Functional characterization of O-methyltransferases used to catalyse site-specific methylation in the post-tailoring steps of pradimicin biosynthesis

J. W. Han, B. G. Ng, J. K. Sohng, Y. J. Yoon, G. J. Choi, B. S. Kim

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

Aims: To identify the roles of the two O-methyltransferase homologous genes pdmF and pdmT in the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2. Methods and Results: Pradimicins are pentangular polyphenol antibiotics synthesized by bacterial type II polyketide synthases (PKSs) and tailoring enzymes. Pradimicins are naturally derivatized by combinatorial O-methylation at two positions (i.e., 7-OH and 11-OH) of the benzo[α]naphthacenequinone structure. PdmF and PdmT null mutants (PFKO and PTKO) were generated. PFKO produced the 11-O-demethyl shunt metabolites 11-O-demethylpradimicinone II (1), 11-O-demethyl-7-methoxypradimicinone II (2), 11-O-demethylpradimicinone I (3) and 11-O-demethylpradimicin A (4), while PTKO generated the 7-O-demethyl derivatives pradimicinone II (5) and 7-hydroxypradimicin A (6). Pradimicinones 1, 2, 3, and 5 were fed to a heterologous host Escherichia coli harbouring expression plasmid pET-22b::pdmF or pET-28a::pdmT. PdmF catalysed 11-O-methylation of pradimicinones 1, 2, and 3 regardless of O-methylation at the C-7 position, while PdmT was unable to catalyse 7-O-methylation when the C-11 hydroxyl group was methylated (5). Conclusions: PdmF and PdmT were involved in 11-O- and 7-O-methylations of the benzo[α]naphthacenequinone moiety of pradimicin, respectively. Methylation of the C-7 hydroxyl group precedes methylation of the C-11 hydroxyl group in pradimicin biosynthesis. Significance and Impact of the Study: This is the first reported demonstration of the functions of PdmF and PdmT for regiospecific O-methylation, which contributes to better understanding of the post-PKS modifications in pradimicin biosynthesis as well as to rational engineering of the pradimicin biosynthetic machinery.

Original languageEnglish
Pages (from-to)144-154
Number of pages11
JournalJournal of Applied Microbiology
Volume124
Issue number1
DOIs
Publication statusPublished - 2018 Jan

Bibliographical note

Funding Information:
J.K. Sohng is thankful for the support from the Agenda Program (200905FP014), Rural Development Administration, Republic of Korea. Y.J. Yoon gives thanks to the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT and Future Planning (2016R1A2A1A05005078). J.W. Han is thankful for the support by a Korea University Grant. This research was partly supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning (2014R1A2A2A01005461).

Publisher Copyright:
© 2017 The Society for Applied Microbiology

Keywords

  • O-methyltransferase
  • aromatic polyketide
  • methyltransferase
  • polyketide
  • pradimicin
  • pradimicin biosynthesis
  • pradimicinone

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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