Advertisement

Applied Biochemistry and Biotechnology

, Volume 149, Issue 1, pp 67–78 | Cite as

Isolation of New Polyketide Synthase Gene Fragments and a Partial Gene Cluster from East China Sea and Function Analysis of a New Acyltransfrase

  • Yu-Liang Jiao
  • Liang-Hua Wang
  • Xiao-Yi Dong
  • Yu-Feng Chen
  • Ying Zong
  • Yun Gao
  • Na Ren
  • Ai-Yun Guo
  • Xin-Qun Zhang
  • Bing-Hua JiaoEmail author
Article

Abstract

Using the consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction method, 26 new ketoacyl synthase (KS) fragments were isolated from a marine sediment sample in the East China Sea (ECS) and analyzed by construction of a phylogenetic tree. With a digoxigenin-labeled KS gene fragment used as a probe, a partial polyketide synthase (PKS) gene cluster was isolated and identified by hybridization screening of a marine sediment sample metagenome fosmid library constructed for this study. A new acyltransferase (AT) gene was cloned from the PKS gene cluster and heterogeneously expressed as a protein fused to maltose-binding protein (MBP). Ultraviolet spectrophotometry was used to study the binding of the MBP–AT fusion protein and single AT domain to substrates using MBP and bovine serum albumin as control proteins. Binding constants (Ka, per micromolar) were calculated and used to analyze the substrate specificity of the acyltransferase. We concluded that there are many unrevealed new PKS gene clusters in marine sediments in the ECS. The acyltransferase is presumably an acetyltransferase from a new PKS gene cluster.

Keywords

Acetyltransferase Acyltransferase Ketoacyl synthase Polyketide synthase Substrate specificity 

Abbreviations

CODEHOP PCR

consensus-degenerate hybrid oligonucleotide primers PCR

BSA

bovine serum albumin

PKS

polyketide synthase

ECS

the East China Sea

CoA

coenzyme A

DIG

digoxigenin

KS

ketoacyl synthase

PFGE

pulsed-field gel electrophoresis

AT

acyltransferase

ACP

acyl carrier protein

MBP

maltose-binding protein

PBS

phosphate-buffered saline

UV

ultraviolet

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

IPTG

isopropyl β-d-1-thiogalactopyranoside

NBT/BCIP

nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate

Notes

Acknowledgment

This work was sponsored by the Natural Science Funds of the People’s Republic of China (grant number 30670043) and the National High Technology Research and Development Program of China (863 Program; grant number 2006AA09Z434).

Jiao, Wang and Dong contributed equally to this work.

References

  1. 1.
    Hopwood, D. A. (1997). Chemical Reviews (Washington, DC), 97, 2465–2497.Google Scholar
  2. 2.
    Staunton, J., & Weissman, K. J. (2001). Natural Product Report, 18, 380–416.CrossRefGoogle Scholar
  3. 3.
    Shen, B. (2000). Topics in Current Chemistry, 209, 1–51.CrossRefGoogle Scholar
  4. 4.
    Wawrik, B., Kerkhof, L., Zylstra, G. J., & Kukor, J. J. (2005). Applied Environmental Microbiology, 71(5), 2232–2238.CrossRefGoogle Scholar
  5. 5.
    Metsa-Ketela, M., Salo, V., Halo, L., Hautala, A., Hakala, J., & Mantsala, P., et al. (1999). FEMS Microbiology Letters, 180, 1–6.CrossRefGoogle Scholar
  6. 6.
    O’Hagan, D. (1991). Polyketides. Chichester, UK: Horwood.Google Scholar
  7. 7.
    Sogin, M. L., Morrison, H. G., Huber, J. A., Welch, D. M., Huse, S. M., Neal, P. R. et al. (2006). Proceeding of the National Academy of Science of the United States of America, 103(32), 12115–12120.CrossRefGoogle Scholar
  8. 8.
    Ward, A. C., & Bora, N. (2006). Current Opinion of Microbiology, 9, 279–286.CrossRefGoogle Scholar
  9. 9.
    Pathom-Aree, W., Stach, J. E., Ward, A. C., Horikoshi, K., Bull, A. T., & Goodfellow, M. (2006). Extremophiles, 10(3), 181–189.CrossRefGoogle Scholar
  10. 10.
    Kim, T. K., & Fuerst, J. A. (2006). Environmental Microbiology, 8(8), 1460–1470.CrossRefGoogle Scholar
  11. 11.
    Davidson, S. K., Allen, S. W., Lim, G. E., Anderson, C. M., & Haygood, M. G. (2001). Applied Environmental Microbiology, 67, 4531–4537.CrossRefGoogle Scholar
  12. 12.
    Lopanik, N. B., Targett, N. M., & Lindquist, N. (2006). Applied Environmental Microbiology, 72(12), 7941–7944.CrossRefGoogle Scholar
  13. 13.
    Floss, H. G. (2006). Journal of Biotechnology, 124(1), 242–257.CrossRefGoogle Scholar
  14. 14.
    Cheng, Y. Q., Tang, G. L., & Shen, B. (2003). Proceeding of the National Academy of Science of the United States of America, 100(6), 3149–3154.CrossRefGoogle Scholar
  15. 15.
    Zhang, W. J., Ames, B. D., Tsai, S. C., & Tang, Y. (2006). Applied Environmental Microbiology, 72(4), 2573–2580.CrossRefGoogle Scholar
  16. 16.
    Strohl, W. R. (2000). Drug Discovery Today, 5, 39–41.CrossRefGoogle Scholar
  17. 17.
    Strohl, W. R. (1997). Industrial antibiotics: today and the future. In W. R. Strohl (Ed.) Biotechnology of antibiotics (2nd ed.). Marcel DekkerNewYork, NY.Google Scholar
  18. 18.
    Ginolhac, A., Jarrin, C., Gillet, B., Robe, P., Pujic, P., & Tuphile, K., et al. (2004). Applied Environmental Microbiology, 70(9), 5522–5527.CrossRefGoogle Scholar
  19. 19.
    Kroken, S., Glass, N. L., Taylor, J. W., Yoder, O. C., & Turgeon, B. G. (2003). Proceeding of the National Academy of Science of the United States of America, 100(26), 15670–15675.CrossRefGoogle Scholar
  20. 20.
    Lopez, J. V. (2003). Mol Genet Genomics, 270, 420–431.CrossRefGoogle Scholar
  21. 21.
    Rose, T. M., & Henikoff, J. G. (2003). Nucleic Acids Research, 31(13), 3763–3766.CrossRefGoogle Scholar
  22. 22.
    Crawford, J. M., Dancy, B. C., Hill, E. A., Udwary, D. W., & Townsend, C. A. (2006). Proceeding of the National Academy of Science of the United States of America, 103(45), 16728–16733.CrossRefGoogle Scholar
  23. 23.
    Reeves, C. D., Chung, L. M., Liu, Y., Xue, Q., Carney, J. R., & Revill, W. P., et al. (2002). Journal of Biological Chemistry, 277(11), 9155–9159.CrossRefGoogle Scholar
  24. 24.
    Kim, B. S., Sherman, D. H., & Reynolds, K. A. (2004). Protein Engineering Design and Selection, 17(3), 277–284.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Yu-Liang Jiao
    • 1
  • Liang-Hua Wang
    • 1
  • Xiao-Yi Dong
    • 1
  • Yu-Feng Chen
    • 2
  • Ying Zong
    • 1
  • Yun Gao
    • 1
  • Na Ren
    • 1
  • Ai-Yun Guo
    • 1
  • Xin-Qun Zhang
    • 1
  • Bing-Hua Jiao
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyCollege of Basic Medical ScienceShanghaiPeople’s Republic of China
  2. 2.Department of Pharmacognosy, School of PharmacySecond Military Medical UniversityShanghaiPeople’s Republic of China

Personalised recommendations