Skip to main content

Advertisement

SpringerLink
Log in

Titer improvement of iso-migrastatin in selected heterologous Streptomyces hosts and related analysis of mRNA expression by quantitative RT–PCR

  • Biotechnological Products and Process Engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

iso-Migrastatin (iso-MGS) has been actively pursued recently as an outstanding candidate of antimetastasis agents. Having characterized the iso-MGS biosynthetic gene cluster from its native producer Streptomyces platensis NRRL 18993, we have recently succeeded in producing iso-MGS in five selected heterologous Streptomyces hosts, albeit the low titers failed to meet expectations and cast doubt on the utility of this novel technique for large-scale production. To further explore and capitalize on the production capacity of these hosts, a thorough investigation of these five engineered strains with three fermentation media for iso-MGS production was undertaken. Streptomyces albus J1074 and Streptomyces lividans K4-114 were found to be preferred heterologous hosts, and subsequent analysis of carbon and nitrogen sources revealed that sucrose and yeast extract were ideal for iso-MGS production. After the initial optimization, the titers of iso-MGS in all five hosts were considerably improved by 3–18-fold in the optimized R2YE medium. Furthermore, the iso-MGS titer of S. albus J1074 (pBS11001) was significantly improved to 186.7 mg/L by a hybrid medium strategy. Addition of NaHCO3 to the latter finally afforded an optimized iso-MGS titer of 213.8 mg/L, about 5-fold higher than the originally reported system. With S. albus J1074 (pBS11001) as a model host, the expression of iso-MGS gene cluster in four different media was systematically studied via the quantitative RT–PCR technology. The resultant comparison revealed the correlation of gene expression and iso-MGS production for the first time; synchronous expression of the whole gene cluster was crucial for optimal iso-MGS production. These results reveal new insights into the iso-MGS biosynthetic machinery in heterologous hosts and provide the primary data to realize large-scale production of iso-MGS for further preclinical studies.

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

Similar content being viewed by others

References

  • Feng ZY, Wang LY, Rajski SR, Xu ZN, Coeffet-LeGal MF, Shen B (2009) Engineered production of iso-migrastatin in heterologous Streptomyces hosts. Bioorgan Med Chem 17(6):2147–2153

    Article  CAS  Google Scholar 

  • Fleige S, Walf V, Huch S, Prgomet C, Sehm J, Pfaffl MW (2006) Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT–PCR. Biotechnol Lett 28:1601–1613

    Article  CAS  Google Scholar 

  • Galm U, Shen B (2006) Expression of biosynthetic gene cluster in heterologous hosts for natural product production and combinatorial biosynthesis. Expert Opin Drug Discov 1(5):409–437

    Article  CAS  Google Scholar 

  • Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf H (2000) Genetic manipulation of Streptomyces: a laboratory manual. The John Innes Foundation, Norwich

    Google Scholar 

  • Ju JH, Lim SK, Jiang H, Shen B (2005) Migrastatin and dorrigocins are shunt metabolites of iso-migrastatin. J Am Chem Soc 127:1622–1623

    Article  CAS  Google Scholar 

  • Ju JH, Rajski SR, Lim SK, Seo JW, Peters NR, Hoffmann FM, Shen B (2008) Evaluation of new migrastatin and dorrigocin congeners unveils cell migration inhibitors with dramatically improved potency. Bioorg Med Chem Lett 18:5951–5954

    Article  CAS  Google Scholar 

  • Ju JH, Rajski SR, Lim SK, Seo JW, Peters NR, Hoffmann FM, Shen B (2009) Lactimidomycin, iso-migrastatin and related glutarimide-containing 12-membered macrolides are extremely potent inhibitors of cell migration. J Am Chem Soc 131(4):1370–1371

    Article  CAS  Google Scholar 

  • Lim SK, Ju JH, Zazopoulos E, Jiang H, Seo JW, Chen YH, Feng ZY, Rajski SR, Farnet CM, Shen B (2009) iso-Migrastatin, migrastatin, and dorrigocin production in Streptomyces platensis NRRL 18993 is governed by a single biosynthetic machinery featuring an acyltransferase-less type I polyketide synthase. J Biol Chem 284(43):29746–29756

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen KJ (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Martinez A, Kolvek SJ, Yip CLT, Hopke J, Brown KA, MacNeil IA, Osburne MS (2004) Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts. Appl Environ Microb 70(4):2452–2463

    Article  CAS  Google Scholar 

  • Moss J, Lane MD (1972) Acetyl coenzyme A carboxylase III: further studies on the relation of catalytic activity to polymeric state. J Biol Chem 247(16):4944–4951

    CAS  Google Scholar 

  • Nakae K, Yoshimoto Y, Sawa T, Homma Y, Hamada M, Takeuchi T, Imoto M (2000) Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929-43F1. Taxonomy, fermentation, isolation and biological activities. J Antibiot 53(10):1130–1136

    CAS  Google Scholar 

  • Nakae K, Nishimura Y, Ohba S, Akamatsu Y (2006) Migrastatin acts as a muscarinic acetylcholine receptor antagonist. J Antibiot 59:685–692

    Article  CAS  Google Scholar 

  • Newman DJ (2008) Natural products as leads to potential drugs: an old process or the new hope for drug discovery? J Med Chem 51(9):2589–2599

    Article  CAS  Google Scholar 

  • Olano C, Lombó F, Méndez C, Salas JA (2008) Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab Eng 10(5):281–292

    Article  CAS  Google Scholar 

  • Reymond S, Cossy J (2008) Migrastatin and analogues: new anti-metastatic agents. CR Chim 11:1447–1462

    Article  CAS  Google Scholar 

  • Sosio M, Giusino F, Cappellano C, Bossi E, Puglia AM, Donadio S (2000) Artificial chromosomes for antibiotic-producing actinomycetes. Nat Biotechnol 18:343–345

    Article  CAS  Google Scholar 

  • Takemoto Y, Tashiro E, Imoto M (2006) Suppression of multidrug resistance by migrastatin. J Antibiot 59:435–438

    Article  CAS  Google Scholar 

  • Wenzel SC, Muller R (2005) Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. Curr Opin Biotech 16(6):594–606

    Article  CAS  Google Scholar 

  • Woo EJ, Starks CM, Carney JR, Arslanin R, Cadapan L, Zavala S, Licari P (2002) Migrastatin and a new compound, isomigrastatin, from Streptomyces platensis. J Antibiot 55(2):141–146

    CAS  Google Scholar 

  • Zhang HR, Wang Y, Pfeifer BA (2008) Bacterial hosts for natural product production. Mol Pharmaceut 5(2):212–225

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by NIH of US (grants CA106150 and CA113297 to B.S.) and The Nature Science Foundation of China (Grant No. 20736008), The Ministry of Science and Technology (Grant No. 2007AA021702), and The Natural Science Foundation of Zhejiang, China, (R4090041), all to Z.X.

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Institute of Bioengineering, Zhejiang University, Hangzhou, 310027, Zhejiang, China

    Dong Yang, Xiangcheng Zhu, Xueyun Wu, Lei Huang & Zhinan Xu

  2. Division of Pharmaceutical Science, School of Pharmacy, University of Wisconsin–Madison, 777 Highland Avenue, Madison, WI, 53705, USA

    Zhiyang Feng & Ben Shen

Authors
  1. Dong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiangcheng Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xueyun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhiyang Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lei Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ben Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhinan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Ben Shen or Zhinan Xu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, D., Zhu, X., Wu, X. et al. Titer improvement of iso-migrastatin in selected heterologous Streptomyces hosts and related analysis of mRNA expression by quantitative RT–PCR. Appl Microbiol Biotechnol 89, 1709–1719 (2011). https://doi.org/10.1007/s00253-010-3025-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-3025-1

Keywords

Search

Navigation