Skip to main content
SpringerLink
Log in

Identification of a type III polyketide synthase involved in the biosynthesis of spirolaxine

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

Abstract

Spirolaxine is a natural product isolated from Sporotrichum laxum ATCC 15155, which has shown a variety of biological activities including promising anti-Helicobacter pylori property. To understand how this compound is biosynthesized, the genome of S. laxum was sequenced. Analysis of the genome sequence revealed two putative type III polyketide synthase (PKS) genes in this strain, Sl-pks1 and Sl-pks2, which are located adjacent to each other (~2.0 kb apart) in a tail-to-tail arrangement. Disruption of these two genes revealed that Sl-PKS2 is the dedicated PKS involved in the biosynthesis of spirolaxine. The intron-free Sl-pks2 gene was amplified from the cDNA of S. laxum and ligated into the expression vector pET28a for expression in Escherichia coli BL21-CodonPlus (DE3)-RIL. The major products of Sl-PKS2 in E. coli were characterized as alkylresorcinols that contain a C13–C17 saturated or unsaturated hydrocarbon side chain based on the spectral data. This enzyme was purified and reacted with malonyl-CoA and a series of fatty acyl-SNACs (C6–C10). Corresponding alkylresorcinols were formed from the decarboxylation of the synthesized tetraketide resorcylic acids, together with fatty acyl-primed triketide and tetraketide pyrones as byproducts. This work provides important information about the PKS involved in the biosynthesis of spirolaxine, which will facilitate further understanding and engineering of the biosynthetic pathway of this medicinally important molecule.

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

  • Arnone A, Assante G, Nasini G, Vajna de Pava O (1990) Secondary mold metabolites. Part 28. Spirolaxine and sporotricale: two long-chain phthalides produced by Sporotrichum laxum. Phytochemistry 29:613–616

    Article  CAS  Google Scholar 

  • Baerson SR, Schröder J, Cook D, Rimando AM, Pan Z, Dayan FE, Noonan BP, Duke SO (2010) Alkylresorcinol biosynthesis in plants: new insights from an ancient enzyme family? Plant Signal Behav 5:1286–1289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bava A, Clericuzio M, Giannini G, Malpezzi L, Meille SV, Nasini G (2005) Absolute configuration of the fungal metabolite spirolaxine. Eur J Org Chem 2005(11):2292–2296

    Article  Google Scholar 

  • Blaser MJ (1992) Helicobacter pylori: its role in disease. Clin Infect Dis 15(3):386–391

    Article  CAS  PubMed  Google Scholar 

  • Dekker KA, Inagaki T, Gootz TD, Kaneda K, Nomura E, Sakakibara T, Sakemi S, Sugie Y, Yamauchi Y, Yoshikawa N, Kojima N (1997) CJ-12,954 and its congeners, new anti-Helicobacter pylori compounds produced by Phanerochaete velutina: fermentation, isolation, structural elucidation and biological activities. J Antibiot 50(10):833–839

    Article  CAS  PubMed  Google Scholar 

  • Dimitrov I, Furkert DP, Fraser JD, Radcliff FJ, Finch O, Brimble MA (2012) Synthesis and anti-Helicobacter pylori activity of analogues of spirolaxine methyl ether. MedChemComm 3(8):938–943

    Article  CAS  Google Scholar 

  • Funa N, Awakawa T, Horinouchi S (2007) Pentaketide resorcylic acid synthesis by type III polyketide synthase from Neurospora crassa. J Biol Chem 282(19):14476–14481

    Article  CAS  PubMed  Google Scholar 

  • Giannini G, Pisano C, Riccioni T, Marcellini M, Chiarucci I, Bava A, Nasini G (2004) Spirolaxine, a fungal metabolite from Sporotrichum laxum: determination of relative stereochemistry and biological activity in vitro. Proc Amer Assoc Cancer Res 45:1011

    Google Scholar 

  • Hashimoto M, Koen T, Takahashi H, Suda C, Kitamoto K, Fujii I (2014) Aspergillus oryzae CsyB catalyzes the condensation of two beta-ketoacyl-CoAs to form 3-acetyl-4-hydroxy-6-alkyl-alpha-pyrone. J Biol Chem 289(29):19976–19984

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ishikawa J, Hotta K (1999) FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content. FEMS Microbiol Lett 174(2):251–253

    Article  CAS  PubMed  Google Scholar 

  • Jeya M, Kim TS, Tiwari MK, Li J, Zhao H, Lee JK (2012) The Botrytis cinerea type III polyketide synthase shows unprecedented high catalytic efficiency toward long chain acyl-CoAs. Mol BioSyst 8(11):2864–2867

    Article  CAS  PubMed  Google Scholar 

  • Knodler M, Conrad J, Wenzig EM, Bauer R, Lacorn M, Beifuss U, Carle R, Schieber A (2008) Anti-inflammatory 5-(11’Z-heptadecenyl)- and 5-(8’Z,11’Z-heptadecadienyl)-resorcinols from mango (Mangifera indica L.) peels. Phytochemistry 69(4):988–993

    Article  PubMed  Google Scholar 

  • Kozubek A, Tyman JH (1999) Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem Rev 99(1):1–26

    Article  CAS  PubMed  Google Scholar 

  • Li J, Luo Y, Lee JK, Zhao H (2011) Cloning and characterization of a type III polyketide synthase from Aspergillus niger. Bioorg Med Chem Lett 21(20):6085–6089

    Article  CAS  PubMed  Google Scholar 

  • Mejía R, Gómez-Eichelmann MC, Fernández MS (1999) Fatty acid profile of Escherichia coli during the heat-shock response. IUBMB Life 47(5):835–844

    Article  Google Scholar 

  • Miyanaga A, Horinouchi S (2010) Type III polyketide synthases responsible for phenolic lipid synthesis. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology. Springer-Verlag, Berlin, Heidelberg, pp. 519–525

    Chapter  Google Scholar 

  • Robinson JE, Brimble MA (2007) Synthesis of the anti-Helicobacter pylori agent (+)-spirolaxine methyl ether and the unnatural (2″S)-diastereomer. Org Biomol Chem 5(16):2572–2582

    Article  CAS  PubMed  Google Scholar 

  • Seshime Y, Juvvadi PR, Fujii I, Kitamoto K (2005) Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae. Biochem Biophys Res Commun 331(1):253–260

    Article  CAS  PubMed  Google Scholar 

  • Shaw MK, Ingraham JL (1965) Fatty acid composition of Escherichia coli as a possible controlling factor of the minimal growth temperature. J Bacteriol 90(1):141–146

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stanke M, Morgenstern B (2005) AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res 33(Web Server):W465-W467

  • Suzuki Y, Esumi Y, Uramoto M, Kono Y, Sakurai A (1997) Structural analyses of carbon chains in 5-alk(en)ylresorcinols of rye and wheat whole flour by tandem mass spectrometry. Biosci Biotechnol Biochem 61(3):480–486

    Article  CAS  Google Scholar 

  • Tsukamoto M, Sano H, Kadota Y, Ojiri K, Suda H (1998) Anticancer spirolaxine and its fermentative manufacture. JP10007557A,

  • Wang S, Zhang S, Zhou T, Zhan J (2013) Three new resorcylic acid derivatives from Sporotrichum laxum. Bioorg Med Chem Lett 23(21):5806–5809

    Article  CAS  PubMed  Google Scholar 

  • White SW, Zheng J, Zhang Y-M, Rock CO (2005) The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem 74:791–831

    Article  CAS  PubMed  Google Scholar 

  • Xu Y, Zhou T, Espinosa-Artiles P, Tang Y, Zhan J, Molnar I (2014a) Insights into the biosynthesis of 12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae. ACS Chem Biol 9(5):1119–1127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu Y, Zhou T, Zhang S, Espinosa-Artiles P, Wang L, Zhang W, Lin M, Gunatilaka AAL, Zhan J, Molnar I (2014b) Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases. Proc Natl Acad Sci U S A 111(34):12354–12359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu Y, Zhou T, Zhang S, Xuan L-J, Zhan J, Molnar I (2013a) Thioesterase domains of fungal nonreducing polyketide synthases act as decision gates during combinatorial biosynthesis. J Am Chem Soc 135(29):10783–10791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu Y, Zhou T, Zhou Z, Su S, Roberts SA, Montfort WR, Zeng J, Chen M, Zhang W, Lin M, Zhan J, Molnar I (2013b) Rational reprogramming of fungal polyketide first-ring cyclization. Proc Natl Acad Sci U S A 110(14):5398–5403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu D, Zeng J, Chen D, Zhan J (2010) Characterization and reconstitution of a new fungal type III polyketide synthase from Aspergillus oryzae. Enzyme Microb Tech 46(7):575–580

    Article  CAS  Google Scholar 

  • Zhou H, Zhan J, Watanabe K, Xie X, Tang Y (2008) A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae. Proc Natl Acad Sci U S A 105(17):6249–6254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by a Grant-In-Aid from the American Heart Association (16GRNT26430067), grants from the National Natural Science Foundation of China (31170763, 31470787), a Hunan Science and Technology Key Project (2014FJ1007), and the Hundred Talents Program of Hunan Province, China.

Author information

Authors and Affiliations

  1. Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT, 84322-4105, USA

    Lei Sun, Siyuan Wang, Shuwei Zhang, Dayu Yu & Jixun Zhan

  2. Department of Applied Chemistry and Biological Engineering, College of Chemical Engineering, Northeast Dianli University, Jilin, Jilin, 132012, China

    Dayu Yu

  3. TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan, 410208, China

    Yuhui Qin, Huiyong Huang, Wei Wang & Jixun Zhan

Authors
  1. Lei Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Siyuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuwei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dayu Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuhui Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Huiyong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jixun Zhan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Wei Wang or Jixun Zhan.

Ethics declarations

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

Conflict of interest

The authors declare that they have no competing interests.

Electronic supplementary material

ESM 1

(PDF 473 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, L., Wang, S., Zhang, S. et al. Identification of a type III polyketide synthase involved in the biosynthesis of spirolaxine. Appl Microbiol Biotechnol 100, 7103–7113 (2016). https://doi.org/10.1007/s00253-016-7444-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7444-5

Keywords

Search

Navigation