Microbial production of methyl anthranilate, a grapeflavor compoundZi Wei Luoa,b,1, Jae Sung Choa,b,1, and Sang Yup Leea,b,c,d,2

aMetabolic 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, 34141 Daejeon, Republic of Korea; bSystems Metabolic Engineering andSystems Healthcare Cross-Generation Collaborative Laboratory, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea;cBioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; and dBioInformaticsResearch Center, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea

Contributed by Sang Yup Lee, April 5, 2019 (sent for review March 6, 2019; reviewed by Jay D. Keasling and Blaine A. Pfeifer)

Methyl anthranilate (MANT) is a widely used compound to givegrape scent and flavor, but is currently produced by petroleum-basedprocesses. Here, we report the direct fermentative production ofMANT from glucose by metabolically engineered Escherichia coli andCorynebacterium glutamicum strains harboring a synthetic plant-derived metabolic pathway. Optimizing the key enzyme anthranilicacid (ANT) methyltransferase1 (AAMT1) expression, increasing thedirect precursor ANT supply, and enhancing the intracellular avail-ability and salvage of the cofactor S-adenosyl-L-methionine requiredby AAMT1, results in improvedMANT production in both engineeredmicroorganisms. Furthermore, in situ two-phase extractive fermen-tation using tributyrin as an extractant is developed to overcomeMANT toxicity. Fed-batch cultures of the final engineered E. coliand C. glutamicum strains in two-phase cultivation mode led tothe production of 4.47 and 5.74 g/L MANT, respectively, in minimalmedia containing glucose. The metabolic engineering strategies de-veloped here will be useful for the production of volatile aromaticesters including MANT.

metabolic engineering | Escherichia coli | Corynebacterium glutamicum |methyl anthranilate | two-phase fermentation

Methyl anthranilate (MANT), which gives grape scent andflavor, has been extensively used in flavoring foods (e.g.,

candy, chewing gum, soft drinks, and alcoholic drinks, etc.) anddrugs (as a flavor enhancer and/or mask). Due to its pleasantaroma, MANT is an important component in perfumes and cos-metics. MANT also has other important industrial applications as abird and goose repellent for crop protection, as an oxidation in-hibitor or a sunscreen agent, and as an intermediate for the syn-thesis of a wide range of chemicals, dyes, and pharmaceuticals (1).MANT is a natural metabolite giving the characteristic odor of

Concord grapes and occurs also in several essential oils (e.g.,neroli, ylang ylang, and jasmine) (1). It has been challenging andeconomically infeasible to directly extract MANT from theseplants due to low yields. Currently, MANT is commerciallymanufactured by petroleum-based chemical processes, whichmainly rely on esterification of anthranilic acid (ANT) withmethanol or isatoic anhydride with methanol, using homogeneousacids as catalysts (2). These processes, however, suffer from sev-eral disadvantages, for example, the requirement of acid catalystsin large quantities and problems with disposal of these toxic liquidacids after the reaction (2). Moreover, MANT produced by suchchemical methods is labeled “artificial flavor,” which does notmeet the increasing demand by consumers for natural flavors.Taking another important flavoring agent vanillin as an example,market preference for natural vanillin has led to a far higher priceof $1,200–$4,000/kg over $15/kg for artificial vanillin (3). Such amarket for natural MANT is also highly desirable, but unfortu-nately there have so far been no promising methods for preparingMANT from natural sources and/or by natural means. Severalenzymatic and microbial whole-cell biotransformation approacheshave been attempted for MANT production by esterification ofANT (4) or N-demethylation of N-methyl methyl anthranilate (5).

While these biotransformation procedures are considered morenatural and ecofriendly compared with chemical synthesis, theiractual use is limited due to low yields, long reaction times, andformation of byproducts (5). In addition, the chemical and bio-transformation processes mentioned above depend on substrates ofpetroleum origin. For these reasons, we were motivated to produceMANT through one-step microbial fermentation of renewablefeedstocks (e.g., glucose), which would offer 100% biobased nat-ural MANT in an ecofriendly manner.To our knowledge, there have been rare attempts on the de

novo microbial production of MANT, except for two reportsnearly 30 y ago describing MANT biosynthesis from simple sugars(i.e., maltose) by the wild-type fungi, Poria cocos (6) and Pycno-porus cinnabarinus (7). Unfortunately, the productivities achievedin these two studies were extremely low (18.7 mg/L MANT pro-duced after 5 d of culture). Also, the underlying biosyntheticmechanisms, including the biosynthesis genes, enzymes, andpathways, in these two fungal species have not been elucidated.Thus, it is a prerequisite to first identify a metabolic pathwayleading to the biosynthesis of MANT from simple carbon sources(e.g., glucose), before implementing various metabolic engineeringstrategies to develop microbial strains capable of efficiently pro-ducing MANT based on the reconstituted biosynthetic pathway.

Significance

Methyl anthranilate (MANT) is widely used in the flavoring andcosmetics industry to give grape scent and flavor. In an effortto replace the conventional petroleum-based synthesis ofMANT, we report the direct fermentative production of MANTfrom glucose in metabolically engineered Escherichia coli andCorynebacterium glutamicum strains. A synthetic plant-derivedmetabolic pathway was introduced and extensive metabolicengineering was performed including fine-tuning key enzymelevels and increasing the availability of precursor and cofactormetabolites. A two-phase extractive cultivation was developedusing an extractant solvent to recover MANT in situ, which ledto high levels of MANT production. This work demonstrates apromising sustainable alternative to MANT production andpresents strategies applicable toward production of othervaluable natural compounds.

Author contributions: S.Y.L. designed research; Z.W.L. and J.S.C. performed research;Z.W.L., J.S.C., and S.Y.L. analyzed data; and Z.W.L., J.S.C., and S.Y.L. wrote the paper.

Reviewers: J.D.K., University of California, Berkeley; and B.A.P., University at Buffalo.

The authors declare no conflict of interest.

Published under the PNAS license.1Z.W.L. and J.S.C. contributed equally to this work.2To whom correspondence should be addressed. Email: leesy@kaist.ac.kr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1903875116/-/DCSupplemental.

Published online May 13, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1903875116 PNAS | May 28, 2019 | vol. 116 | no. 22 | 10749–10756

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In this study, we report the development of metabolicallyengineered Escherichia coli and Corynebacterium glutamicumstrains capable of producing MANT directly from glucosethrough fermentation (Fig. 1). E. coli was initially chosen as amodel organism for metabolic engineering toward efficientproduction of MANT. In addition, C. glutamicum, a generallyrecognized as safe (GRAS) strain, was also engineered for theproduction of MANT for its consequent human consumption togive grape flavor and scent in food and cosmetics industries. Weapplied multiple strategies to produce MANT by optimizing itsbiosynthesis in both E. coli and C. glutamicum (Fig. 2). First, a

synthetic metabolic pathway originated from plant for de novoMANT biosynthesis was constructed in both E. coli and C. gluta-micum. Next, MANT production in both engineered organismswas improved through optimization of the key enzyme level, in-crease of flux to the direct precursor metabolite, increase of theavailability of cosubstrate required in MANT synthesis, and es-tablishment of in situ two-phase extractive cultivation process.Finally, two-phase fed-batch cultures of the best E. coli and C.glutamicum strains were performed to demonstrate their po-tential for large-scale production of MANT from glucose inminimal media.

Fig. 1. The metabolic network related to MANT biosynthesis from glucose in (A) E. coli and (B) C. glutamicum, as well as metabolic engineering strategiesemployed in this study. Abbreviations: ANT, anthranilate; ASP, L-aspartate; CHA, chorismate; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; E4P, erythrose 4-phosphate; EPSP, 5-enolpyruvyl-shikimate 3-phosphate; G6P, glucose 6-phosphate; Gln, glutamine;Glu, glutamate; HCYS, L-homocysteine; ILE, isoleucine; L-PHE, L-phenylalanine; L-TRP, L-tryptophan; L-TYR, L-tyrosine; MANT, methyl anthranilate; MET, L-methionine;PCA, protocatechuate; PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway; PRANT, N-(5-phosphoribosyl)-anthranilate; PTS, phosphotransferase system;PYR, pyruvate; QA, quinate; S3P, shikimate-3-phosphate; SAH, S-adenosyl-L-homocysteine; SAM, S-adenosyl-L-methionine; SER, L-serine; SHK, shikimate; SRH,S-ribosyl-L-homocysteine; TCA, tricarboxylic acid cycle. Genes that encode enzymes: ppsA, PEP synthetase; pykF, PYR kinase I; pykA, PYR kinase II; tktA, trans-ketolase I; aroGfbr, feedback-inhibition resistant mutant of DAHP synthase; aroB, DHQ synthase; aroD, DHQ dehydratase; aroE and ydiB, SHK dehydrogenase;aroK, SHK kinase I; aroL, SHK kinase II; aroA, 3-phosphoshikimate-1-carboxyvinyltransferase; aroC, CHA synthase; tyrA and pheA, TyrA and PheA subunits of CHAmutase, respectively; trpEfbr and trpD, ANT synthase component I (feedback inhibition-resistant mutant) and II, respectively; glnA, Gln synthetase; cysEfbr, SERacetyltransferase (feedback inhibition-resistant mutant);metAfbr, homoserineO-succinyltransferase (feedback inhibition-resistant mutant); luxS, S-ribosylhomocysteinelyase; mtn, 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase; metK, MET adenosyltransferase; aamt1, ANT methyltransferase1; pgi, G6P isomerase;zwf, G6P 1-dehydrogenase; tkt, transketolase; hdpA, dihydroxyacetone phosphate phosphatase; qsuB, DHS dehydratase; qsuD, QA/SHK dehydrogenase; metB,cystathionine-γ-synthase; sahH, SAH hydrolase; mcbR and Ncgl2640, transcriptional regulator. The solid arrows indicate single metabolic reaction, and the dashedarrows indicate multiple reactions. Overexpressed genes are marked in red, and red X indicates gene deletions. In situ two-phase extractive cultivation process isschematically illustrated, in which the MANT produced in the aqueous phase is extracted into the organic phase.

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Fig. 2. The overview of engineering strategies to optimize MANT production in E. coli and C. glutamicum. (A) Engineering strategies applied to produceMANT from glucose in E. coli, which include (1) initial construction of the de novo MANT biosynthesis pathway, (2) establishing an in situ two-phase extractivecultivation process to address MANT toxicity, (3) optimizing the key enzyme AAMT1 expression, (4) increasing the precursor ANT supply by metabolic en-gineering, (5) enhancing the availability of cosubstrate SAM required by the AAMT1-catalyzed reaction, and (6) in situ two-phase extractive fed-batch culturefor bioreactor-scale MANT production. The successive increase in MANT titer with each strategy is also illustrated. The pink bars represent the concentrationof MANT (in grams per liter). Values and error bars represent means and SDs of biological duplicates. The white circles represent individual data points. Errorbars represent mean ± SD (n = 2). The P values were computed by two-tailed Student’s t test (**P < 0.01; ***P < 0.001; ****P < 0.0001). (B) Engineeringstrategies to produce MANT from glucose using C. glutamicum, which include (1) initial construction of the de novo MANT biosynthesis pathway, (2) opti-mizing the key enzyme AAMT1 expression, (3) increasing precursor ANT supply by metabolic engineering, (4) establishing an in situ two-phase extractivecultivation process to address MANT toxicity, (5) salvaging the availability of cosubstrate SAM required by the AAMT1-catalyzed reaction, and (6) in situ two-phase extractive fed-batch culture for bioreactor-scale MANT production. The successive increase in MANT titer with each strategy is also illustrated. The pinkbars represent the concentration of MANT (in grams per liter). Values and error bars represent means and SDs of biological triplicates. The white circlesrepresent individual data points. Error bars represent mean ± SD (n = 3). The P values were computed by two-tailed Student’s t test (****P < 0.0001).

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Results and DiscussionConstructing a MANT Biosynthetic Pathway in E. coli.Due to the lackof knowledge on the MANT metabolism in the two fungal speciesdescribed above, we focused on the mechanism of MANT bio-synthesis in plants to identify a MANT biosynthetic pathway. In-triguingly, literature survey (8) suggested two different MANTbiosynthetic mechanisms present in plants (SI Appendix, Fig. S1).Both routes share the same precursor metabolite ANT, whichis derived from the L-tryptophan (L-TRP) biosynthesis pathwayin plants. For further conversion of ANT to MANT, one routeinvolves two reaction steps: CoA activation catalyzed byanthranilate-CoA ligase followed by acyl transfer by anthraniloyl-CoA:methanol acyltransferase using CoA, ATP, and methanol ascosubstrates. The other route uses a single-step conversion ofANT to MANT catalyzed by S-adenosyl-L-methionine (SAM)-dependent methyltransferase. In this study, we chose the latterroute as it is a simpler reaction requiring less cofactors, and moreimportantly, it does not require toxic methanol. Three such SAM-dependent methyltransferases have previously been identified inthe plant maize (Zea mays) (8). Among them, the one calledanthranilic acid methyltransferase1 (AAMT1; encoded by aamt1)was selected in this work as it was characterized to be the mostactive for MANT formation in maize (8).Based on the selected SAM-dependent methylation-mediated

MANT synthesis pathway, we expressed AAMT1 in an ANT-overproducing E. coli W3110 trpD9923 strain (9) (SI Appendix,Table S1) by introducing pTrcT that contained an E. coli codon-optimized version of aamt1 gene, designated as aamt1opt (SI Ap-pendix, Table S2), under the control of trc promoter. The successfulexpression of AAMT1 in the recombinant W3110 trpD9923 strainharboring pTrcT was confirmed by SDS/PAGE (SI Appendix, Fig.S2A). In a shake-flask culture using glucose as a sole carbon source,the W3110 trpD9923 strain harboring pTrcT successfully produced35.8 ± 3.0 mg/L MANT (Fig. 2A and SI Appendix, Fig. S2B), alongwith the accumulation of 215.5 ± 7.5 mg/L ANT (SI Appendix, Fig.S2B). No MANT production was detected in the wild-type W3110trpD9923, although it produced a higher amount of ANT (305.4 ±2.5 mg/L) (SI Appendix, Fig. S2B).

MANT Toxicity to E. coli. After the construction of a functionalMANT biosynthesis pathway in E. coli, we next evaluated thetoxicity of MANT to E. coli cells before further experiments.Exposing the wild-type E. coli W3110 cells to different concen-trations of MANT (0.1, 0.2, 0.3, 0.5, 0.7, 0.8, and 1.0 g/L) in-dicated a dose-dependent growth inhibition by MANT (SIAppendix, Fig. S3). In the presence of 0.3 g/L MANT, the finaloptical density at the wavelength of 600 nm (OD600) was only ahalf of that obtained with the control experiment without MANTexposure (SI Appendix, Fig. S3). When exposed in 1.0 g/LMANT, E. coli cell growth was completely inhibited (SI Appen-dix, Fig. S3).

Designing an in Situ Two-Phase Extractive Culture Process. To cir-cumvent this product toxicity limitation, a two-phase (aqueous/organic) cultivation system was designed, where MANT isextracted in situ from the culture medium using an organic solvent(Fig. 1). Tributyrin is one organic solvent nontoxic to microor-ganisms including E. coli (10) and is also used in the food industry,which can facilitate downstream purification processes for pre-paring food-grade MANT. Tributyrin was observed to extractMANT very efficiently as evidenced by its high partition co-efficient (420.1 ± 7.6) between aqueous medium phase andtributyrin phase (SI Appendix, Table S3). Tributyrin could alsoextract ANT from the aqueous phase, but to a far lesser extentcompared with that for MANT; the partition coefficients forANT were 4.7 ± 0.2 and 0.1 ± 0.0 at pH 5.0 and 7.0, respectively(SI Appendix, Table S3). The low partitioning of ANT intotributyrin phase can be explained by its pKa value of 2.14.

Using this two-phase system in shake-flask cultivation (a5:1 aqueous-to-organic phase ratio) (SI Appendix, Fig. S4A),W3110 trpD9923 strain harboring pTrcT produced 65.6 ± 0.4 mg/L MANT (Fig. 2A and SI Appendix, Fig. S4B; see SI Appendix,Supplementary Information Materials and Methods for the cal-culation of MANT concentration), which was 83.2% higher thanthat obtained in the single-phase culture. Also, almost all of theproduced MANT was extracted into the solvent phase, whileMANT was undetectable in the aqueous phase (SI Appendix,Fig. S4B).

Optimizing AAMT1 Expression for MANT Production in E. coli. Fol-lowing the establishment of in situ two-phase extractive cultivationprocess, we set out to optimize MANT production by manipulatingexpression of the key enzyme AAMT1 (Fig. 1A). Expression levelsof AAMT1 were varied by expressing the aamt1opt gene, either onthe medium-copy pTrc99A vector under a set of six synthetic consti-tutive promoters with different strengths (BBa_J23117, BBa_J23114,BBa_J23105, BBa_J23118, BBa_J23101, and BBa_J23100) (SI Ap-pendix, Table S4), or on the low-copy pTac15K vector under thesame set of synthetic promoters as well as the stronger isopropylβ-D-1-thiogalactopyranoside (IPTG)-inducible tac promoter (SIAppendix, Fig. S5A). When these AAMT1 constructs, pTrc99A-series or pTac15K-series, were introduced to W3110 trpD9923strain and tested in two-phase flask culture, a clearly positivecorrelation of MANT production with the strengths of the pro-moters employed within the same vector series was observed,while ANT accumulation was negatively correlated (SI Appendix,Fig. S5B). Also, it was found that under each of the six syntheticpromoters, AAMT1 expressed on the medium-copy pTrc99Avector enabled higher MANT production compared with thoseon the low-copy pTac15K (SI Appendix, Fig. S5B). Among theserecombinant strains, W3110 trpD9923 strain harboring pTacT(aamt1opt expressed on pTac15K under tac promoter) producedMANT to the highest titer of 297.3 ± 0.7 mg/L (Fig. 2A and SIAppendix, Fig. S5B), while accumulating ANT to the leastamount of 117.1 ± 2.2 mg/L (SI Appendix, Fig. S5B). Based onthese results, we further cloned aamt1opt on pTac15K underT5 promoter with stronger strength than tac promoter (11),which however resulted in less MANT production (219.1 ±10.9 mg/L) (SI Appendix, Fig. S5B).

Increasing ANT Supply for MANT Production in E. coli.Next, we aimedat increasing intracellular flux to the direct precursor ANT tofurther improve MANT production. ANT is a native metabolite inL-TRP biosynthesis pathway in E. coli (Fig. 1A). Previous studies onengineering E. coli for ANT overproduction have suggested severalpromising strategies for increasing ANT production (12, 13). First,a feedback inhibition-resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (encoded by aroGfbr) catalyzing thecondensation of erythrose 4-phosphate (E4P) and phosphoenol-pyruvate (PEP) was expressed, by constructing pTrcGfbr (aroGfbr

cloned on medium-copy pTrc99A under the strong trc promoter)and pBBR1Gfbr (aroGfbr cloned on low-copy pBBR1MCS underthe less strong lac promoter). W3110 trpD9923 strain harboringpBBR1Gfbr produced 731.7 ± 7.4 mg/L ANT (SI Appendix, Fig.S6A), which was 2.3- and 1.4-fold higher than those obtained withW3110 trpD9923 strain harboring pTrcGfbr (SI Appendix, Fig. S6A)and wild-type W3110 trpD9923 strain (SI Appendix, Fig. S2B), re-spectively. Second, we attempted to increase the availability of twokey precursors E4P and PEP of the aromatic amino acid pathwayfor improving ANT production. To increase E4P, the tktA geneencoding transketolase I was overexpressed by constructingpBBR1Gfbr-A. W3110 trpD9923 strain harboring pBBR1Gfbr-Aproduced 760.4 ± 12.5 mg/L ANT (SI Appendix, Fig. S6A), whichwas only slightly higher than that obtained with W3110 trpD9923strain harboring pBBR1Gfbr. To increase PEP, several differentstrains were constructed: ZWA1 (trc promoter exchange for ppsA

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overexpression), ZWA2 (pykF knockout), ZWA3 (pykF and pykAdouble-knockout), and ZWA4 (trc promoter exchange for ppsAoverexpression and pykF knockout). However, flask cultures ofthese four engineered strains each harboring pBBR1Gfbr-A showedno further increase in ANT titer (SI Appendix, Fig. S6A). Thespecific ANT production per OD600 of ZWA1, ZWA2, ZWA3,ZWA4, and the parental strain (W3110 trpD9923) harboringpBBR1Gfbr-A was calculated to be 113.4, 114.6, 117.0, 112.0, and122.7 mg/L OD600

−1, respectively. Thus, ZWA4 strain was chosenfor further engineering. Third, two additional overexpression targetsinvolved in L-TRP pathway (Fig. 1A), aroL (encoding shikimatekinase II) (14) and trpEfbr (encoding a feedback inhibition-resistantANT synthase) (15), were examined by constructing pTacLEfbr.ZWA4 strain harboring pBBR1Gfbr-A and pTacLEfbr produced820.5 ± 30.3 mg/L ANT (SI Appendix, Fig. S6A), which was 8.7%higher than that obtained with ZWA4 harboring pBBR1Gfbr-A.The above-engineered strains overproducing ANT were

combined with AAMT1 to produce MANT. To this end, threecombination strains were constructed: ZWA4 strain harboringpBBR1Gfbr-A and LEfbr-pTacT, ZWA4 harboring pBBR1Gfbr-Aand pTacT, and ZWA4 harboring pBBR1Gfbr and pTacT. Two-phase flask cultures of these engineered strains showed thatZWA4 strain harboring pBBR1Gfbr-A and pTacT produced thehighest MANT titer (392.0 ± 1.7 mg/L) (Fig. 2A and SI Appendix,Fig. S6B).

Enhancing SAM Availability for MANT Production in E. coli. Apartfrom MANT production in the strains described above, ANT wasalso formed as a byproduct. For example, ZWA4 strain harboringpBBR1Gfbr-A and pTacT, which so far produced the highestMANT titer (392.0 ± 1.7 mg/L), accumulated 416.4 ± 7.8 mg/LANT at the same time (SI Appendix, Fig. S6B). Such a high-levelaccumulation of ANT indicated a potential bottleneck present inthe conversion of ANT to MANT. One possible reason wasthought to be limited availability of the cosubstrate SAM requiredby the AAMT1 reaction. The following strategies were applied toincrease SAM availability by optimizing its biosynthesis (SI Ap-pendix, Fig. S7) and consequently to further increase MANTproduction while reducing ANT formation. First, the metAfbr geneencoding feedback inhibition-resistant homoserine succinyl-transferase and cysEfbr encoding L-serine O-acetyltransferase (16)were overexpressed for enhancing SAM biosynthesis (Fig. 1A). Asa result, ZWA4 strain harboring pBBR1GfbrAfbrEfbr and pTacTproduced MANT to a 6.8% higher concentration of 388.3 ±6.0 mg/L and ANT to a 16.9% lower concentration of 346.3 ±3.3 mg/L (SI Appendix, Fig. S8) than those obtained with the pa-rental strain ZWA4 harboring pBBR1Gfbr and pTacT. Next, themetK gene encoding SAM synthetase was also overexpressed byconstructing pTacTK. ZWA4 strain harboring pBBR1Gfbr-A andpTacTK produced MANT to an 8.4% higher concentration of424.8 ± 5.0 mg/L and ANT to a 16.4% lower concentration of348.3 ± 7.8 mg/L (SI Appendix, Fig. S8) than those obtained withZWA4 harboring pBBR1Gfbr-A and pTacT. These three over-expression targets were combined to construct ZWA4 harboringpBBR1GfbrAfbrEfbr and pTacTK, which produced ANT to thelowest concentration of 274.2 ± 8.4 mg/L. However, MANT titerwas intriguingly decreased to 404.3 ± 5.4 mg/L compared with thebest strain (ZWA4 harboring pBBR1Gfbr-A and pTacTK) so far(SI Appendix, Fig. S8). Thus, L-methionine (MET), the directprecursor to SAM synthesis, was further supplemented to seewhether MANT production can be increased; this is thought towork because the metAfbr and cysEfbr genes we employed encodefeedback inhibition-resistant enzymes in MET biosynthetic path-way. The three strains constructed above were cultured again intwo-phase flasks with addition of 20 mM MET. As a result, thehighest MANT titer of 489.0 ± 7.4 mg/L was obtained inZWA4 harboring pBBR1GfbrAfbrEfbr and pTacT (Fig. 2A and SIAppendix, Fig. S8), which was 25.9% higher than that without

MET supplementation. This MANT titer corresponded to 12.7-fold increase compared with that obtained with the initial W3110trpD9923 strain harboring pTrcT in single-phase flask culturewithout MET supplementation. This strain still accumulated346.7 ± 4.2 mg/L ANT. Also, ZWA4 harboring pBBR1GfbrAfbr

Efbr and pTacTK produced a similarly high level of MANT(486.8 ± 4.8 mg/L) to that achieved with ZWA4 harboringpBBR1GfbrAfbrEfbr and pTacT, but accumulated much less ANT(258.9 ± 7.6 mg/L) (SI Appendix, Fig. S8). Thus, these two strainswere selected and further evaluated in fed-batch cultures.

MANT Production in E. coli Two-Phase Fed-Batch Fermentations. Fed-batch cultures of the two engineered E. coli strains above wereperformed in two-phase mode for in situ extraction of MANT in aglucose minimal medium. The pH-stat feeding strategy wasemployed for nutrient feeding. Ammonium sulfate was added toprovide additional nitrogen source. As it was found beneficial,MET (20 mM) was supplemented to enhance SAM availability.Under these conditions, ZWA4 strain harboring pBBR1GfbrAfbr

Efbr and pTacTK produced 4.12 g/L MANT and 3.74 g/L ANT (SIAppendix, Fig. S9), while ZWA4 harboring pBBR1GfbrAfbrEfbr

and pTacT produced 4.47 g/L MANT (Figs. 2 and 3A) and 2.26 g/LANT (Fig. 3A). For the latter strain, the yield and productivity ofMANT were 0.045 g/g glucose and 0.062 g·L−1·h−1, respectively.

Selecting C. glutamicum Chassis for Food-Grade MANT Production.Having accomplished the proof-of-concept fermentative pro-duction of MANT from glucose by metabolically engineered E.coli, we pursued to produce food-grade MANT by using otherindustrial GRAS microbial strains as MANT is mainly applied infood and cosmetic industries. Two such bacterial hosts, Pseudo-monas putida KT2440 (Gram-negative) (17) and C. glutamicumATCC 13032 (Gram-positive) (18), were examined for their po-tential to produce MANT by comparing their tolerance levels toMANT toxicity. The toxicity test showed that both strains had thegrowth profiles exhibiting concentration-dependent inhibition byMANT (SI Appendix, Fig. S10), similar to E. coli. However, P.putida KT2440 cells could not grow in the presence of 1.0 g/LMANT (SI Appendix, Fig. S10A). On the other hand, completegrowth inhibition of C. glutamicum was observed when 2.0 g/LMANT was applied (SI Appendix, Fig. S10B). These results sug-gest that C. glutamicum has a higher tolerance to MANT com-pared with P. putida KT2440 and also E. coli. Thus, C. glutamicumwas selected as the host for food-grade MANT production (Fig.1B). Similar metabolic engineering strategies were applied tooptimize MANT production in C. glutamicum (Fig. 2B).

Tuning AAMT1 Levels for MANT Production in C. glutamicum. Toconstruct MANT synthesis pathway, we expressed the aamt1opt

gene in the wild-type C. glutamicum ATCC 13032 strain and op-timized its expression by constructing the following AAMT1 ex-pression plasmids (SI Appendix, Table S5): pEKT (aamt1opt clonedon pEKEx1 vector under tac promoter), pL10T, pI16T and pH36T(aamt1opt cloned on pCES208 vector under L10, I16 andH36 promoters), pL10HT, pI16HT and pH36HT (aamt1opt clonedon pCES208 vector under L10, I16, and H36 promoters with a N-terminal 6xHis-tag) (SI Appendix, Fig. S11A). These promoters (tac,L10, I16, and H36) have been reported to have different strengths ofgene expression (19), and the use of a His-tag was previously dem-onstrated to enhance gene expression in C. glutamicum (20). C.glutamicum ATCC 13032 strains harboring these plasmids weretested in single-phase shake-flask cultures supplemented with 0.8 g/LANT. As a result, C. glutamicum ATCC 13032 strain harboringpH36HT producedMANT to the highest titer of 97.2± 8.6 mg/L (SIAppendix, Fig. S11B). No MANT was detected in the wild-type C.glutamicum ATCC 13032 strain (SI Appendix, Fig. S11B).Plasmid pH36HT was thus introduced to C. glutamicum

YTM1 strain (SI Appendix, Table S5), in which the trpD gene was

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disrupted to block the L-TRP biosynthesis pathway in C. gluta-micum after ANT (Fig. 1B). In flask culture without external ANTfeeding and using glucose as a sole carbon source, YTM1 strainharboring pH36HT produced 45.3 ± 1.6 mg/L MANT (Fig. 2Band SI Appendix, Fig. S12) as well as 408.2 ± 4.2 mg/L ANT (SIAppendix, Fig. S12). Furthermore, we tested a C. glutamicumcodon-optimized version of aamt1 gene, designated as aamt1opt-Cgl

(SI Appendix, Table S6), by cloning it on pCES208 underH36 promoter with a N-terminal 6×His-tag in the same configu-ration as for pH36HT, generating pH36HTc. Flask culture ofYTM1 strain harboring pH36HTc produced MANT to a 1.6-foldhigher concentration of 117.1 ± 2.0 mg/L (Fig. 2B and SI Ap-pendix, Fig. S12) and ANT to a 55.0% lower concentration of183.8 ± 3.1 mg/L (SI Appendix, Fig. S12), compared with thoseobtained by YTM1 strain harboring pH36HT. We further deletedthe qsuB and qsuD genes in YTM1 strain, which are involved incompetitive pathways toward protocatechuate and quinate syn-thesis, respectively, in C. glutamicum (Fig. 1B), generatingYTM2 strain. As a result, YTM2 harboring pH36HTc producedMANT to an increased titer of 130.4 ± 0.8 mg/L (SI Appendix, Fig.S12) compared with the parental strain (YTM1 harboringpH36HTc).

Increasing ANT Supply for MANT Production in C. glutamicum. Next,further metabolic engineering approaches were taken to increasethe level of ANT and consequently to improve MANT productionin C. glutamicum. Although reported in E. coli (12, 13) and P.putida (21), ANT overproduction has never been explored in C.glutamicum. To increase ANT production in C. glutamicum, sev-eral target genes were manipulated: e.g., aroG, aroB, and aroKknown as potential limiting steps in the shikimate pathway (Fig.2B), and pgi, zwf, tkt, opcA, pgl, and tal known to enhance fluxthrough the pentose phosphate pathway (SI Appendix, Fig. S13),which were target genes similarly manipulated for the over-production of shikimic acid, 4-hydroxybenzoic acid, L-ornithine,and L-arginine in C. glutamicum (22–25). To this end, the fol-lowing strains were constructed: YTM3 (with the native promotersof aroK and aroB replaced with the strong constitutive sod pro-moter in YTM2 strain), YTM4 (with the start codon of pgichanged from ATG to GTG for down-regulation and of zwfchanged from GTG to ATG for up-regulation in YTM3 strain),YTM5 (with the native promoter of tkt replaced with sod pro-moter in YTM4 strain), as well as plasmid pEKG (overexpressingaroGS180F encoding a feedback-resistant DAHP synthase). In flask

cultures, YTM3 strain produced 15.7% less ANT than did theparental YTM2 strain; YTM3 strain harboring pEKG produced4.8-fold more ANT than did YTM3 strain without pEKG;YTM4 and YTM5 strains harboring pEKG produced similarlevels of ANT to that obtained with the parental YTM3 strainharboring pEKG (SI Appendix, Fig. S14A). These results collec-tively suggested that only aroGS180F overexpression by pEKGcould increase ANT production. Thus, aroGS180F was overex-pressed in the best MANT producer strain YTM2 harboringpH36HTc. Since pH36HTc and pEKG harbor the same antibiotic(kanamycin) marker, we changed the antibiotic marker ofpH36HTc from kanamycin to spectinomycin/streptomycin, gen-erating pSH36HTc. Flask culture of YTM2 strain harboringpSH36HTc produced 131.1 ± 9.0 mg/L MANT (SI Appendix, Fig.S14B), which was almost the same as that obtained withYTM2 harboring pH36HTc (130.4 ± 0.8 mg/L). Plasmid pEKGwas then successfully introduced to YTM2 strain harboringpSH36HTc, which produced MANT to a 91.6% higher concen-tration of 251.2 ± 2.5 mg/L (Fig. 2B and SI Appendix, Fig. S14B)and ANT to a 13.8-fold higher concentration of 3.38 ± 0.07 g/L (SIAppendix, Fig. S14B), compared with those obtained withYTM2 strain harboring pSH36HTc alone. Up to this point, allflask cultures of C. glutamicum strains were conducted in single-phase cultivation mode. Thus, two-phase extractive flask culture ofYTM2 strain harboring pSH36HTc and pEKG was performed,which produced MANT to a 44.9% higher concentration of364.1 ± 5.4 mg/L (Fig. 2B and SI Appendix, Fig. S14B) and ANT toa 21.0% lower concentration of 2.67 ± 0.03 g/L (SI Appendix, Fig.S14B), compared with those obtained in single-phase culture.

Enhancing SAM Salvage for MANT Production in C. glutamicum. Tofurther improve MANT production in C. glutamicum, thecosubstrate SAM availability was also considered. SAM over-production in C. glutamicum has been reported in a previous study(26), which focused on enhancing the flux to SAM biosynthesis(Fig. 1B and SI Appendix, Fig. S15). Several effective strategies inthat report were selected and applied for SAM production in thisstudy. First, the metK gene encoding methionine adenosyl-transferase was overexpressed by constructing pEKGK. As a result,YTM2 strain harboring pSH36HTc and pEKGK in two-phase flaskculture produced 377.0 ± 16.2 mg/L MANT (SI Appendix, Fig.S16), which was only slightly higher than that obtained withYTM2 strain harboring pSH36HTc and pEKG. Next, the mcbRand Ncgl2640 genes encoding transcriptional regulators involved in

Fig. 3. In situ two-phase extractive fed-batch culture profiles of (A) the engineered E. coli ZWA4 strain harboring pBBR1GfbrAfbrEfbr and pTacT and (B) theengineered C. glutamicum YTM8 strain harboring pSH36HTc and pEKGH under the condition of elevated DO level (50% air saturation). Symbols: blue circle,cell growth (OD600); red rectangle, residual glucose concentration (in grams per liter); orange diamond, ANT concentration (in grams per liter); magentatriangle, MANT concentration (in grams per liter).

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regulation of MET biosynthesis were deleted in YTM2 strain,generating YTM6. Furthermore, the metB gene encoding cys-tathionine-γ-synthase involved in competitive synthesis of L-isoleucine was deleted in YTM6 strain, generating YTM7. However,two-phase flask cultures of YTM6 and YTM7 strains harboringpSH36HTc and pEKG showed severely retarded cell growth, andconsequently produced 80.0 ± 4.2 and 72.3 ± 5.5 mg/L MANT,respectively (SI Appendix, Fig. S16), which were far lower than thatobtained with YTM2 strain harboring pSH36HTc and pEKG. Inaddition to genetic engineering for SAM synthesis, we directlysupplemented the SAM precursor MET (10 mM) in two-phaseflask culture of YTM2 strain harboring pSH36HTc and pEKG asit had increased MANT titer in E. coli. Unfortunately, the directsupplementation of MET resulted in the production of 326.8 ±3.9 mg/L MANT (SI Appendix, Fig. S16), lower than that withoutMET supplementation. From these results, it was speculated thatSAM availability might not be a limiting factor for MANT pro-duction in C. glutamicum.Thus, a different strategy of engineering the SAM salvage

pathway (Fig. 1B) was applied for recycling the SAM reactionproduct S-ribosyl-L-homocysteine (SAH) back to SAM synthesispathway catalyzed by SAH hydrolase (encoded by sahH) (27). ThesahH gene was overexpressed from pEKGH. To our pleasantsurprise, two-phase flask culture of YTM2 strain harboringpSH36HTc and pEKGH produced MANT to a 63.9% higherconcentration of 596.9 ± 16.7 mg/L (Fig. 2B and SI Appendix, Fig.S16) and ANT to a 5.2% lower concentration of 2.53 ± 0.01 g/L(SI Appendix, Fig. S16), compared with those obtained withYTM2 strain harboring pSH36HTc and pEKG. This MANT titeris 12.3-fold higher than that obtained with the initial YTM1 strainharboring pH36HT in single-phase flask culture.

MANT Production in C. glutamicum Two-Phase Fed-Batch Fermentations.Since C. glutamicum showed higher tolerance to MANT toxicity,we first performed a single-phase fed-batch culture using thebest C. glutamicum MANT producer strain YTM2 harboringpSH36HTc and pEKGH in glucose minimal medium withoutMET supplementation, which led to production of 1.70 g/L MANTand 14.11 g/L ANT (SI Appendix, Fig. S17). Next, in situ two-phaseextractive fed-batch culture of YTM2 strain harboring pSH36HTcand pEKGH under the same settings was performed, which pro-duced MANT to a 1.36-fold higher concentration of 4.01 g/L andANT to an 86.1% lower concentration of 1.96 g/L (SI Appendix,Fig. S18), compared with those obtained in single-phase fermen-tation. In this two-phase fermentation, succinic acid was also ac-cumulated to 10.06 g/L. Compared with single-phase fermentation,the two-phase fermentation resulted in an emulsion-like environ-ment, which might interfere with oxygen transferred to the cells. Itwas hypothesized that the high succinic acid accumulation was dueto oxygen limitation. Thus, the dissolved oxygen (DO) concentra-tion was increased by setting the control DO value from 30 to 50%of air saturation and also by changing agitation speed from thefixed at 600 rpm to automatic increase from 600 to 1,000 rpmaccording to DO. By increasing the DO level, the two-phase fed-batch culture of YTM2 strain harboring pSH36HTc and pEKGHproduced MANT to a 30.9% higher concentration of 5.25 g/L atthe 110-h mark and ANT to a 2.0-fold higher concentration of5.90 g/L (SI Appendix, Fig. S19), with succinic acid reduced to5.09 g/L. Glycerol formation was also observed in the fermentationbroth, and thus the hdpA gene involved in glycerol formationpathway was also deleted (SI Appendix, Fig. S20) (22, 23) inYTM2 strain, generating YTM8. As a result, two-phase fed-batchculture of YTM8 strain harboring pSH36HTc and pEKGH pro-duced MANT to a 9.3% higher concentration of 5.74 g/L at 110 h(Figs. 2B and 3B) and ANT to a 33.7% higher concentration of7.89 g/L (Fig. 3B). The resultant yield and productivity of MANTwere 0.020 g/g glucose and 0.052 g·L−1·h−1, respectively.

ANT Production. Although it is not the major subject of this study,ANT is also an important industrial platform chemical with manyapplications (21), and its biobased production is an active re-search subject (12, 13, 21). During the two-phase extractivefermentation at neutral pH performed in this study, it is no-ticeable that all of the MANT was extracted into the tributyrinphase, while almost all ANT (e.g., 96.9% in the case of fed-batchculture of E. coli ZWA4 harboring pBBR1GfbrAfbrEfbr andpTacT, 98.4% in the case of fed-batch culture of C. glutamicumYTM8 harboring pSH36HTc and pEKGH) was retained inaqueous phase. This is particularly beneficial for downstreamprocesses to purify MANT and also ANT from the fermentationbroth. From a process engineering perspective, this could beregarded as a coproduction of both MANT and ANT. To explorethe capacity for sole ANT production, we further performed asingle-phase fed-batch culture using one of the engineered ANToverproducers developed in this study, i.e., C. glutamicumYTM5 strain harboring pEKG. The single-phase fermentation ofthis strain led to the production of 26.40 g/L ANT at 84 h inglucose minimal medium (SI Appendix, Fig. S21), representingnot only the production of this compound by C. glutamicum, butalso the highest ANT production titer reported in any microbialhost to date. On the other hand, the residual amount of thisprecursor metabolite indicates that there is still some room forimprovement of MANT production. One promising strategywould be to engineer the ANT methyltransferase by enzymeevolution for greater catalytic activity, thereby more efficientlyconverting the residual ANT to MANT.In summary, we report the development of metabolically

engineered E. coli and C. glutamicum strains capable of pro-ducing MANT by direct fermentation in minimal media con-taining glucose as a sole carbon source. A synthetic metabolicpathway for MANT biosynthesis was constructed in both E. coliand C. glutamicum, and MANT production was optimized bytuning the key enzyme AAMT1 expression, increasing flux to-ward precursor ANT, and increasing availability (in E. coli) andsalvage (C. glutamicum) of cosubstrate SAM required by theAAMT1 reaction. In situ two-phase extractive fed-batch fer-mentation process was also developed for MANT production bythe engineered E. coli and C. glutamicum, which led to pro-duction of 4.47 and 5.74 g/L MANT from glucose, respectively.These titers are significantly high for natural compounds pro-duced by microbial fermentation, as most natural compoundsfrom engineered microbes are produced at levels of milligramsor micrograms per liter (28). Also, all fermentations described inthis study were performed in minimal media, which further helpdecrease operation and separation costs (29). This methanol-freebiobased production of MANT paves the way toward a sustain-able, consumer-friendly process to produce MANT as a “natu-ral” flavoring agent, which for the past 100 y has only beenproduced industrially through chemical synthesis. The metabolicengineering strategies and methodologies described and theengineered microbial systems established here will contribute tothe development of engineered strains for the sustainable pro-duction of other chemicals with similar properties and charac-teristics as MANT.

Materials and MethodsAll of the materials and methods conducted in this study are detailed in SIAppendix, Supplementary Information Materials and Methods, includingbacterial strains and media, construction of E. coli expression plasmids, E. coligenome manipulation, construction of C. glutamicum expression plasmids,construction of CRISPR-based recombineering plasmids for gene knockout inC. glutamicum, construction of genetic engineering plasmids for promoterexchange and for further gene knockout in C. glutamicum YTM2, C. gluta-micum genome manipulation, MANT toxicity test, SDS/PAGE analysis, de-termination of partition coefficients, cultivation condition, and analyticalprocedures. The data supporting the findings of this study are available inSI Appendix.

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ACKNOWLEDGMENTS. We thank Prof. Ki Jun Jeong for generously pro-viding us with plasmids pCES-L10-M18, pCES-I16-M18, and pCES-H36-M18 forcloning in this study. We also thank Tae Hee Han for providing plasmidpSY06b. This work was supported by the Technology Development Program

to Solve Climate Changes on Systems Metabolic Engineering for Biorefi-neries from the Ministry of Science and ICT through the National ResearchFoundation (NRF) of Korea (Grants NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557).

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