Skip to main content

Advertisement

SpringerLink
Log in

Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Ashenhurst JA (2010) Intermolecular oxidative cross-coupling of arenes. Chem Soc Rev 39:540–548. doi:10.1039/b907809f

    Article  CAS  PubMed  Google Scholar 

  • Beaudoin GAW, Facchini PJ (2014) Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240:19–32. doi:10.1007/s00425-014-2056-8

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Brunel JM (2005) BINOL: A versatile chiral reagent. Chem Rev 105:857–897. doi:10.1021/cr040079g

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Keshmiri-Neghab H, Goliaei B (2014) Therapeutic potential of gossypol: an overview. Pharmaceut Biol 52:124–128. doi:10.3109/13880209.2013.832776

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li C, Lei X (2014) Strategies toward the biomimetic syntheses of oligomeric sesquiterpenoids. J Org Chem 79:3289–3295. doi:10.1021/jo5002092

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Petersen M, Alfermann AW (2001) The production of cytotoxic lignans by plant cell cultures. Appl Microbiol Biotechnol 55:135–142. doi:10.1007/s002530000510

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Chapter  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

    PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Zhou QL (2011) Privileged chiral ligands and catalysts. Wiley, New York

    Book  Google Scholar 

  • 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

    CAS  Google Scholar 

Download references

Acknowledgements

We thank Jutta Babo for cloning of the GhDIR4 expression construct and Benedikt Fabry for its transformation into Pichia X33.

Author information

Authors and Affiliations

  1. Institute of Plant Physiology and Biotechnology, University of Hohenheim, 70593, Stuttgart, Germany

    Isabelle Effenberger, Michael Harport & Andreas Schaller

  2. Core Facility Hohenheim, Mass Spectrometry Unit, University of Hohenheim, 70593, Stuttgart, Germany

    Jens Pfannstiel & Iris Klaiber

Authors
  1. Isabelle Effenberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Harport
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jens Pfannstiel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Iris Klaiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas Schaller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreas Schaller.

Ethics declarations

Funding

This study was funded by Deutsche Forschungsgemeinschaft (DFG; grant number SCHA 591/10-1).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

ESM 1

(PDF 997 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7997-3

Keywords

Search

Navigation