inhibition on 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (encoded by aroG) by over-expressing a feedback-resistant variant, enhancement of the precursor phosphoenolpyruvate supply by changing the nativepromoter of the ppsA gene with the strong trc promoter, and reducing accumulation of the major byproduct DHS byoverexpression of a DHS importer (encoded by shiA). Furthermore, cofactor (NADP+/NADPH) utilization was manipulatedthrough genetic modifications of the E. coli soluble pyridine nucleotide transhydrogenase (encoded by sthA), and the resultantimpact on PDC production was investigated. Fed-batch fermentation of the final engineered E. coli strain allowed production of16.72 g/L of PDC from glucose with the yield and productivity of 0.201 g/g and 0.172 g/L/h, respectively, representing thehighest PDC production performance indices reported to date.

KEYWORDS: 2-pyrone-4,6-dicarboxylic acid, protocatechuic acid, biomonomer, metabolic engineering, Escherichia coli

2-Pyrone-4,6-dicarboxylic acid (PDC) is a dicarboxylic acidwith a polar pseudoaromatic moiety, which naturally

occurs during microbial degradation of lignin-derived aromaticcompounds by Sphingobium sp. SYK-6 (previously charac-terized as Shingomonas paucimobilis SYK-6).1 Given thestructural similarity between PDC and terephthalic acid(TPA), which is an industrially important platform chemicalfor the production of various polyesters, e.g., polyethyleneterephthalate (PET),2 PDC has been successfully polymerizedwith various diols or hydroxyacids by direct dehydrationpolycondensation reactions, generating diverse polymers of anovel category possessing unique and promising character-istics.3,4 Due to the pseudoaromatic nature of PDC, the PDC-derived polymers are thermally stable, possess higher Young’smodulus and undergo easier degradation in the environmentcompared to PET.3 For these reasons, PDC has beenconsidered as a potential replacement of TPA in aromaticpolyesters.

To the best of our knowledge, synthesis of PDC by means ofchemical methods has never been reported. Over the pastdecades, a wide range of chemicals and materials have beenproduced from renewable resources by metabolically engi-neered microorganisms.5,6 Production of aromatic compoundsincluding aromatic amino acids has been well documented.7,8

However, microbial production of PDC has rarely beenreported except for the following two studies. In the first study,whole-cell biotransformation of protocatechuic acid (PCA)into PDC was achieved in a recombinant Pseudomonas putidastrain by introducing PCA 4,5-dioxygenase (encoded by ligAand ligB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde(CHMS) dehydrogenase (encoded by ligC) from S. paucimo-bilis SYK-6.9 In the second study more recently reported, de

Received: July 3, 2018Published: August 10, 2018

Research Article

pubs.acs.org/synthbioCite This: ACS Synth. Biol. 2018, 7, 2296−2307

© 2018 American Chemical Society 2296 DOI: 10.1021/acssynbio.8b00281ACS Synth. Biol. 2018, 7, 2296−2307

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novo PDC biosynthesis from glucose was realized byconstructing a PDC biosynthetic pathway that stems from

the shikimate (SHK) pathway.10 However, the PDC titerobtained was rather low (350 mg/L). The authors in this

Figure 1. Biosynthetic pathway of PDC from glucose and the metabolic engineering strategies used in this study. Abbreviations: PTS,phosphotransferase system; TCA, tricarboxylic acid; PP, pentose phosphate; G6P, glucose 6-phosphate; G3P, glyceraldehydes 3-phosphate; PEP,phosphoenolpyruvate; PYR, pyruvate; OAA, oxaloacetate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate;DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvyl-shikimate 3-phosphate;CHA, chorismate; 4HBA, 4-hydroxybenzoate; PCA, protocatechuate; CHMS, 4-carboxy-2-hydroxymuconate-6-semialdehyde; PDC, 2-pyrone-4,6-dicarboxylic acid. Genes that encode enzymes: ppsA, PEP synthetase; pykF, pyruvate kinase I; pykA, pyruvate kinase II; pckA, PEP carboxykinase;ppc, PEP carboxylase; tktA, transketolase I; aroGfbr, feedback-inhibition resistant mutant of DAHP synthase; aroB, DHQ synthase; aroD, DHQdehydratase; aroE, SHK dehydrogenase; aroK, SHK kinase I; aroL, SHK kinase II; aroA, 3-phosphoshikimate-1-carboxyvinyltransferase; aroC, CHAsynthase; ubiC, CHA lyase; asbF, dehydroshikimate dehydratase; pmdAB, PCA 4,5-dioxygenase; pmdC, CHMS dehydrogenase; shiA, SHK:H+

symporter. Overexpressed genes are marked in blue, and red fork indicates gene deletion. SHK:H+ symporter, two recombinant plasmids andgenome manipulations for promoter replacement employed in this study are illustrated.

Figure 2. Comparison of two distinct pathways for PDC biosynthesis from glucose via in silico flux analysis. (A) Schematic for the two distinct PDCpathways: single-step and six-step, as well as the six-step pathway with cofactors intentionally removed. Cofactors (NADPH and ATP) areindicated. Abbreviations: DHS, 3-dehydroshikimate; SHK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvyl-shikimate 3-phosphate;CHA, chorismate; 4HBA, 4-hydroxybenzoate; PCA, protocatechuate; PDC, 2-pyrone-4,6-dicarboxylic acid. Black arrows indicate E. coli nativemetabolic reactions, red arrows indicate heterologous enzymatic reactions, and dotted arrows indicate multiple steps of enzymatic reactions. (B)Performance of the different PDC pathways during in silico simulation experiments. The orange line corresponds to the result of the single-steppathway, the magenta line corresponds to the result of the six-step pathway, and the blue line corresponds to the result of the six-step pathwaywithout cofactors requirement.

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report simultaneously adopted two different pathway routes toobtain the precursor PCA, which might be less than optimaldue to the potential carbon flux loss at multiple nodes.Additionally, all the genes in the above study were overex-pressed using the strong T7 promoter from plasmid, which isnot necessarily optimal for the production of metabolitesincluding PDC.11,12

To develop a bacterial strain capable of efficiently producingPDC from simple carbon sources, we aimed at establishing asynthetic pathway allowing efficient carbon flux throughout thepathway from glucose to PDC (Figure 1) aided by in silicometabolic flux simulation studies. After successful de novobiosynthesis of PDC from glucose by introducing the necessaryand screened heterologous enzymes, PDC production wasfurther improved through elimination of feedback repressionon the key enzyme in the aromatic pathway, increase ofprecursors availability and importer engineering for byproductreduction. In addition, the redox cofactor (NADP+/NADPH)regeneration for optimal PDC biosynthesis was considered,which revealed an important role of redox cofactor balance onPDC production. Ultimately, fed-batch culture of the best-performing engineered strain was performed to demonstrate itspotential for the production of PDC from glucose.

■ RESULTS AND DISCUSSIONComparison of Different PDC Biosynthetic Pathways

via in Silico Flux Response Analysis. The biosyntheticpathway from glucose to PDC comprises two parts (Figure 1).At first, carbon flow is directed from glucose to PCA, the firstheterologous metabolite in the PDC biosynthetic pathway.Next, PCA is converted to PDC by an aromatic ring cleavageoperon. In E. coli, PCA can be produced through two differentroutes (Figure 2A). One route is from 3-dehydroshikimate(DHS) to PCA via a single heterologous enzymatic reactioncatalyzed by DHS dehydratase, which is designated as thesingle-step route in this study. Several previous studies haveemployed this single-step PCA route for building syntheticmetabolic pathways leading to production of various PCA-derived chemicals, including adipic acid,13 cis,cis-muconic acidand catechol.14−16 To promote the availability of DHS to beconverted into PCA using this single-step route, formation ofSHK from DHS was prevented by the inactivation of SHKdehydrogenase.13 The other route to PCA synthesis is from 4-hydroxybenzoate (4HBA), which is derived from the endproduct of SHK pathway and converted into PCA by 4HBA

hydroxylase. Combing the conversions of DHS to 4HBA and4HBA to PCA, it takes totally six enzymatic steps to obtainPCA from DHS through 4HBA, which is designated as the six-step route. Recently, this six-step PCA route has been utilizedin a number of studies for constructing novel biosynthesispathways for value-added aromatic products.17,18

Despite both the PCA-supplying routes described abovehave been reported and used for production of variousaromatic chemicals of interest, a systematic comparisonbetween the two has not been made. In order to select moreefficient biosynthetic pathway for PDC production, we firstperformed in silico flux analyses on both the genome-scalemetabolic networks employing the single-step or six-steppathway for PDC production from glucose in E. coli. Thesimulation results indicated that the single-step PDC pathwaywas more efficient for PDC production than the six-steppathway, as reflected by the higher specific PDC productionrate (Figure 2B). The theoretical maximum PDC yieldsobtainable by employing the single-step pathway and the six-step pathway were calculated to be 0.906 and 0.738 g/gglucose, respectively, suggesting again that the single-steppathway is more efficient for PDC production. It washypothesized that the six-step pathway is less efficient due tothe requirement for cofactors (e.g., 1 molecule of ATP and 2molecules of NADPH) (Figure 2A). To verify this assumption,another simulation was performed for the six-step pathwaywhere cofactor requirement was artificially removed (Figure2A). This artificial six-step pathway without cofactor require-ment showed increased specific PDC production rate (Figure2B), which proved our hypothesis. Based on these results, thesingle-step pathway was selected for constructing the PDCbiosynthetic pathway from glucose in E. coli.

Biosynthesis of PDC from Glucose. To construct thePDC biosynthetic pathway based on the single-step PCAroute, we first focused on constructing the upstream pathwaymodule from glucose to PCA. As described earlier, PCA can beproduced from glucose via the single-step conversion of DHSto PCA catalyzed by DHS dehydratase. Naturally occurringand structurally diverse DHS dehydratases have beendiscovered in different microbial species (SupplementaryTable S1),19−23 and employed in many reports for microbialproduction of PCA and PCA-derived products.10,13,14,16

To identify a suitable DHS dehydratase candidate forefficient production of PCA from glucose, three DHSdehydratases including AroZ of Klebsiella pneumonia KCTC

Figure 3. Flask cultures for PCA production from glucose by introducing different DHS dehydratases (A) and for PDC production from glucoseusing the selected DHS dehydratase (B). Symbols are gray box, cell growth (OD600); blue box, PCA concentration (g/L); red box, PDCconcentration (g/L).

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2208, AsbF of Bacillus thuringiensis ATCC 10792 and QsuB ofCorynebacterium glutamicum ATCC 13032 were examined forthe following reasons. The DHS dehydratase AroZ (encodedby aroZ) has been used in an earlier study to construct a cis,cis-muconic acid biosynthetic pathway, which produced as high as36.8 g/L of cis,cis-muconic acid in fed-batch culture.13 In amore recent study, the DHS dehydratase AsbF (encoded byasbF) was compared with two other DHS dehydratases forproviding PCA as a precursor, showing better performance.14

The DHS dehydratase QsuB (encoded by qsuB) was found inthe SHK/quinate degradation pathway of C. glutamicum,24 andit has been expressed in a recombinant plant Arabidopsis toengineer the lignin deposition in cell walls.25 However, theapplication potential of QsuB for production of PCA and PCAderivatives in E. coli host has not been examined to date.To compare these three DHS dehydratases, the aroZ, asbF

and qsuB genes were cloned into plasmid pTrc99A under trcpromoter, resulting in plasmids pTrcZ, pTrcF and pTrcB,respectively. These plasmids were transformed individuallyinto E. coli GYT0, which is DHS dehydrogenase-deficientstrain of E. coliW3110 constructed by disrupting the aroE geneto block the downstream SHK pathway. Flask cultures showedthat the E. coli GYT0 strain harboring pTrcF produced thehighest titer of PCA (1.14 g/L), which was 4.4-fold and 1.2-fold that of PCA produced by the recombinant GYT0 strainsharboring pTrcZ and pTrcB, respectively (Figure 3A). Thus,the DHS dehydratase AsbF was selected for further experi-ments that include assembling with the downstream pathwaymodule for converting PCA to PDC.After the successful validation of upstream pathway

conversion from glucose to PCA, we then set out to introducethe downstream pathway module from PCA to PDC. Asmentioned, this aromatic ring opening process is catalyzed byPCA 4,5-dioxygenase and CHMS dehydrogenase along with anonenzymatic, spontaneous conversion step. In previousstudies on microbial PDC production,9,10 a gene operoncontaining ligA and ligB genes for PCA 4,5-dioxygenase andligC for CHMS dehydrogenase from S. paucimobilis SYK-6were employed. In this study, we employed an alternativeoperon comprising pmdA, pmdB and pmdC genes fromComamonas testosteroni ATCC 11996 for converting PCA toPDC. The pmdA, pmdB and pmdC genes are functionallyhomologous to ligA, ligB and ligC,26 with amino acid sequenceidentities of 48.4%, 57.1% and 75.0%, respectively. It wasreported that the PCA 4,5-dioxygenase encoded by pmdABshows less promiscuous activity compared with the oneencoded by ligAB.27 Thus, it is of interest to examine theperformance of pmdABC operon in biocatalysis of PCA intoPDC. To investigate the proper expression levels of pmdA,pmdB and pmdC for PDC production, three genes were clonedas the native operon into plasmids pTac15K and pET-22b(+)under the tac and T7 promoters, giving plasmids pTacABCand pETABC, respectively. Two recombinant strains, W3110harboring pTacABC and BL21(DE3) harboring pETABC,were cultured in flasks supplemented with 2.50 g/L of PCA.The results showed that PCA was successfully converted intoPDC by both the E. coli transformants (Supplementary FigureS1). No bioconversion was detected in the recombinant E. colicells harboring the respective empty vectors. Furthermore,when induction levels were varied by applying differentconcentrations (0.1, 0.5, and 1 mM) of the inducer isopropylβ-D-1-thiogalactopyranoside (IPTG), distinct profiles of thebioconversion from PCA to PDC were observed for the two

transformants (Supplementary Figure S1). The E. coli W3110harboring pTacABC achieved complete conversion of PCAunder conditions of all the three IPTG concentrations tested.Particularly under the condition of 1 mM IPTG, the highestPDC titer of 2.67 g/L was obtained from 2.50 g/L of PCAsupplemented, which corresponded to a conversion yield of89.4 mol %. In contrast, the E. coli BL21(DE3) harboringpETABC achieved only partial conversion of PCA, and thefinal PDC titers were declined as the IPTG concentrationsincreased. The highest PDC titer of 2.32 g/L at conversionyield of 78.8 mol % for the E. coli BL21(DE3) harboringpETABC was obtained at the IPTG concentration of 0.1 mM.These PCA-feeding experiments indicated that the tacpromoter was performing better in driving expression of thepmdABC operon for an optimal PCA to PDC conversioncompared to T7 promoter. Also, it is noteworthy thatintermediary metabolites (e.g., CHMS isoforms) might havealso been produced over the course of PCA conversion, as canbe seen from the fact that complete PCA conversion did notlead to a PDC yield of 100 mol %. However, the intermediateswere not investigated in this study due to analytical difficulties.Having constructed both pathway modules from glucose to

PCA and from PCA to PDC, the complete biosynthesispathway from glucose to PDC was then assembled byconstructing the plasmid pTacFABC. The E. coli GYT0 strainharboring pTacFABC successfully produced 0.97 g/L of PDCin flask culture using glucose as the sole carbon source (Figure3B). Moreover, no PCA formation was detected in the culturebroth, which suggested that the pmdABC operon bearssufficient capability for catalyzing the downstream pathwaymodule from PCA to PDC (Figure 3B). The PDC productionwas also confirmed by GC−MS analysis (SupplementaryFigure S2). Since the key enzyme DHS dehydratase, AsbF, wasof heterologous origin, it was examined whether an E. coli-codon optimized asbF gene, designated as asbFopt (Supple-mentary Table S2), would enhance PDC production; therewas a report showing that a codon-optimized asbF geneexhibited higher AsbF activity.14 The asbFopt gene obtained bygene synthesis service was cloned in the same configuration asfor the asbF gene, giving pTrcFopt and pTrcFoptABC. In flaskculture, however, the E. coli GYT0 strain harboring pTrcFopt

produced 0.32 g/L of PCA (Figure 3A), which was less by71.9% than that obtained with the wild-type asbF; the E. coliGYT0 strain harboring pTrcFoptABC produced 0.55 g/L ofPDC (Figure 3B), less by 43.3% than that obtained with thewild-type asbF. The low activity of asbFopt might be due to theformation of inclusion body, which requires further inves-tigation to confirm. Overall, the de novo production of PDCfrom glucose was successfully accomplished through the use ofthe B. thuringiensis asbF gene and C. testosteroni pmdABCoperon based on the single-step pathway.

Redirecting Flux into the Truncated SHK Pathway forImproved PDC Production. After the single-step PDCbiosynthetic pathway was functionally established in E. coli, wenext focused on engineering the cellular targets that couldredirect central metabolic flux toward PDC biosyntheticpathway, aiming at higher PDC productivity.As the first step in SHK pathway, condensation of erythrose

4-phosphate (E4P) and phosphoenolpyruvate (PEP) generates3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) by theaction of DAHP synthase (Figure 1), which dictates the carbonflux directed into SHK pathway. In E. coli, the total activity ofDAHP synthase is comprised of three isoenzymes, AorG

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(accounting for 80%), AroF (15%) and AroH (5%), which aresubjected to tight regulation imposed by allosteric control andtranscriptional repression.28 Hence, in order to increasecellular DAHP synthase activity, we overexpressed aroGfbr

gene encoding a feedback-inhibition resistant mutant ofAroG,29 by generating plasmid pBBR1Gfbr. E. coli GYT0 strainharboring pTacFABC and pBBR1Gfbr produced dramaticallyincreased PDC titer (2.07 g/L) (Figure 4), 1.1-fold higher thanthat obtained by the parental strain in flask culture.

As the elevated DAHP synthase activity significantly raisedPDC titer, it was speculated that this might in turn cause ahigher demand for the precursor substrates E4P and PEP.Therefore, it is necessary to also enhance the intracellularavailability of these two key aromatic substrates. To enhancesupply of E4P, a commonly utilized approach is to overexpressthe transketolase encoded by tktA, which is a key enzymeresponsible for E4P synthesis.30,31 The E. coli tktA gene wasthus cloned into plasmid pBBR1Gfbr to generate pBBR1Gfbr-A.When pBBR1Gfbr-A was transformed into E. coli GYT0 strainharboring pTacFABC, however, only 1.97 g/L of PDC wasproduced (Figure 4), which was 4.8% lower than that obtainedby GYT0 strain harboring pTacFABC and pBBR1Gfbr. On theother hand, for enrichment of PEP, strategies includingamplification of PEP synthetase (encoded by ppsA) forimproved formation of PEP from pyruvate (PYR),31 andinactivation of PYR kinase I (encoded by pykF) and PYRkinase II (encoded by pykA) for preventing consumption ofPEP to PYR in glycolysis,32,33 have previously proven to beeffective. On the basis of these validated strategies, weconstructed the following engineered strains, GYT1 (carryingppsA overexpression through exchange of its native promoterwith the strong trc promoter), GYT2 (carrying pykF deletion),GYT3 (carrying pykF and pykA double-deletions) and GYT4(carrying both ppsA overexpression by trc promotor exchangeand pykF deletion). When each of these four strains wastransformed with pTacFABC and pBBR1Gfbr-A, and tested inflask cultures, it was found that only GYT1 strain harboringpTacFABC and pBBR1Gfbr-A produced slightly higher PDCtiter (2.12 g/L) (Figure 4) than that obtained by the parentalstrain GYT0 harboring pTacFABC and pBBR1Gfbr-A. All ofthe other three strains harboring pTacFABC and pBBR1Gfbr-Aproduced less PDC (Figure 4). These results suggest that the

availability of precursors E4P and PEP might not be thelimiting factor for PDC production from glucose in ourengineered strain under the tested culture condition. Althoughnot performed in this study, in silico genome-scale metabolicsimulations can be performed for identifying potentialbottlenecks and also new engineering targets in the future.To sum up, among all the gene targets examined above, only

aroGfbr and ppsA overexpression displayed improved PDCproduction. Thus, we combined these two positive targets bygenerating GYT1 strain harboring pTacFABC and pBBR1Gfbr,which produced 2.28 g/L of PDC in flask culture (Figure 5),

showing a cumulatively positive effect on PDC production.Also, this PDC titer represented a 1.4-fold increase over thepreliminary strain GYT0 harboring pTacFABC, suggesting thatcarbon flux was successfully redirected toward PDC biosyn-thesis through the above efforts.

Importer Engineering for Reduced Byproduct DHSAccumulation. During analysis of the culture supernatants ofPDC production strains described above, we observed thatconcentrations of PCA remained very low (0−10 mg/L) (datanot shown), but large amounts of DHS were accumulated. Forinstance, GYT1 strain harboring pTacFABC and pBBR1Gfbr,which produced the highest titer of PDC so far, accumulated2.52 g/L of DHS (Figure 5).To avoid carbon loss via such a surplus extracellular

formation of DHS and, in turn, to reuse it for boosting PDCproduction, we targeted on a membrane-bound transportercalled ShiA in E. coli, which reportedly could assimilateextracellular DHS into the cytosol of cells.34,15 In the attemptto enhance DHS import and thus its intracellular availabilityfor improved PDC production, we tested E. coli native ShiA(EcShiA) as well as a number of ShiA proteins originated fromother different microbial species and showing various aminoacid sequence similarities relative to EcShiA (Supplementary

Figure 4. Effects of overexpression of the aroGfbr, tktA and ppsAgenes, and deletion of the pykF and/or pykA genes on PDCproduction. Symbols are gray box, cell growth (OD600); red box, PDCconcentration (g/L).

Figure 5. Effect of overexpression of DHS importers from differentmicrobial origins on PDC production. Engineered strains indicatedare Control, GYT1 harboring pTacFABC and pBBR1Gfbr; KpShiA,GYT1 harboring pTacFABC and pBBR1Gfbr-KpA; RoShiA, GYT1harboring pTacFABC and pBBR1Gfbr-RoA; AsShiA, GYT1 harboringpTacFABC and pBBR1Gfbr-AsA; EcShiA, GYT1 harboring pTac-FABC and pBBR1Gfbr-EcA; PtrcShiA, GYT5 harboring pTacFABCand pBBR1Gfbr; EcShiA + PtrcShiA, GYT5 harboring pTacFABC andpBBR1Gfbr-EcA. Symbols are gray box, cell growth (OD600); red box,PDC concentration (g/L); yellow box, DHS concentration (g/L).

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Table S3). Genes encoding K. pneumonia ShiA (KpShiA),Rhodococcus opacus PD630 ShiA (RoShiA) and Acinetobactersp. ADP1 ShiA (AsShiA) along with EcShiA, were inserted intoplasmid pBBR1Gfbr to generate pBBR1Gfbr-KpA, pBBR1Gfbr-RoA, pBBR1Gfbr-AsA and pBBR1Gfbr-EcA, respectively.Introduction of these four plasmids individually into E. coliGYT1 strain harboring pTacFABC led to distinct profiles ofDHS formation and PDC production in flask cultures (Figure5). Among these four ShiA importers tested, AsShiA andEcShiA showed reduced DHS formation, whereas KpShiA andRoShiA resulted in elevated DHS accumulation (Figure 5).Particularly, GYT1 strain harboring pTacFABC andpBBR1Gfbr-EcA produced the least DHS (0.42 g/L), 83.3%lower than that obtained by the parental strain GYT1harboring pTacFABC and pBBR1Gfbr. However, such aconsiderable decrease in DHS accumulation improved PDCproduction by only 5.7%, reaching 2.41 g/L. On the otherhand, coexpression of AsShiA also lowered DHS formation (by40.5%) compared to the control strain without expressingAsShiA, probably thanks to its relatively high amino acidsequence similarity to EcShiA (65.3%) (Supplementary TableS3), but the PDC titer was also decreased (1.97 g/L)surprisingly.Given that overexpression of EcShiA had the best effect on

reducing byproduct DHS formation and improving PDCproduction, we sought to further increase the expression levelof EcShiA, in order to further reduce extracellular DHSaccumulation thereby increasing PDC production. It wasreported that although its chromosomal transcription isconstitutive, E. coli shiA gene is repressed under normalgrowth conditions via a small RNA-mediated translationregulation mechanism, and the small RNA base-pairinginteraction region is located upstream of the coding sequencein the mRNA of shiA gene.35 As such, we determined toreplace the native promoter of E. coli shiA gene with the strongtrc promoter. By doing so, not only could the transcription ofshiA gene be strengthened, but also its translation could bederegulated as the small RNA binding region would beeliminated after the promoter exchange. GYT5 strain was thusgenerated, which carried trc promoter-driven shiA gene in thechromosome. At first, we examined effect of the promoterexchange of shiA alone on reducing DHS formation. Asexpected, flask culture of GYT5 strain harboring pTacFABCand pBBR1Gfbr showed reduced accumulation of DHS (1.86g/L) by 26.2% compared to the parental strain GYT1harboring pTacFABC and pBBR1Gfbr (Figure 5). However,GYT5 strain harboring pTacFABC and pBBR1Gfbr producedless PDC (2.18 g/L). Furthermore, combined effect of theplasmid-borne and chromosomal trc promoter-driven over-expression of E. coli shiA was investigated. Flask culture ofGYT5 strain harboring pTacFABC and pBBR1Gfbr-EcAshowed further reduced accumulation of DHS (0.37 g/L) by11.9% compared to GYT1 strain harboring pTacFABC andpBBR1Gfbr-EcA (Figure 5). But similarly, only 2.16 g/L ofPDC was produced, which was less than that by GYT1 strainharboring pTacFABC and pBBR1Gfbr-EcA.Taken together, we successfully reduced accumulation of the

major byproduct DHS to a minimum level through over-expression of DHS importer. And GYT1 strain harboringpTacFABC and pBBR1Gfbr-EcA, which produced the highestPDC titer (2.41 g/L) along with the second least DHSformation (0.42 g/L), was selected as base strain for furtherengineering.

Effect of Genetic Manipulations of the SolubleTranshydrogenase (SthA) on PDC Production. In orderto further optimize PDC production, it is necessary to considerthe cofactor utilization for the following reason. In the last stepof PDC pathway (Figure 1), one molecule of the cofactorNADP+ is converted to NADPH accompanying one moleculeof PDC produced; this might interfere with cellular redoxcofactor balance. Thus, we targeted on E. coli soluble pyridinenucleotide transhydrogenase (encoded by sthA) as SthA hasbeen reported to be responsible for reoxidation of NADPH toNADP+, particularly under metabolic conditions with excessNADPH formation.36,37

To enhance SthA activity, we decided to overexpress sthAgene through two layers of manipulations. First, we overex-pressed it by promoter exchange with the strong trc promoterin GYT1 strain, generating GYT6 strain. Second, based onGYT6, we additionally constructed GYT7 strain, in whichthree silent point mutations (C15T, T18C, C21T) wereintroduced into sthA gene in the chromosome, for thefollowing reason. In E. coli, a small regulatory RNA calledSpot 42 (encoded by spf), highly abundant in the presence ofglucose, was reported to repress sthA expression by transla-tional regulation through inhibitory base-pairing with the sthAmRNA, and the binding site also localized within the beginningregion of the coding sequence of sthA.38 Thus, we attempted toderegulate this repression mechanism by introducing the abovethree silent point mutations that would affect the comple-mentary base-pairing interaction as seen from the binding freeenergy increase from −14.52 to −1.57 kcal/mol (Figure 6).

These two newly constructed strains GYT6 and GYT7 wereexamined in flask cultures using GYT1 strain as a control.Among these three strains tested, distinct time profiles of cellgrowth, PDC production and byproducts (i.e., PCA and DHS)formation were observed (Figure 7). Throughout the course offlask culture, GYT6 strain harboring pTacFABC andpBBR1Gfbr-EcA produced less PDC than the control strainGYT1 harboring same plasmids (Figure 7B). In the case ofGYT7 strain harboring pTacFABC and pBBR1Gfbr-EcA, alsoless amount of PDC was produced than the control as seenfrom the final titer (2.21 g/L) (Figure 7B). However, at timepoint of 36 h, GYT7 strain harboring pTacFABC andpBBR1Gfbr-EcA produced higher titer of PDC (2.05 g/L)

Figure 6. Schematic representation of the chromosomal manipu-lations of the sthA gene: (1) promoter replacement with the strong trcpromoter and (2) further introduction of three silent point mutations(C15T, T18C, C21T) in the coding sequence of sthA, which leads toweakened base-pairing interaction of the sthA mRNA with the smallregulatory RNA spf, as seen from the binding free energy increasefrom −14.52 to −1.57 kcal/mol.

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versus that by the control (1.82 g/L) (Figure 7B), which wasaccomplished apparently due to the higher maximum PDCproductivity of GYT7 strain harboring pTacFABC andpBBR1Gfbr-EcA (0.103 g/L/h) over that of the control(0.082 g/L/h). Moreover, it was interestingly noticeable thatGYT7 strain harboring pTacFABC and pBBR1Gfbr-EcA hadhigher byproducts formation (Figure 7C and D), particularlyDHS, than the control, which indicated a stronger total fluxredirected into the aromatic biosynthetic pathway. Accordingto these results, GYT7 strain harboring pTacFABC andpBBR1Gfbr-EcA, and GYT1 strain harboring pTacFABC andpBBR1Gfbr-EcA, were selected and further evaluated inbioreactor fermentations as discussed below.Bioreactor Fermentations. Following development of

engineered E. coli strains for PDC production, bioreactorfermentations were conducted to demonstrate their potentialsfor scale-up production.Batch fermentations were first carried out to optimize

cultivation conditions and select the best-performing PDCproduction strain for subsequent fed-batch culture study. Asoxygen is required by PmdAB in PDC biosynthesis (Figure 1),we thus tested effects of different dissolved oxygen (DO) levels

(25%, 40% and 80% of air saturation) on PDC fermentation,using GYT1 strain harboring pTacFABC and pBBR1Gfbr-EcA.Batch fermentation profiles under the three DO conditions aredepicted (Supplementary Figure S3) and the results aresummarized (Table 1). It was observed that the highest PDCproduction (4.78 g/L) was obtained under DO level of 40%(Supplementary Figure S3B and Table 1). When DO level wasset as 25% or 80%, the PDC titer was slightly decreased(Supplementary Figure S3A and S3C, and Table 1).Using the selected DO setting of 40% of air saturation,

GYT7 strain harboring pTacFABC and pBBR1Gfbr-EcA wasexamined in batch fermentation with all other conditions sameas for GYT1 strain harboring pTacFABC and pBBR1Gfbr-EcA.Unexpectedly, GYT7 strain harboring pTacFABC andpBBR1Gfbr-EcA produced only 0.38 g/L of PDC withaccumulations of PCA and DHS as high as 4.74 g/L and9.37 g/L, respectively (Supplementary Figure S4A and Table1). Out of curiosity, we further examined GYT6 strainharboring pTacFABC and pBBR1Gfbr-EcA in batch fermenta-tion under the same conditions, which produced 2.32 g/L ofPDC with formations of 1.86 g/L of PCA and 4.57 g/L ofDHS (Supplementary Figure S4B and Table 1).

Figure 7. Time profiles of cell growth (A), PDC (B), PCA (C) and DHS (D) of engineered E. coli strains in flask cultures. Symbols are blue square,GYT1 harboring pTacFABC and pBBR1Gfbr-EcA; red circle, GYT6 harboring pTacFABC and pBBR1Gfbr-EcA; green triangle, GYT7 harboringpTacFABC and pBBR1Gfbr-EcA.

Table 1. Batch Fermentation Performances of the Engineered Strains for PDC Production

strain maximum OD600 PDC (g/L) PCA (g/L) DHS (g/L) productivity (g/L/h) yield (g/g)

GYT1/pTacFABC + pBBR1Gfbr-EcAa 11.90 4.28 0.14 1.71 0.104 0.209GYT1/pTacFABC + pBBR1Gfbr-EcAb 12.45 4.78 0.18 2.79 0.118 0.235GYT1/pTacFABC + pBBR1Gfbr-EcAc 12.25 4.27 0.70 1.70 0.101 0.221GYT7/pTacFABC + pBBR1Gfbr-EcAb 12.85 0.38 4.74 9.37 0.007 0.016GYT6/pTacFABC + pBBR1Gfbr-EcAb 11.90 2.32 1.86 4.57 0.052 0.107

aDO level was set as 25% of air saturation during fermentation. bDO level was set as 40% of air saturation during fermentation. cDO level wasinitially set as 40% of air saturation and then shifted to 80% of upon induction.

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Comparing the results in bioreactor batch fermentationswith those in flask cultures for GYT1, GYT6 and GYT7 strainsharboring pTacFABC and pBBR1Gfbr-EcA, it was observedthat GYT1 and GYT6 strains harboring pTacFABC andpBBR1Gfbr-EcA had relatively consistent performances in PDCproduction when they were transferred from flask cultivationto bioreactor cultivation. By contrast, GYT7 strain harboringpTacFABC and pBBR1Gfbr-EcA exhibited a dramatic decay inPDC production capability when moved from flask culture tobioreactor culture. Such a huge change in PDC productionperformance suggested an intriguing insight into the importantrole of cellular redox cofactor in affecting PDC production andthe flux toward DHS in the context of different cultivationconditions. Yet, further detailed investigation is needed toclarify the underlying mechanisms that have resulted in suchoutcomes. Although the three silent point mutations of sthAgene in GYT7 strain failed to increase PDC production in thisstudy, this genetic modification strategy might potentially serveas a useful approach to perturb the SthA activity in E. coli whenneeded.On the basis of batch fermentation results above, GYT1

strain harboring pTacFABC and pBBR1Gfbr-EcA was selectedas the final strain and examined in a fed-batch culture underDO level of 40% of air saturation and with a pH-stat nutrientfeeding strategy. The fed-batch fermentation resulted inproduction of 16.72 g/L of PDC with the productivity of0.172 g/L/h and the yield of 0.201 g/g glucose (Figure 8). At

the same time, 9.76 g/L of DHS was accumulated, while only0.36 g/L of PCA was formed. It is speculated that the highaccumulation of DHS during fed-batch culture might becaused by relatively limited activity of DHS dehydratase and/or decay of DHS importer activity over the course offermentation. This suggests potential room for furtherenhancement of PDC production level. Thus, future studiesshould be focused on improving the activity of DHSdehydratase and/or maintaining of DHS importer activityduring large-scale fermentation processes.

■ CONCLUSIONS

In this paper, we developed metabolically engineered E. coliplatform strains capable of producing PDC using glucose as acarbon source. An efficient metabolic pathway toward PDCwas suggested on the basis of in silico flux simulation, and theselected PDC pathway was reconstructed through introductionof screened, efficient heterologous enzymes. Removal offeedback inhibition of a key enzyme, elevated precursor supplyand overexpression of DHS importer were important inimproving PDC production and reducing byproduct for-mation. Furthermore, manipulation of the cofactor (NADPH/NADP+) utilization through perturbing small regulatory RNA-based soluble pyridine nucleotide transhydrogenase geneexpression provided insight into the impact of redox cofactorbalancing on PDC production. Finally, fed-batch culture of thebest-performing engineered strain produced 16.72 g/L of PDCwith an overall productivity of 0.172 g/L/h and yield of 0.201g/g glucose, which represented the highest titer for PDCproduction from glucose reported to date. Further improve-ment in PDC production would be possible by takingadvantage of various systems metabolic engineering tools,39

such as systematic flux optimization, synthetic regulatorysRNA-mediated large-scale gene target identification coupledwith high-throughput techniques,40 as well as bioprocessoptimization.

■ METHODS

Bacterial Strains and Media. The bacterial strains used inthis study are listed in Table 2. E. coli DH5α was employed forgene cloning and plasmid propagation, and E. coli W3110 andits derivatives were used for PDC production as host strains.For general cultures during plasmid construction and bacterialgenome manipulation, E. coli cells were cultured in Luria−Bertani (LB) broth or on LB plates (1.5%, w/v, agar) withproper antibiotics supplemented as follows: 100 μg/mL ofampicillin, 50 μg/mL of kanamycin, and/or 34 μg/mL ofchloramphenical. Bacterial species Comamonas testosteroniATCC 11996, Bacillus thuringiensis ATCC 10792, Klebsiellapneumonia KCTC 2208, Corynebacterium glutamicum ATCC13032, Rhodococcus opacus PD630 and Acinetobacter sp. ADP1served as genetic sources for heterologous enzymes ortransporters employed in this study. Cultivation of thesewild-type strains was carried out under the conditions specifiedby the corresponding suppliers.

Plasmid Construction. All plasmids used in this study arelisted in Table 2. The basic molecular biology experimentsincluding PCR, gel electrophoresis, and bacterial trans-formation for plasmid and strain construction were conductedaccording to standard procedures.41 Commercial kits wereused for preparation of bacterial genomic DNA, isolation ofplasmid DNA and purification of DNA fragments from agarosegels following the supplier’s instructions (Genotech, Daejeon,South Korea). Services of primer synthesis and DNAsequencing were also provided by Genotech. All theoligonucleotide primers utilized in this work are listed inSupplementary Table S4. To construct pETABC, the pmdABCoperon from the genome of C. testosteroni ATCC 11996 wasamplified using pmdABC(pET)-f and pmdABC(pET)-rprimers, and cloned into pET-22b(+) vector amplified usingpET(pmdABC)-f and pET(pmdABC)-r primers by Gibsonassembly method.42 To construct pTacABC, the pmdABCoperon was amplified using pmdABC(pTac)-f and pmdABC-

Figure 8. Fed-batch fermentation profile of GYT1 harboringpTacFABC and pBBR1Gfbr-EcA. Symbols are blue circle, cell growth(OD600); red square, residual glucose concentration (g/L); greendiamond, PDC concentration (g/L); magenta triangle, PCAconcentration (g/L); orange inverted triangle, DHS concentration(g/L).

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(pTac)-r primers, and cloned into pTac15K vector amplifiedusing pTac(pmdABC)-f and pTac(pmdABC)-r primers byGibson assembly. To construct pTrcZ, the aroZ gene fromK. pneumonia KCTC 2208 was amplified using aroZ(pTrc)-fand aroZ(pTrc)-r primers, and cloned into pTrc99A vector

amplified using pTrc(aroZ)-f and pTrc(aroZ)-r primers byGibson assembly. To construct pTrcB, the qsuB gene fromC. glutamicum ATCC 13032 was amplified using qsuB-f andqsuB-r primers, and cloned into pTrc99A vector at SacI andXbaI sites. To construct pTrcF, the asbF gene fromB. thuringiensis ATCC 10792 was amplified using asbF-f andasbF-r primers, and cloned into pTrc99A vector at EcoRI andPstI sites. To construct pTrcFopt, an E. coli codon-optimizedversion of asbF gene (synthesized by GenScript) was amplifiedusing asbFopt-f and asbFopt-r primers, and cloned into pTrc99Avector at EcoRI and PstI sites. To construct pTacFABC, the trc-asbF-rrnBT1T2 cassette was amplified from pTrcF using trc-asbF-f and trc-asbF-r primers, and assembled by Gibsonassembly with pTacABC linearized by enzyme digest at NheIsite. To construct pTacFoptABC, the trc-asbFopt-rrnBT1T2cassette was amplified from pTrcFopt using trc-asbF-f and trc-asbF-r primers, and assembled by Gibson assembly withpTacABC linearized by enzyme digest at NheI site. Toconstruct pBBR1Gfbr, the feedback inhibition-resistant versionof the E. coli aroG gene was amplified using aroGfbr(pBBR1)-fand aroGfbr(pBBR1)-f primers, and cloned into pBBR1MCSvector amplified using pBBR1(aroGfbr)-f and pBBR1(aroGfbr)-rprimers by Gibson assembly. To construct pBBR1Gfbr-A, theRBS-tktA cassette was amplified using RBS-tktA-f and RBS-tktA-r primers, and assembled by Gibson assembly withpBBR1Gfbr linearized by enzyme digest at BamHI site. Toconstruct pBBR1Gfbr-EcA, the RBS-shiA cassette was amplifiedfrom E. coli using RBS-EcA-f and EcA-r primers, and clonedinto pBBR1Gfbr at HindIII and BamHI sites. To constructpBBR1Gfbr-KpA, the RBS-shiA cassette was amplified fromK. pneumonia KCTC 2208 using RBS-KpA-f and RBS-KpA-rprimers, and cloned into pBBR1Gfbr at HindIII and SpeI sites.To construct pBBR1Gfbr-RoA, the RBS-shiA cassette wasamplified from R. opacus PD630 using RBS-RoA-f and RBS-RoA-r primers, and cloned into pBBR1Gfbr at HindIII andBamHI sites. To construct pBBR1Gfbr-AsA, the RBS-shiAcassette was amplified from A. sp. ADP1 using RBS-AsA-f andRBS-AsA-r primers, and cloned into pBBR1Gfbr at HindIII andBamHI sites. All the recombinant plasmids were confirmed bycolony PCR and DNA sequencing.

Genome Manipulation. One-step homologous recombi-nation-based inactivation method43 was used for gene deletionand promoter replacement experiments. For deleting aroE, thefirst knockout PCR product was amplified using aroE-KO-fand aroE-KO-r primers having 50 bp nucleotide extensionhomologous to the upstream and downstream regions of aroEwithin the genome. The plasmid pECmulox bearing a lox71-chloramphenical resistance gene (cat)-lox66 cassette was usedas a template for PCR amplifying the first knockout fragment.The homology sequence was elongated to 100 bp via a secondPCR using the first PCR product as template and using aroE-KOEX-f and aroE-KOEX-r primers. For the promoter changeof ppsA, the pMtrc9 plasmid containing the strong trcpromoter and the upstream lox71-cat-lox66 cassette wasemployed as a template in the PCR reactions using primerpairs of Ptrc(ppsA)-f/Ptrc(ppsA)-r and Ptrc(ppsA)-EX-f/Ptrc(ppsA)-EX-r, instead of pECmulox. PCR products fordeletion or promoter replacement of the remaining genes wereprepared in the same manner using primers listed inSupplementary Table S4. For construction of the GYT7 strain,the three point mutations (C15T, T18C, C21T) wereintroduced into the promoter replacement primers. Regardlessof gene deletion or promoter replacement, the amplified

Table 2. Strains and Plasmids Used in This Studya

name relevant genotype reference

StrainsDH5α F− endA1 gln V44 thi-1 recA1 relA1 gyrA96

deoR nupG purB20 φ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(rK−mK

+),λ−

Invitrogen

BL21(DE3) F− ompT gal dcm lon hsdSB (rB−mB

−) (DE3) InvitrogenW3110 Coli genetic stock center strain No. 4474 CGSCb

GYT0 W3110 ΔaroE This studyGYT1 W3110 ΔaroE PppsA::Ptrc This studyGYT2 W3110 ΔaroE ΔpykF This studyGYT3 W3110 ΔaroE ΔpykF ΔpykA This studyGYT4 W3110 ΔaroE ΔpykF PppsA::Ptrc This studyGYT5 W3110 ΔaroE PppsA::Ptrc PshiA::Ptrc This studyGYT6 W3110 ΔaroE PppsA::Ptrc PsthA::Ptrc This studyGYT7 W3110 ΔaroE PppsA::Ptrc PsthA::Ptrc

(C15T, T18C, C21T)This study

PlasmidspTrc99A ApR, trc promoter, pBR322 origin, lacIq,

4.2 kbLab stock

pTac15K KmR, tac promoter, p15A origin, 4.0 kb Lab stockpET-22b(+) ApR, T7 promoter, pBR322 origin, 5.5 kb NovagenpBBR1MCS CmR, lac, T3 and T7 promoters, 4.7 kb 49

pTrcZ pTrc99A derivative containing aroZ genefrom K. pneumonia

This study

pTrcF pTrc99A derivative containing asbF genefrom B. thuringiensis

This study

pTrcFopt pTrc99A derivative containing E. coli-codonoptimized asbF gene

This study

pTrcB pTrc99A derivative containing qsuB genefrom C. glutamicum ATCC13032

This study

pTacABC pTac15K derivative containing pmdABCoperon from C. testosteroni

This study

pETABC pET-22b(+) derivative containing pmdABCoperon from C. testosteroni

This study

pTacFABC pTacABC derivative containing trc-asbF-rrnBT1T2 cassette

This study

pTacFoptABC pTacABC derivative containing trc-asbFopt-rrnBT1T2 cassette

This study

pBBR1Gfbr pBBR1CS derivative containing aroGfbr geneunder the lac promoter

This study

pBBR1Gfbr-A pBBR1Gfbr derivative containing RBS-tktAcassette

This study

pBBR1Gfbr-EcA

pBBR1Gfbr derivative containing RBS-shiAcassette from E. coli

This study

pBBR1Gfbr-KpA

pBBR1Gfbr derivative containing RBS-shiAcassette from K. pneumoniae

This study

pBBR1Gfbr-RoA

pBBR1Gfbr derivative containing RBS-shiAcassette from R. opacus PD630

This study

pBBR1Gfbr-AsA

pBBR1Gfbr derivative containing RBS-shiAcassette from A. sp. ADP1

This study

pKD46 ApR, λ-Red recombinase under arabinose-inducible araBAD promoter, ts origin,6.3 kb

43

pJW168 ApR, Cre-recombinase under IPTG-induciblelacUV5 promoter, ts origin, 5.5 kb

44

pECmulox ApR, CmR, lox66-cat-lox71, 3.5 kb 50

pMtrc9 Modified pECmulox containing trc promoterdownstream of lox66-cat-lox71 cassette

Lab stock

aAbbreviations: Ap, ampicillin; Km, kanamycin; Cm, chlorampheni-col; R, resistance; ts, temperature sensitive. bColi Genetic StockCenter, New Haven, CT.

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knockout PCR products were introduced into E. coli cellsexpressing the Red recombinase from pKD46 induced by 10mM L-arabinose. Colonies were selected on LB agar platessupplemented with chloramphenical, and the successfulgenome manipulation was confirmed by colony PCR.Subsequently, the helper plasmid pJW168 that expressed theCre recombinase by 1 mM IPTG was introduced to removethe chloramphenical marker gene.44 With the temperature-sensitive replication origins, both pKD46 and pJW168 wereeasily cured through temperature shift between 30 and 42 °C.The excision of the marker gene was further confirmed byperforming colony PCR. For promoter replacement, thepositive clones were further sequenced to validate the correctmanipulation.In Silico Flux Simulation Experiments. To perform in

silico flux response analysis for comparing two biosyntheticpathway routes (single-step and six-step pathways) for PDC,we employed the genome-scale metabolic model EcoM-BEL979,45 which is a modified model of iJR9043046 andcomprises 979 metabolic reactions and 814 metabolites. AsPDC biosynthetic pathways are not native to E. coli, theheterologous pathway reactions are required to be additionallyrecruited to the EcoMBEL979 model. To obtain PCA, thefollowing reaction “DHS ↔ PCA + H2O” was added to theEcoMBEL979 model for the single-step biosynthetic pathway,whereas the following reaction “4HBA + NADPH + O2 + H+

↔ PCA + NADP+ + H2O” was added for the six-stepbiosynthetic pathway. From PCA to PDC, the subsequentreactions were introduced into the EcoMBEL979 model forboth pathways: “PCA + O2 ↔ CHMS + H+”, “CHMS ↔CHMS (hemiacetal form)”, and “CHMS (hemiacetal form) +NADP+ ↔ PDC + NADPH + H+”. Next, we investigated thetheoretical impacts of the two different biosynthetic pathways(single-step and six-step) on PDC production, throughconstraint-based flux simulation with the hypothesis of pseudosteady-state. Cell growth rate was maximized as an objectivefunction as the PDC biosynthetic flux was gradually increasedfrom minimum to maximum values. Over the course ofsimulation, the glucose uptake rate was set at 10 mM/gDCW/h.Cultivation. Shake-flask cultivations were performed in the

baffled flasks (300 mL), which contained a working volume of50 mL MR minimal salts medium (pH 7.0) supplemented with10 g/L glucose. The MR medium (1 L) contained: 6.67 gKH2PO4, 4 g (NH4)2HPO4, 0.8 g MgSO4·7H2O, 0.8 g citricacid, and 5 mL trace metal solution.47 Glucose, MgSO4·7H2O,MR salts and trace metal solution were prepared andautoclaved separately. To deal with the auxotroph caused bythe disruption of aroE gene, the MR medium (1 L) was alsosupplemented with L-phenylalanine (40 mg), L-tyrosine (40mg), L-tryptophan (40 mg), 4-hydroxybenzoic acid (10 mg),4-aminobenzoic acid (10 mg), 2,3-dihydroxybenzoic acid (10mg) and thiamine hydrochloride (10 mg),13 stock solutions ofwhich were separately sterilized through 0.22 μm membrane.To prepare the inoculums for flask cultivation, glycerol stocksof the engineered strains were first inoculated into a 25 mL testtube containing 5 mL of LB medium and cultured at 200 rpmand 37 °C for 12 h. Aliquots of 1 mL of the preculture wereused for inoculation. After inoculation, flasks were placed in arotary incubator at 200 rpm and 37 °C for initial growth. After6 h of cultivation when cells grew up to the OD600 value of0.6−0.8, 1 mM IPTG was added for induction, and then cellcultures were immediately transferred to a rotary incubator at

200 rpm and 30 °C for additional 54 h of cultivation.Appropriate antibiotics were added to the medium whennecessary. All flask cultivations described in this study wereconducted in biological duplicates, and the results weredepicted as mean values plus standard deviations in the graph.Batch reactor fermentations were conducted using a 6.6-L

jar fermentor (Bioflo 3000; New Brunswick Scientific Co.,Edison, NJ) containing 1.8 L of the MR mediumsupplemented with 20 g/L of glucose, 3 g/L yeast extract,40 mg/L of L-phenylalanine, 40 mg/L of L-tyrosine, 40 mg/Lof L-tryptophan and 10 mg/L of 4-hydroxybenzoic acid under30 °C. To prepare seed cultures, 2 mL of overnight LB testtube cultures were transferred into the Erlenmeyer nonbaffledflasks (300 mL) containing 100 mL of the same medium andcultivated in a shaking incubator for 10 h at 200 rpm and 37°C. A total volume of 200 mL of seed cultures were used toinoculate the fermentor to make a total working volume of 2 L.The fermentation culture pH was maintained at 7.0 with theaddition of ammonia solution (28%, v/v). The dissolvedoxygen (DO) was maintained at 25%, 40% or 80% of airsaturation as indicated by automatically changing the agitationspeed from 200 to 1000 rpm and additional supply of pureoxygen when necessary with a constant air flow of 2.0 L/min.Cells were induced with 1 mM IPTG at the OD600 value of2.0−3.0. All batch fermentations with each of engineeredstrains were performed twice with reproducible results, and theresults of one representative batch fermentation are presented.Fed-batch fermentation was performed under the same

settings except the followings. The initial fermentation mediumcontained 1.8 L of the MR medium supplemented with 20 g/Lof glucose, 3 g/L yeast extract, 100 mg/L of L-phenylalanine,100 mg/L of L-tyrosine, 100 mg/L of L-tryptophan and 25mg/L of 4-hydroxybenzoic acid. Cells were induced with 1mM IPTG at the OD600 value of 4.0−5.0. When glucose wasdepleted at the end of batch culture, fed-batch mode wasinitiated by a pH-stat nutrient feeding strategy. The pH-statloop was whenever the medium pH ≥ 7.02, the nutrientsolution was pumped into the fermentor at the rate of 100%.The nutrient solution comprised 700 g/L of glucose, 8 g/L ofMgSO4·7H2O, 5 mL trace metal solution, 3 g/L of yeastextract, 100 mg/L of L-phenylalanine, 100 mg/L of L-tyrosine,100 mg/L of L-tryptophan, 25 mg/L of 4-hydroxybenzoic acid,1 mM IPTG and appropriate antibiotics. Foam formation wasrepressed by manually adding Antifoam 289 (Sigma ChemicalCo., St. Louis, MO, USA). Fed-batch fermentation wasperformed three times, and the result of one representativefed-batch culture is presented.

Analytical Procedures. Cell growth was monitored bymeasuring the optical density at the wavelength of 600 nm(OD600) using the Ultrospec 3100 spectrophotometer(Amersham Biosciences, Uppsala, Sweden). The residualglucose concentration in culture broth was measured usinghigh-performance liquid chromatography (HPLC) (Waters1515/2414/2707, Waters, Milford, MA). The concentrationsof PDC, DHS and PCA were analyzed according to a modifiedmethod,48 which employed a HPLC (1100 Series, Agilent)equipped with a Zorbax SB-Aq column (4.6 × 250 mm,Agilent) operating at 30 °C. The mobile phase consisting ofbuffer A (25 mM potassium phosphate buffer, pH 2.0) andbuffer B (acetonitrile), flowed at 0.8 mL/min according to thefollowing program: 0−1 min, 0% B; 1−8 min, a linear gradientof B from 0% to 70%; 8−11 min, 70% B; 11−18 min, a lineargradient of B from 70% to 0%; 18−20 min, 0% B. The

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injection volume of 10 μL was used and photodiode arraydetector was used to monitor the signal at 280 nm for PDC,DHS and PCA. Samples for HPLC analysis were prepared asfollows: cells and debris in the fermentation broth were firstpelleted by centrifugation at 13 200g for 10 min, and then thesupernatant was diluted properly and filtered through a 0.22μm membrane. The quantification was made according to astandard calibration curve established with each authenticcompound. To further confirm the production of PDC, culturesample was centrifuged to remove cells and the supernatantwas acidified to pH 1.0 by concentrated HCl. An aliquot (1mL) of the resulting solution was extracted twice with thesame volume of ethyl acetate. The collected extract wasvacuum-dried on a rotary evaporator. The residue wasdissolved in pyridine and incubated with bistrimethylsilyl-trifluoroacetamide to prepare trimethylsilyl derivatives, andthen subjected to gas chromatography (GC)−mass spectrom-etry as described previously.9

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssyn-bio.8b00281.

Supporting tables and figures (PDF)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: leesy@kaist.ac.kr. Tel: +82-42-350-3930. Fax: +82-42-350-3910.ORCIDZi Wei Luo: 0000-0001-6018-9368Sang Yup Lee: 0000-0003-0599-3091Author ContributionsZ.W.L. and S.Y.L. designed the research; Z.W.L. performed theexperiments; W.J.K. performed in silico simulation experi-ments; and Z.W.L. and S.Y.L. wrote the manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Dr. Masaya Nakamura at Forestry and ForestProducts Research Institute (FFPRI) in Japan for generouslyproviding us the authentic PDC compound used as ananalytical standard in this study. This work was supported bythe Intelligent Synthetic Biology Center through the GlobalFrontier Project (2011-0031963) and also by the TechnologyDevelopment Program to Solve Climate Changes on SystemsMetabo l ic Eng ineer ing for B iorefiner ies (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) fromthe Ministry of Science and ICT through the NationalResearch Foundation of Korea.

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