Skip to main content
SpringerLink
Log in

Genetic manipulation of the biosynthetic process leading to phoslactomycins, potent protein phosphatase 2A inhibitors

  • Review
  • Published:
Journal of Industrial Microbiology and Biotechnology

Abstract

Phoslactomycins (PLMs) represent an unusual structural class of natural products secreted by various streptomycetes, containing an α,β-unsaturated δ-lactone, an amino group, phosphate ester, conjugated diene and a cyclohexane ring. Phosphazomycins, phospholines and leustroducsins contain the same structural moieties, varying only in the acyl substituent at the C-18 hydroxyl position. These compounds possess either antifungal or antitumor activities or both. The antitumor activity of the PLM class of compounds has been attributed to a potent and selective inhibition of protein phosphatase 2A (PP2A). The cysteine-269 residue of PP2Ac-subunit has been shown to be the site of covalent modification by PLMs. In this article, we review previous work on the isolation, structure elucidation and biological activities of PLMs and related compounds and current status of our work on both PLM stability and genetic manipulation of the biosynthetic process. Our work has shown that PLM B is surprisingly stable in solution, with a pH optimum of 6. Preliminary biosynthetic studies utilizing isotopically labeled shikimic acid and cyclohexanecarboxylic acid (CHC) suggested PLM B to be a polyketide-type antibiotic synthesized using CHC as a starter unit. Using a gene (chcA) from a set of CHC-CoA biosynthesis genes from Streptomyces collinus as a probe, a 75 kb region of 29 ORFs encoding PLM biosynthesis was located in the genome of Streptomyces sp. strain HK803. Analysis and subsequent manipulation of plmS 2 and plmR 2 in the gene cluster has allowed for rational engineering of a strain that produces only one PLM analog, PLM B, at ninefold higher titers than the wild type strain. A strain producing PLM G (the penultimate intermediate in PLMs biosynthesis) has also been generated. Current work is aimed at selective in vitro acylation of PLM G with various carboxylic acids and a precursor-directed biosynthesis in a chcA deletion mutant with the aim of generating novel PLM analogs.

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
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Boger DL, Ichikawa S, Zhong W (2001) Total synthesis of fostriecin (CI-920). J Am Chem Soc 123:4161–4167

    Article  PubMed  CAS  Google Scholar 

  2. Bradford D (1996) Molecular mechanisms of the protein serine/threonine phosphatases. Trends Biochem Sci 21:407–412

    Article  PubMed  Google Scholar 

  3. Buck SB, Hardouin C, Ichikawa S, Soenen DR, Gauss C-M, Hwang I, Swingle MR, Bonness KM, Honkanen RE, Boger DL (2003) Fundamental role of the fostriecin unsaturated lactone and implications for selective protein phosphatase inhibition. J Am Chem Soc 125:15694–15695

    Article  PubMed  CAS  Google Scholar 

  4. Cropp TA, Wilson DJ, Reynolds KA (2000) Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin. Nat Biotechnol 18:980–983

    Article  PubMed  CAS  Google Scholar 

  5. Das Choudhuri S, Ayers S, Soine WH, Reynolds KA (2005) A pH-stability study of phoslactomycin B and analysis of the acid and base degradation products. J Antibiot 58:573–582

    Article  PubMed  Google Scholar 

  6. Esumi T, Okamoto N, Hatakeyama S (2002) Versatile enantiocontrolled synthesis of (+)-fostriecin. Chem Commun (Camb) 24:3042–3043

    Article  CAS  Google Scholar 

  7. Fujii K, Maki K, Kanai M, Shibasaki M (2003) Formal catalytic asymmetric total synthesis of fostriecin. Org Lett 5:733–736

    Article  PubMed  CAS  Google Scholar 

  8. Fushimi S, Nishikawa S, Shimazu A, Seto H (1989) Studies on new phosphate ester antifungal antibiotics phoslactomycins. I. Taxonomy, fermentation, purification and biological activities. J Antibiot 42:1019–1025

    PubMed  CAS  Google Scholar 

  9. Fushimi S, Furihata K, Seto H (1989) Studies on new phosphate ester antifungal antibiotics phoslactomycins. II. Structure elucidation of phoslactomycins A to F. J Antibiot 42:1026–1036

    PubMed  CAS  Google Scholar 

  10. Ghatge MS, Reynolds KA (2005) The plmS2-encoded Cytochrome P450 monooxygenase mediates hydroxylation of phoslactomycin B in Streptomyces sp. strain HK803. J Bacteriol 187:7970–7976

    Article  PubMed  CAS  Google Scholar 

  11. Gust B, Challis GL, Fowler K, Kieser T, Chater KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100:1541–1546

    Article  CAS  Google Scholar 

  12. de Jong RS, Mulder NH, Uges DR, Sleijfer DT, Hoppener FJ, Groen HJ, Willemse PH, van der Graaf WT, de Vries EG (1999) Phase I and pharmacokinetic study of the topoisomerase II catalytic inhibitor fostriecin. Br J Cancer 79:882–887

    Article  PubMed  Google Scholar 

  13. Katz L (1997) Manipulation of modular polyketide synthases. Chem Rev 97:2557–2575

    Article  PubMed  CAS  Google Scholar 

  14. Kawada M, Kawatsu M, Masuda T, Ohba S, Amemiya M, Kohama T, Ishizuka M, Takeuchi T (2003) Specific inhibitors of protein phosphatase 2A inhibit tumor metastasis through augmentation of natural killer cells. Int Immunopharmacol 3:179–188

    Article  PubMed  CAS  Google Scholar 

  15. Kim BS, Cropp TA, Beck BJ, Sherman DH, Reynolds KA (2002) Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin. J Biol Chem 277:48028–48034

    Article  PubMed  CAS  Google Scholar 

  16. Kohama T, Enokita R, Okazaki T, Miyaoka H, Torikata A, Inukai M, Kaneko I, Kagasaki T, Sakaida Y, Satoh A, Shiraishi A (1993) Novel microbial metabolites of the phoslactomycins family induce production of colony-stimulating factors by bone marrow stromal cells. I. Taxonomy, fermentation and biological properties. J Antibiot 46:1503–1511

    PubMed  CAS  Google Scholar 

  17. Kohama T, Nakamura T, Kinoshita T, Kaneko I, Shiraishi A (1993) Novel microbial metabolites of the phoslactomycins family induce production of colony-stimulating factors by bone marrow stromal cells. II. Isolation, physico-chemical properties and structure determination. J Antibiot 46:1512–1519

    PubMed  CAS  Google Scholar 

  18. Kohama T, Katayama T, Inukai M, Maeda H, Shiraishi A (1994) Augmentation of host resistance against bacterial infection by treatment with leustroducsin B, a new CSF inducer. Microbiol Immunol 38:741–745

    PubMed  CAS  Google Scholar 

  19. Kohama T, Maeda H, Sakai JI, Shiraishi A, Yamashita K (1996) Leustroducsin B, a new cytokine inducer derived from an actinomycetes, induces thrombocytosis in mice. J Antibiot (Tokyo) 49:91–94

    CAS  Google Scholar 

  20. Leopold WR,Shillis JL, Mertus AE, Nelson JM, Roberts BJ, Jackson RC (1984) Anticancer activity of the structurally novel antibiotic CI-920 and its analogues. Cancer Res 44:1928–1932

    PubMed  CAS  Google Scholar 

  21. Leopold WR, Mertus AE, Nelson JM, Roberts BJ (1983) In vivo activity of the novel anticancer antibiotic PD 110,161. Proc Am Assoc Cancer Res 24:320

    Google Scholar 

  22. Lewy DS, Gauss CM, Soenen DR, Boger DL (2002) Fostriecin: chemistry and biology. Curr Med Chem 9:2005–2032

    PubMed  CAS  Google Scholar 

  23. Lowden PA, Bohm GA, Staunton J, Leadlay PF (1996) The nature of the starter unit for the rapamycin polyketide synthase. Angew Chem Int Ed Eng 35:2249–2251

    Article  CAS  Google Scholar 

  24. Lowden PA, Wilkinson B, Bohm GA, Handa S, Floss HG, Leadlay PF, Staunton J (2001) Origin and true nature of the starter unit for the rapamycin polyketide synthase. Angew Chem Int Ed Engl 40:777–779

    Article  PubMed  CAS  Google Scholar 

  25. Lu H, Tsai SC, Khosla C, Cane DE (2002) Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase. Biochemistry 41:12590–12597

    Article  PubMed  CAS  Google Scholar 

  26. Miyashita K, Ikejiri M, Kawasaki H, Maemura S, Imanishi T (2002) Total synthesis of fostriecin (CI-920) via a convergent route. Chem Commun 7:742–743

    Article  CAS  Google Scholar 

  27. Moore BS, Cho H, Casati R, Kennedy E, Reynolds KA, Mocek U, Beale JM, Floss HG (1993) Biosynthetic studies on ansatrienin A. Formation of the cyclohexanecarboxylic acid moiety. J Am Chem Soc 115:5254–5266

    Article  CAS  Google Scholar 

  28. Moore BS, Poralla K, Floss HG (1993) Biosynthesis of the cyclohexanecarboxylic acid starter unit of ω-cyclohexyl fatty acids in Alicyclobacillus acidocaldarius. J Am Chem Soc 115:5267–5274

    Article  CAS  Google Scholar 

  29. Ozasa T, Suzuki K, Sasamata M, Tanaka K, Kobori M, Kadota S, Nagai K, Saito T, Watanabe S, Iwanami M (1989) Novel antitumor antibiotic phospholine. 1. Production, isolation and characterization. J Antibiot 42:1331–1338

    PubMed  CAS  Google Scholar 

  30. Ozasa T, Tanaka K, Sasamata M, Kaniwa H, Shimizu M, Matsumoto H, Iwanami M (1989) Novel antitumor antibiotic phospholine. 2. Structure determination. J Antibiot 42:1339–1343

    PubMed  CAS  Google Scholar 

  31. Palaniappan N, Kim BS, Sekiyama Y, Osada H, Reynolds KA (2003) Enhancement and selective production of phoslactomycin B, a protein phosphatase IIa inhibitor, through identification and engineering of the corresponding biosynthetic gene cluster. J Biol Chem 278:35552–35557

    Article  PubMed  CAS  Google Scholar 

  32. Patton SM, Ashton T. Cropp, Reynolds KA (2000) A novel Δ3,Δ2 enoyl CoA isomerase involved in the biosynthesis of the cyclohexanecarboxylic acid-derived moiety of the polyketide ansatrienin A. Biochemistry 39:7595–7604

    Article  PubMed  CAS  Google Scholar 

  33. Reddy YK, Falck JR (2002) Asymmetric total synthesis of (+)-fostriecin. Org Lett 4:969–971

    Article  PubMed  CAS  Google Scholar 

  34. Reynolds KA, Wallace KK, Handa S, Brown MS, McArthur HAI, Floss HG (1997) Biosynthesis of the shikimate-derived starter unit of the immunosuppressant ascomycin: Stereochemistry of the 1,4-conjugate elimination. J Antibiot 50:701–703

    PubMed  CAS  Google Scholar 

  35. Sekiyama Y, Palaniappan N, Reynolds KA, Osada H (2003) Biosynthesis of phoslactomycins: cyclohexanecarboxylic acid as the starter unit. Tetrahedron 59:7465–7471

    Article  CAS  Google Scholar 

  36. Shen B (2004) Accessing natural products by combinatorial biosynthesis. Sci STKE 225:pe14

    Article  Google Scholar 

  37. Shibata T, Kurihara S, Oikawa T, Ohkawa N, Shimazaki N, Sasagawa K, Kobayashi T, Kohama T, Asai F, Shiraishi A, Sugimura Y (1995) Preparation of leustroducsin H and the structure-activity relationship of its derivatives. J Antibiot (Tokyo) 48:1518–1520

    CAS  Google Scholar 

  38. Shibata T, Kurihara S, Yoda K, Haruyama H (1995) Absolute configuration of leustroducsins. Tetrahedron 51:11999–12012

    Article  CAS  Google Scholar 

  39. Shimada K, Kaburagi Y, Fukuyama T (2003) Total synthesis of leustroducsin B. J Am Chem Soc 125:4048–4049

    Article  PubMed  CAS  Google Scholar 

  40. Shimada K, Koishi R, Kohama T (2004) Leustroducsins, low molecular weight thrombopoietic agents: discovery, biological properties and total synthesis. Annu Rep Sankyo Res Lab 56:11–28

    CAS  Google Scholar 

  41. Stampwala SS, Bunge RH, Hurley TR, Willmer NE, Brankiewicz AJ, Steinman CE, Smitka TA, French JC (1983) Novel antitumor agents CI-920, PD 113,270 and PD 113,271. II. Isolation and characterization. J Antibiot 36:1601–1605

    PubMed  CAS  Google Scholar 

  42. Teruya T, Simizua S, Kanoha N, Osada H (2005) Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells. FEBS Lett 579:2463–2468

    Article  PubMed  CAS  Google Scholar 

  43. Tomiya T, Uramoto M, Isono K (1990) Isolation and structure of phosphazomycin C. J Antibiot 43:118–121

    PubMed  CAS  Google Scholar 

  44. Tsai SC, Lu H, Cane DE, Khosla C, Stroud RM (2002) Insights into channel architecture and substrate specificity from crystal structures of two macrocycle-forming thioesterases of modular polyketide synthases. Biochemistry 41:12598–12606

    Article  PubMed  CAS  Google Scholar 

  45. Tunac JB, Graham BD, Dobson WE (1983) Novel antitumor agents CI-920, PD 113,270 and PD 113,271. I. Taxonomy, fermentation and biological properties. J Antibiot 36:1595–1600

    PubMed  CAS  Google Scholar 

  46. Uramoto M, Shen YC, Takizawa N, Kusakabe H, Isono K (1985) A new antifungal antibiotic, phosphazomycin A. J Antibiot 38:665–668

    PubMed  CAS  Google Scholar 

  47. Usui T, Marriott G, Inagaki M, Swarup G, Osada H (1999) Protein phosphatase 2A inhibitors, phoslactomycins. Effects on the cytoskeleton in NIH/3T3 cells. J Biochem 125:960–965

    PubMed  CAS  Google Scholar 

  48. Wallace KK, Reynolds KA, Koch K, McArthur HAI, Brown MS, Wax RG, Moore BS (1994) Biosynthetic studies of ascomycin (FK520): formation of the (1R,3R,4R)-3,4-dihydroxycyclohexanecarboxylic acid-derived moiety. J Am Chem Soc 116:11600–11601

    Article  CAS  Google Scholar 

  49. Wang P, Denoya CD, Morgenstern MR, Skinner DD, Wallace KK, DiGate R, Patton S, Banavali N, Schuler G, Speedie MK, Reynolds KA (1996) Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl CoA reductase from Streptomyces collinus. J Bacteriol 178:6873–6881

    PubMed  CAS  Google Scholar 

  50. Wang YG, Kobayashi Y (2002) Formal total synthesis of fostriecin. Org Lett 4:4615–4618

    Article  PubMed  CAS  Google Scholar 

  51. Xue Y, Wilson D, Zhao L, Liu H, Sherman DH (1998) Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae. Chem Biol 5:661–667

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Our work on PLM stability and biosynthesis is supported by grant AI51629 from the National Institutes of Health. Streptomyces sp. strain HK803 was kindly provided by Hiroyuki Osada (Riken, Japan).

Author information

Authors and Affiliations

  1. Department of Chemistry, Portland State University, P.O. Box 751, Portland, OR, 97207-0751, USA

    Mohini Ghatge, Nadaraj Palaniappan & Kevin Reynolds

  2. Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, 23298, USA

    Suparna Das Choudhuri

Authors
  1. Mohini Ghatge
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nadaraj Palaniappan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suparna Das Choudhuri
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kevin Reynolds
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin Reynolds.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ghatge, M., Palaniappan, N., Das Choudhuri, S. et al. Genetic manipulation of the biosynthetic process leading to phoslactomycins, potent protein phosphatase 2A inhibitors. J IND MICROBIOL BIOTECHNOL 33, 589–599 (2006). https://doi.org/10.1007/s10295-006-0116-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-006-0116-1

Keywords

Search

Navigation