Abstract
Biological synthesis of therapeutic drugs beneficial for human health using microbes offers an alternative production strategy to the methods that are commonly employed such as direct extraction from source organisms or chemical synthesis. In this study, we evaluated the potential for yeast (Saccharomyces cerevisiae) to be used as a catalyst for the synthesis of tranilast and various tranilast analogs (cinnamoyl anthranilates). Several studies have demonstrated that these phenolic amides have antioxidant properties and potential therapeutic benefits including antiinflammatory, antiproliferative, and antigenotoxic effects. The few cinnamoyl anthranilates naturally produced in plants such as oats and carnations result from the coupling of various hydroxycinnamoyl-CoAs to anthranilic acid. In order to achieve the microbial production of tranilast and several of its analogs, we engineered a yeast strain to co-express a 4-coumarate/CoA ligase (4CL, EC 6.2.1.12) from Arabidopsis thaliana and a hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC 2.3.1.144) from Dianthus caryophyllus. This modified yeast strain allowed us to produce tranilast and 26 different cinnamoyl anthranilate molecules within a few hours after exogenous supply of various combinations of cinnamic acids and anthranilate derivatives. Our data demonstrate the feasibility of rapidly producing a wide range of defined cinnamoyl anthranilates in yeast and underline a potential for the biological designed synthesis of naturally and non-naturally occurring molecules.
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Notes
Vector name derived from the Gateway manual, Invitrogen.
References
Azuma H, Banno K, Yoshimura T (1976) Pharmacological properties of N-(3′,4′-dimethoxycinnamoyl) anthranilic acid (N-5′), a new anti-atopic agent. Br J Pharmacol 58:483–488
Balderas-Hernández VE, Sabido-Ramos A, Silva P, Cabrera-Valladares N, Hernández-Chávez G, Báez-Viveros JL, Martínez A, Bolívar F, Gosset G (2009) Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli. Microb Cell Fact 2:8–19
Bhuiya MW, Liu CJ (2010) Engineering monolignol 4-O-methyltransferases to modulate lignin biosynthesis. J Biol Chem 285:277–285
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Branduardi P, Fossati T, Sauer M, Pagani R, Mattanovich D, Porro D (2007) Biosynthesis of vitamin C by yeast leads to increased stress resistance. PLoS ONE 2:e1092
Bratt K, Sunnerheim K, Bryngelsson S, Fagerlund A, Engman L, Andersson RE, Dimberg LH (2003) Avenanthramides in oats (Avena sativa L.) and structure-antioxidant activity relationships. J Agric Food Chem 51:594–600
Chakrabarti R, Subramaniam V, Abdalla S, Jothy S, Prud’homme GJ (2009) Tranilast inhibits the growth and metastasis of mammary carcinoma. Anticancer Drugs 20:334–345
Chang MC, Keasling JD (2006) Production of isoprenoid pharmaceuticals by engineered microbes. Nat Chem Biol 2:674–681
Collins FW (1989) Oat phenolics: avenanthramides, novel substituted N-cinnamoylanthranilate alkaloids from oat groats and hulls. J Agric Food Chem 37:60–66
Collins FW, Mullin WJ (1988) High performance liquid chromatographic determination of avenanthramides, N-aroylanthranilic acid alkaloids from oats. J Chromatogr 445:363–370
Cui H, Gensini M, Kataria R, Twaddle T, Zhang J, Wadsworth S, Petrilli J, Rodgers K, diZerega G, Cooper K (2009) Reducing post-surgical adhesions utilizing a drug-enhanced device: sodium carboxymethylcellulose aqueous gel/poly(p-dioxanone) and tranilast. Biomed Mater 4:015001
Engels B, Dahm P, Jennewein S (2008) Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards Taxol (paclitaxel) production. Metab Eng 10:201–206
Fagerlund A, Sunnerheim K, Dimberg LH (2009) Radical-scavenging and antioxidant activity of avenanthramides. Food Chem 113:550–556
Fosdick LS, Starke AC Jr (1940) Some alkamine esters of 4-acetylferulic and 3, 4-dimethoxycinnamic acids. J Am Chem Soc 62:3352–3355
Funk C, Brodelius PE (1990) Phenylpropanoid metabolism in suspension cultures of Vanilla planifolia Andr.: III. Conversion of 4-methoxycinnamic acids into 4-hydroxybenzoic acids. Plant Physiol 94:102–108
Gietz RD, Woods RA (2002) Tranformation of yeast by the LiAc/SS carrier DNA/PEG method. Methods Enzymol 350:87–96
Guo W, Wise ML, Collins FW, Meydani M (2008) Avenanthramides, polyphenols from oats, inhibit IL-1β-induced NF-kB activation in endothelial cells. Free Radical Biol Med 44:415–429
Guo T, Chen WQ, Zhang C, Zhao YX, Zhang Y (2009) Chymase activity is closely related with plaque vulnerability in a hamster model of atherosclerosis. Atherosclerosis 207:59–67
Hamberger B, Hahlbrock K (2004) The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc Natl Acad Sci USA 101:2209–2214
Heuschkel S, Wohlrab J, Schmaus G, Neubert RH (2008) Modulation of dihydroavenanthramide D release and skin penetration by 1,2-alkanediols. Eur J Pharm Biopharm 70:239–247
Heuschkel S, Wohlrab J, Neubert RH (2009) Dermal and transdermal targeting of dihydroavenanthramide D using enhancer molecules and novel microemulsions. Eur J Pharm Biopharm 72:552–560
Horwitz SB (1994) How to make taxol from scratch. Nature 367:593–594
Isaji M, Miyata H, Ajisawa Y (1998) Tranilast: a new application in the cardiovascular field as an antiproliferative drug. Cardiovasc Drug Rev 16:288–299
Ji LL, Lay D, Chung E, Fu Y, Peterson DM (2003) Effects of avenanthramides on oxidant generation and antioxidant enzyme activity in exercised rats. Nutr Res 23:1579–1590
Kliebenstein DJ, D’Auria JC, Behere AS, Kim JH, Gunderson KL, Breen JN, Lee G, Gershenzon J, Last RL, Jander G (2007) Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana. Plant J 51:1062–1076
Knobloch KH, Hahlbrock K (1975) Isoenzymes of p-coumarate: CoA ligase from cell suspension cultures of Glycine max. Eur J Biochem 52:311–320
Komatsu H, Kojima M, Tsutsumi N, Hamano S, Kusama H, Ujiie A, Ikeda S, Nakazawa M (1988) Study of the mechanism of inhibitory action of tranilast on chemical mediator release. Jpn J Pharmacol 46:43–51
Konneh M (1998) Tranilast, kissei pharmaceuticals. IDrugs 1:141–146
Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82:488–492
Lalonde S, Sero A, Pratelli R, Pilot G, Chen J, Sardi MI, Parsa SA, Kim D-Y, Acharya BR, Stein EV, Hu H-C, Villiers F, Takeda K, Yang Y, Han YS, Schwacke R, Chiang W, Kato N, Loqué D, Assmann SM, Kwak JM, Schroeder JI, Rhee SY, Frommer WB (2010) A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2. Front Physio 1:24. doi:10.3389/fphys.2010.00024
Lee-Manion AM, Price RK, Strain JJ, Dimberg LH, Sunnerheim K, Welch RW (2009) In vitro antioxidant activity and antigenotoxic affects of avenanthramides and related compounds. J Agric Food Chem 57:10619–10624
Limem I, Guedon E, Hehn A, Bourgaud F, Ghedira L, Engasser J-M, Ghoul M (2008) Production of phenylpropanoid compounds by recombinant microorganisms expressing plant-specific biosynthesis genes. Process Biochem 43:463–479
Lindermayr C, Fliegmann J, Ebel J (2003) Deletion of a single amino acid residue from different 4-coumarate:CoA ligases from soybean results in the generation of new substrate specificities. J Biol Chem 278:2781–2786
Liu L, Zubik L, Collins FW, Marko M, Meydani M (2004) The antiatherogenic potential of oat phenolic compounds. Artherosclerosis 175:39–49
Loqué D, Lalonde S, Looger LL, von Wirén N, Frommer WB (2007) A cytosolic trans-activation domain essential for ammonium uptake. Nature 446:195–198
Lv N, Song MY, Lee YR, Choi HN, Kwon KB, Park JW, Park BH (2009) Dihydroavenanthramide D protects pancreatic beta-cells from cytokine and streptozotocin toxicity. Biochem Biophys Res Commun 387:97–102
Moglia A, Comino C, Lanteri S, de Vos R, de Waard P, van Beek TA, Goitre L, Retta SF, Beekwilder J (2010) Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast. Metab Eng 12:223–232
Mukai N, Masaki K, Fujii T, Kawamukai M, Lefuji H (2010) PAD1 and FDC1 are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. J Biosci Bioeng 109:564–569
Nair RB, Xia Q, Kartha CJ, Kurylo E, Hirji RN, Datla R, Selvaraj G (2002) Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation. Developmental regulation of the gene, and expression in yeast. Plant Physiol 130:210–220
Nelson JT, Lee J, Sims JW, Schmidt EW (2007) Characterization of SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis. Appl Environ Microbiol 73:3575–3580
Nie L, Wise ML, Peterson DM, Meydani M (2006) Avenanthramide, a polyphenol from oats, inhibits vascular smooth muscle cell proliferation and enhances nitric oxide production. Atherosclerosis 186:260–266
Ogawa Y, Dogru M, Uchino M, Tatematsu Y, Kamoi M, Yamamoto Y, Ogawa J, Ishida R, Kaido M, Hara S, Matsumoto Y, Kawakita T, Okamoto S, Tsubota K (2010) Topical tranilast for treatment of the early stage of mild dry eye associated with chronic GVHD. Bone Marrow Transplant 45:565–569
Okazaki Y, Isobe T, Iwata Y, Matsukawa T, Matsuda F, Miyagawa H, Ishihara A, Nishioka T, Iwamura H (2004) Metabolism of avenanthramide phytoalexins in oat. Plant J 39:560–572
Okuda M, Ishikawa T, Saito Y, Shimizu T, Baba S (1984) A clinical evaluation of N-5′ with perennial-type allergic rhinitis—a test by the multi-clinic, intergroup, double-blind comparative method. Ann Allergy 53:178–185
Oshitani N, Yamagami H, Watanabe K, Higuchi K, Arakawa T (2007) Long-term prospective pilot study with tranilast for the prevention of stricture progression in patients with Crohn’s disease. Gut 56:599–600
Pae HO, Jeong SO, Koo BS, Ha HY, Lee KM, Chung HT (2002) Tranilast, an orally active anti-allergic drug, up-regulates the anti-inflammatory heme oxygenase-1 expression but down-regulates the pro-inflammatory cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW264.7 macrophages. Biochem Biophys Res Commun 371:361–365
Park M, Kang K, Park S, Kim YS, Ha S-H, Lee SW, Ahn M-J, Bae J-M, Back K (2008) Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes. Appl Microbiol Biotechnol 81:43–49
Platten M, Ho PP, Youssef S, Fontoura P, Garren H, Hur EM, Gupta R, Lee LY, Kidd BA, Robinson WH, Sobel RA, Selley ML, Steinman L (2005) Treatment of autoimmune neuroinflammation with a synthetic tryptophan metabolite. Science 310:850–855
Ponchet M, Favre-Bonvin J, Hauteville M, Ricci P (1988) Dianthramides (N-benzoyl and N-paracoumarylanthranilic acid derivatives) from elicited tissues of Dianthus caryophyllus. Phytochemistry 27:725–730
Prud’homme GJ (2007) Pathobiology of transforming growth factor beta in cancer, fibrosis and immunologic disease, and therapeutic considerations. Lab Invest 87:1077–1091
Reinhard K, Matern U (1989) The biosynthesis of phytoalexins in Dianthus caryophyllus L. cell cultures: induction of benzoyl-CoA:anthranilate N-benzoyltransferase activity. Arch Biochem Biophys 275:295–301
Ro DK, Paradise EM, Ouellet M, Fisher KJ, Newman KL, Ndungu JM, Ho KA, Eachus RA, Ham TS, Kirby J, Chang MC, Withers ST, Shiba Y, Sarpong R, Keasling JD (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440:940–943
Schmaus G, Joppe H, Herrmann M, Sabater-Luntzel C, Vossing T (2006) Anthranilic acid amides and derivatives thereof as cosmetic and pharmaceutical agents. U.S. Patent 20060089413
Shiota N, Kovanen PT, Eklund KK, Shibata N, Shimoura K, Niibayashi T, Shimbori C, Okunishi H (2010) The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice. Br J Pharmacol 159:626–635
Sun X, Suzuki K, Nagata M, Kawauchi Y, Yano M, Ohkoshi S, Matsuda Y, Kawachi H, Watanabe K, Asakura H, Aoyagi Y (2010) Rectal administration of tranilast ameliorated acute colitis in mice through increased expression of heme oxygenase-1. Pathol Int 60:93–101
Sur R, Nigam A, Grote D, Liebel F, Southall MD (2008) Avenanthramides, polyphenols from oats, exhibit anti-inflammatory and anti-itch activity. Arch Dermatol Res 25:1–6
Szczebara FM, Chandelier C, Villeret C, Masurel A, Bourot S, Duport C, Blanchard S, Groisillier A, Testet E, Costaglioli P, Cauet G, Degryse E, Balbuena D, Winter J, Achstetter T, Spagnoli R, Pompon D, Dumas B (2003) Total biosynthesis of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol 21:143–149
Tamai H, Katoh K, Yamaguchi T, Hayakawa H, Kanmatsuse K, Haze K, Aizawa T, Nakanishi S, Suzuki S, Suzuki T, Takase S, Nishikawa H, Katoh O (2002) The impact of tranilast on restenosis after coronary angioplasty: the Second Tranilast Restenosis Following Angioplasty Trial (TREAT-2). Am Heart J 143:506–513
Tan SM, Zhang Y, Cox AJ, Kelly DJ, Qi W (2010) Tranilast attenuates the up-regulation of thioredoxin-interacting protein and oxidative stress in an experimental model of diabetic nephropathy. Nephrol Dial Transplant. doi:10.1093/ndt/gfq355
Trantas E, Panopoulos N, Ververidis F (2009) Metabolic engineering of the complete pathway leading to heterologous biosynthesis of various flavonoids and stilbenoids in Saccharomyces cerevisiae. Metab Eng 11:355–366
Vannelli T, Wei Qi W, Sweigard J, Gatenby AA, Sariaslani FS (2007) Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi. Metab Eng 9:142–151
Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP, Boles E (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett 464:123–128
Winzeler EA, Shoemaker DD, Astromoff A et al (1999) Functionnal characterization of the S. cerevisiae genome by deletion and parallel analysis. Science 285:901–906
Yang Q, Reinhard K, Schiltz E, Matern U (1997) Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L. Plant Mol Biol 35:777–789
Yang Q, Trinh HX, Imai S, Ishihara A, Zhang L, Nakayashiki H, Tosa Y, Mayama S (2004) Analysis of the involvement of hydroxyanthranilate hydroxycinnamoyltransferase and caffeoyl-CoA 3-O-methyltransferase in phytoalexin biosynthesis in oat. Mol Plant-Microb Interact 17:81–89
Zammit SC, Cox AJ, Gow RM, Zhang Y, Gilbert RE, Krum H, Kelly DJ, Williams SJ (2009) Evaluation and optimization of antifibrotic activity of cinnamoyl anthranilates. Bioorg Med Chem Lett 19:7003–7006
Acknowledgments
This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org/) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.
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Eudes, A., Baidoo, E.E.K., Yang, F. et al. Production of tranilast [N-(3′,4′-dimethoxycinnamoyl)-anthranilic acid] and its analogs in yeast Saccharomyces cerevisiae . Appl Microbiol Biotechnol 89, 989–1000 (2011). https://doi.org/10.1007/s00253-010-2939-y
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DOI: https://doi.org/10.1007/s00253-010-2939-y