ORIGINAL ARTICLE Identification of the duplicated genes for S-adenosyl-L-methionine synthetase (metK1-sp and metK2-sp) in Streptomyces peucetius var. caesius ATCC 27952 T.-J. Oh, N.P. Niraula, K. Liou and J.K. Sohng Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, Tangjeong-myeon, Asansi, Chungnam, Republic of Korea Introduction S-Adenosyl-l-methionine (SAM) is most commonly used in living systems as a methyl donor involved in methyla- tion reactions. Previous studies have shown that SAM plays a central role in cellular metabolism via its involve- ment in various methylating reactions of proteins, nucleic acids, lipids and polysaccharides. SAM also plays the role Keywords doxorubicin, duplicated gene, S-adenosyl- L-methionine synthetase, secondary metabolism, Streptomyces peucetius var. caesius ATCC 27952. Correspondence Jae Kyung Sohng, Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, #100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea. E-mail: sohng@sunmoon.ac.kr 2009 ⁄ 0968: received 2 June 2009, revised and accepted 23 November 2009 doi:10.1111/j.1365-2672.2010.04688.x Abstract Aims: To characterize the function of both metK1-sp (sp1190) and metK2-sp (sp1566) in vitro and in vivo, and to study the regulation of doxorubicin production by overexpressing the metK. Methods and Results: We cloned two orfs into pET32a(+) respectively, and the formation of S-Adenosyl-l-methionine was clearly observed in the in vitro enzyme assays as functional MetKs. Reverse transcriptase polymerase chain reaction (PCR) analysis indicated that the transcripts for the metK1-sp were repressed as Streptomyces cells entered the decline phase, whereas that of the metK2-sp was induced, suggesting that these MetK proteins may be important for the growth and the regulation of secondary metabolites during the station- ary growth phase, whether considered together or separately. Furthermore, we found that the introduction of high-copy-number plasmids containing the metK1-sp and metK2-sp resulted in 2Æ1- and 1Æ4-fold greater levels of doxorubi- cin production than the control transformants containing only the vector, respectively. We also attempted to disrupt the metK-sp and found that doxoru- bicin production from the metK1-sp-deleted mutant (Streptomyces peuce- tius ⁄ pNN1) was reduced when compared to the parent strain (S. peucetius var. caesius ATCC 27952). Conclusions: The results of this study indicated that two metK are differentially expressed during cell growth, and that the expressions of the two metK genes are differentially regulated under the same conditions. Significance and Impact of the Study: Streptomyces peucetius var. caesius con- tains two genes, metK1-sp and metK2-sp, which encode functional S-adenosyl- l-methionine synthetase (MetK). The degree of homology (90% identity) found between the two genes shows that metK1-sp and metK2-sp are duplicated genes. Although there is currently no evidence for the relationship of the dupli- cated metK genes involved in the regulation of doxorubicin production, metK1-sp and metK2-sp may play a role in controlling the stimulation of anti- biotic production during secondary metabolism. Journal of Applied Microbiology ISSN 1364-5072 398 Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 398–407 ª 2010 The Authors