Abstract
We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan5 strain to prevent hyperglycosylation. The two (His)6-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L−1 of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man5GlcNAc2 with, however, a significant contribution of Man4–10GlcNAc2. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O2) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.
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Aldemir H, Richarz R, Gulder TAM (2014) The biocatalytic repertoire of natural biaryl formation. Angew Chem Int Ed Engl 53:8286–8293. doi:10.1002/anie.201401075
Ashenhurst JA (2010) Intermolecular oxidative cross-coupling of arenes. Chem Soc Rev 39:540–548. doi:10.1039/b907809f
Beaudoin GAW, Facchini PJ (2014) Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240:19–32. doi:10.1007/s00425-014-2056-8
Bischoff D, Pelzer S, Bister B, Nicholson GJ, Stockert S, Schirle M, Wohlleben W, Jung G, Sussmuth RD (2001) The biosynthesis of vancomycin-type glycopeptide antibiotics—the order of the cyclization steps. Angew Chem Int Ed Engl 40:4688–4691. doi:10.1002/1521-3773(20011217)40:24<4688::AID-ANIE4688>3.0.CO;2-M
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. doi:10.1016/0003-2697(76)90527-3
Bringmann G, Gulder T, Gulder TAM, Breuning M (2011) Atroposelective total synthesis of axially chiral biaryl natural products. Chem Rev 111:563–639. doi:10.1021/cr100155e
Brunel JM (2005) BINOL: A versatile chiral reagent. Chem Rev 105:857–897. doi:10.1021/cr040079g
Cass QB, Oliveira RV, De Pietro AC (2004) Determination of gossypol enantiomer ratio in cotton plants by chiral higher-performance liquid chromatography. J Agr Food Chem 52:5822–5827. doi:10.1021/jf049626p
Cass QB, Tiritan E, Matlin SA, Freire EC (1991) Gossypol enantiomer ratios in cotton seeds. Phytochemistry 30:2655–2657. doi:10.1016/0031-9422(91)85117-I
Cedzich A, Huttenlocher F, Kuhn BM, Pfannstiel J, Gabler L, Stintzi A, Schaller A (2009) The protease-associated (PA) domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3). J Biol Chem 284:14068–14078. doi:10.1074/jbc.M900370200
Dalisay DS, Kim KW, Lee C, Yang H, Rübel O, Bowen BP, Davin LB, Lewis NG (2015) Dirigent protein-mediated lignan and cyanogenic glucoside formation in flax seed: integrated omics and MALDI mass spectrometry imaging. J Nat Prod 78:1231–1242. doi:10.1021/acs.jnatprod.5b00023
Davin LB, Lewis NG (2005) Dirigent phenoxy radical coupling: advances and challenges. Curr Opin Biotech 16:398–406. doi:10.1016/j.copbio.2005.06.010
Davin LB, Wang HB, Crowell AL, Bedgar DL, Martin DM, Sarkanen S, Lewis NG (1997) Stereoselective bimolecular phenoxy radical coupling by an auxiliary (dirigent) protein without an active center. Science 275:362–366. doi:10.1126/science.275.5298.362
Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard J-F, Guindon S, Lefort V, Lescot M, Claverie J-M, Gascuel O (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucl Acids Res 36:W465–W469. doi:10.1093/nar/gkn180
Effenberger I, Zhang B, Li L, Wang Q, Liu Y, Klaiber I, Pfannstiel J, Wang Q, Schaller A (2015) Dirigent proteins from cotton (Gossypium sp.) for the atropselective synthesis of gossypol. Angew Chem Int Ed Engl 54:14660–14663. doi:10.1002/anie.201507543
Fang W, Ji S, Jiang N, Wang W, Zhao GY, Zhang S, Ge HM, Xu Q, Zhang AH, Zhang YL, Song YC, Zhang J, Tan RX (2012) Naphthol radical couplings determine structural features and enantiomeric excess of dalesconols in Daldinia eschscholzii. Nature Comm 3:1039. doi:10.1038/ncomms2031
Frankfater CR, Dowd MK, Triplett BA (2009) Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots. Plant Cell Tiss Organ Cult 98:341–349. doi:10.1007/s11240-009-9568-0
Funatsuki H, Suzuki M, Hirose A, Inaba H, Yamada T, Hajika M, Komatsu K, Katayama T, Sayama T, Ishimoto M, Fujino K (2014) Molecular basis of a shattering resistance boosting global dissemination of soybean. Proc Natl Acad Sci U S A 111:17797–17802. doi:10.1073/pnas.1417282111
Gang DR, Costa MA, Fujita M, Dinkova-Kostova AT, Wang H-B, Burlat V, Martin W, Sarkanen S, Davin LB, Lewis NG (1999) Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis. Chem Biol 6:143–151. doi:10.1016/S1074-5521(99)89006-1
Gao W, Long L, Zhu L-F, Xu L, Gao W-H, Sun L-Q, Liu L-L, Zhang X-L (2013) Proteomic and virus-induced gene silencing (VIGS) analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae. Mol Cell Proteomics 12:3690–3703. doi:10.1074/mcp.M113.031013
Gerardy R, Zenk MH (1992) Formation of salutaridine from (R)-reticuline by a membrane-bound cytochrome P-450 enzyme from Papaver somniferum. Phytochemistry 32:79–86. doi:10.1016/0031-9422(92)80111-Q
Gesell A, Rolf M, Ziegler J, Díaz Chávez ML, Huang F-C, Kutchan TM (2009) CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of morphine biosynthesis in opium poppy. J Biol Chem 284:24432–24442. doi:10.1074/jbc.M109.033373
Gil Girol C, Fisch KM, Heinekamp T, Günther S, Hüttel W, Piel J, Brakhage AA, Müller M (2012) Regio- and stereoselective oxidative phenol coupling in Aspergillus niger. Angew Chem Int Ed 51:9788–9791. doi:10.1002/anie.201203603
Guo W, Jin L, Miao Y, He X, Hu Q, Guo K, Zhu L, Zhang X (2016) An ethylene response-related factor, GbERF1-like, from Gossypium barbadense improves resistance to Verticillium dahliae via activating lignin synthesis. Plant Mol Biol 91:305–318. doi:10.1007/s11103-016-0467-6
Hagenbucher S, Olson DM, Ruberson JR, Wäckers FL, Romeis J (2013) Resistance mechanisms against arthropod herbivores in cotton and their interactions with natural enemies. Crit Rev Plant Sci 32:458–482. doi:10.1080/07352689.2013.809293
Halls SC, Davin LB, Kramer DM, Lewis NG (2004) Kinetic study of coniferyl alcohol radical binding to the (+)-pinoresinol forming dirigent protein. Biochemistry 43:2587–2595. doi:10.1021/bi035959o
Hosmani PS, Kamiya T, Danku J, Naseer S, Geldner N, Guerinot ML, Salt DE (2013) Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root. Proc Natl Acad Sci U S A 110:14498–14503. doi:10.1073/pnas.1308412110
Howard-Jones AR, Walsh CT (2006) Staurosporine and Rebeccamycin aglycones are assembled by the oxidative action of StaP, StaC, and RebC on chromopyrrolic acid. J Am Chem Soc 128:12289–12298. doi:10.1021/ja063898m
Hron R, Kim H, Calhoun M, Fisher G (1999) Determination of (+)-, (−)-, and total gossypol in cottonseed by high-performance liquid chromatography. J Am Oil Chem Soc 76:1351–1355. doi:10.1007/s11746-999-0149-5
Ikezawa N, Iwasa K, Sato F (2008) Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C–C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells. J Biol Chem 283:8810–8821. doi:10.1074/jbc.M705082200
Jacobs P, Inan M, Festjens N, Haustraete J, Van Hecke A, Contreras R, Meagher M, Callewaert N (2010) Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate. Microb Cell Factories 9:93. doi:10.1186/1475-2859-9-93
Jacobs PP, Geysens S, Vervecken W, Contreras R, Callewaert N (2009) Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc 4:58–70
Jaroszewski JW, Strøm-Hansen T, Hansen SH, Thastrup O, Kofod H (1992) On the botanical distribution of chiral forms of gossypol. Planta Med 58:454–458. doi:10.1055/s-2006-961512
Kazenwadel C, Klebensberger J, Richter S, Pfannstiel J, Gerken U, Pickel B, Schaller A, Hauer B (2013) Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function. Appl Microbiol Biotechnol 97:7215–7227. doi:10.1007/s00253-012-4579-x
Keshmiri-Neghab H, Goliaei B (2014) Therapeutic potential of gossypol: an overview. Pharmaceut Biol 52:124–128. doi:10.3109/13880209.2013.832776
Kim K-W, Moinuddin SGA, Atwell KM, Costa MA, Davin LB, Lewis NG (2012) Opposite stereoselectivities of dirigent proteins in Arabidopsis and Schizandra species. J Biol Chem 287:33957–33972. doi:10.1074/jbc.M112.387423
Kim KW, Smith CA, Daily MD, Cort JR, Davin LB, Lewis NG (2015) Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 A resolution with three isolated active sites. J Biol Chem 290:1308–1318. doi:10.1074/jbc.M114.611780
Kim MK, Jeon J-H, Fujita M, Davin LB, Lewis NG (2002) The western red cedar (Thuja plicata) 8-8' DIRIGENT family displays diverse expression patterns and conserved monolignol coupling specificity. Plant Mol Biol 49:199–214. doi:10.1023/A:1014940930703
Kozlowski MC, Morgan BJ, Linton EC (2009) Total synthesis of chiral biaryl natural products by asymmetric biaryl coupling. Chem Soc Rev 38:3193–3207. doi:10.1039/b821092f
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Lan L, Appelman C, Smith AR, Yu J, Larsen S, Marquez RT, Liu H, Wu X, Gao P, Roy A, Anbanandam A, Gowthaman R, Karanicolas J, De Guzman RN, Rogers S, Aubé J, Ji M, Cohen RS, Neufeld KL, Xu L (2015) Natural product (−)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1. Mol Oncol 9:1406–1420. doi:10.1016/j.molonc.2015.03.014
Li C, Lei X (2014) Strategies toward the biomimetic syntheses of oligomeric sesquiterpenoids. J Org Chem 79:3289–3295. doi:10.1021/jo5002092
Li Z, Rupasinghe SG, Schuler MA, Nair SK (2011) Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis. Proteins 79:1728–1738. doi:10.1002/prot.22996
Liu J, Stipanovic RD, Bell AA, Puckhaber LS, Magill CW (2008) Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein. Phytochemistry 69:3038–3042. doi:10.1016/j.phytochem.2008.06.007
Mazzaferro LS, Hüttel W, Fries A, Müller M (2015) Cytochrome P450-catalyzed regio- and stereoselective phenol coupling of fungal natural products. J Am Chem Soc 137:12289–12295. doi:10.1021/jacs.5b06776
Niemetz R, Gross GG (2003a) Ellagitannin biosynthesis: laccase-catalyzed dimerization of tellimagrandin II to cornusiin E in Tellima grandiflora. Phytochemistry 64:1197–1201. doi:10.1016/j.phytochem.2003.08.013
Niemetz R, Gross GG (2003b) Oxidation of pentagalloylglucose to the ellagitannin, tellimagrandin II, by a phenol oxidase from Tellima grandiflora leaves. Phytochemistry 62:301–306. doi:10.1016/S0031-9422(02)00557-5
Petersen M, Alfermann AW (2001) The production of cytotoxic lignans by plant cell cultures. Appl Microbiol Biotechnol 55:135–142. doi:10.1007/s002530000510
Pickel B, Constantin M-A, Pfannstiel J, Conrad J, Beifuss U, Schaller A (2010) An enantiocomplementary dirigent protein for the enantioselective laccase-catalyzed oxidative coupling of phenols. Angew Chem Int Ed Engl 49:202–204. doi:10.1002/anie.200904622
Pickel B, Pfannstiel J, Steudle A, Lehmann A, Gerken U, Pleiss J, Schaller A (2012) A model of dirigent proteins derived from structural and functional similarities with allene oxide cyclase and lipocalins. FEBS J 279:1980–1993. doi:10.1111/j.1742-4658.2012.08580.x
Pickel B, Schaller A (2013) Dirigent proteins: molecular characteristics and potential biotechnological applications. Appl Microbiol Biotechnol 97:8427–8438. doi:10.1007/s00253-013-5167-4
Präg A, Grüning BA, Häckh M, Lüdeke S, Wilde M, Luzhetskyy A, Richter M, Luzhetska M, Günther S, Müller M (2014) Regio- and stereoselective intermolecular oxidative phenol coupling in Streptomyces. J Am Chem Soc 136:6195–6198. doi:10.1021/ja501630w
Puckhaber LS, Dowd MK, Stipanovic RD, Howell CR (2002) Toxicity of (+)- and (−)-gossypol to the plant pathogen, Rhizoctonia solani. J Agr Food Chem 50:7017–7021. doi:10.1021/jf0207225
Raab D, Graf M, Notka F, Schödl T, Wagner R (2010) The GeneOptimizer algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst Synth Biol 4:215–225. doi:10.1007/s11693-010-9062-3
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75:663–670
Rayon C, Lerouge P, Faye L (1998) The protein N-glycosylation in plants. J Exp Bot 49:1463–1472. doi:10.1093/jxb/49.326.1463
Schlauer J, Rückert M, Wiesen B, Herderich M, Assi LA, Haller RD, Bär S, Fröhlich K-U, Bringmann G (1998) Characterization of enzymes from Ancistrocladus (Ancistrocladaceae) and Triphyophyllum (Dioncophyllaceae) catalyzing oxidative coupling of naphthylisoquinoline alkaloids to michellamines. Arch Biochem Biophys 350:87–94. doi:10.1006/abbi.1997.0494
Seneviratne HK, Dalisay DS, Kim KW, Moinuddin SG, Yang H, Hartshorn CM, Davin LB, Lewis NG (2015) Non-host disease resistance response in pea (Pisum sativum) pods: biochemical function of DRR206 and phytoalexin pathway localization. Phytochemistry 113:140–148. doi:10.1016/j.phytochem.2014.10.013
Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858. doi:10.1021/ac950914h
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539–10.1038/msb.2011.75
Smith ET, Perry ET, Sears MB, Johnson DA (2014) Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris. Protein Expr Purif 102:69–75. doi:10.1016/j.pep.2014.08.005
Stipanovic RD, López JD Jr, Dowd MK, Puckhaber LS, Duke SE (2006) Effect of racemic and (+)- and (−)-gossypol on the survival and development of Helicoverpa zea larvae. J Chem Ecol 32:959–968. doi:10.1007/s10886-006-9052-9
Stipanovic RD, López JD Jr, Dowd MK, Puckhaber LS, Duke SE (2008) Effect of racemic, (+)- and (−)-gossypol on survival and development of Heliothis virescens larvae. Environ Entomol 37:1081–1085. doi:10.1603/0046-225X(2008)37[1081:EORAGO]2.0.CO;2
Tretter V, Altmann F, MÄRz L (1991) Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1 → 3 to the asparagine-linked N-acetylglucosamine residue. Eur J Biochem 199:647–652. doi:10.1111/j.1432-1033.1991.tb16166.x
Vassao DG, Kim K-W, Davin LB, Lewis NG (2010) Lignans (neolignans) and allyl/propenyl phenols: biogenesis, structural biology, and biological/human health considerations. In: Townsend CA (ed) Comprehensive natural products II chemistry and biology, vol 1. Elsevier, Amsterdam, pp. 815–928
Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K, Contreras R (2004) In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl Environ Microb 70:2639–2646. doi:10.1128/AEM.70.5.2639-2646.2004
Wagner TA, Liu J, Stipanovic RD, Puckhaber LS, Bell AA (2012) Hemigossypol, a constituent in developing glanded cottonseed (Gossypium hirsutum). J Agr Food Chem 60:2594–2598. doi:10.1021/jf2051366
Wang X, Howell CP, Chen F, Yin J, Jiang Y (2009) Gossypol—a polyphenolic compound from cotton plant. In: Taylor SL (ed) Advances in food and nutrition research, vol 58. Academic Press, New York, pp. 215–263
Wezeman T, Brase S, Masters K-S (2015) Xanthone dimers: a compound family which is both common and privileged. Nat Prod Rep. doi:10.1039/c4np00050a
Wilson IB, Zeleny R, Kolarich D, Staudacher E, Stroop CJ, Kamerling JP, Altmann F (2001) Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha1,3-linked fucose and xylose substitutions. Glycobiology 11:261–274. doi:10.1093/glycob/11.4.261
Zhou M, Zhang C, Wu Y, Tang Y (2013) Metabolic engineering of gossypol in cotton. Appl Microbiol Biotechnol 97:6159–6165. doi:10.1007/s00253-013-5032-5
Zhou QL (2011) Privileged chiral ligands and catalysts. Wiley, New York
Zhu L, Zhang X, Tu L, Zeng F, Nie Y, Guo X (2007) Isolation and characterization of two novel dirigent-like genes highly induced in cotton (Gossypium barbadense and G. hirsutum) after infection by Verticillium dahliae. J Plant Pathol 89:41–45. doi:10.4454/jpp.v89i1.722
Acknowledgements
We thank Jutta Babo for cloning of the GhDIR4 expression construct and Benedikt Fabry for its transformation into Pichia X33.
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This study was funded by Deutsche Forschungsgemeinschaft (DFG; grant number SCHA 591/10-1).
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Effenberger, I., Harport, M., Pfannstiel, J. et al. Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol. Appl Microbiol Biotechnol 101, 2021–2032 (2017). https://doi.org/10.1007/s00253-016-7997-3
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DOI: https://doi.org/10.1007/s00253-016-7997-3