Biotechnology and Bioprocess Engineering 17: 8-15 (2012) DOI 10.1007/s12257-011-0310-5 Functional Analysis of ABC Transporter Genes pdmR1 and pdmR2 in Actinomadura hibisca P-1752 and Enhancement of Pradimicin Production Sarita Paudel, Je Won Park, Joo Ho Lee, Yeo Joon Yoon, and Jae Kyung Sohng Received: 13 July 2011 / Revised: 28 August 2011 / Accepted: 29 August 2011 © The Korean Society for Biotechnology and Bioengineering and Springer 2012 Abstract Two putative ABC transporter genes pdmR1 and pdmR2 are found in the biosynthetic gene cluster of pradimicin. pdmR1 showed a hydrophilic profile with similarity to the daunorubicin-resistant ABC transporter ATP-binding protein family, a characteristic ABC transporter. pdmR2 codes for hydrophobic polypeptides showing a high degree of similarity to several ABC-type multidrug transport systems. To assess the possible roles of these genes in pradimicin biosynthesis, pdmR1 and pdmR2 were introduced into Actinomadura hibisca P-1752. The re- combinant strains Actinomadura hibisca SP1, SP2, and SP3 showed longer life spans compared to the wild-type A. hibisca. Pradimicin production was increased by 3.5, 2.4, and 5.4-fold at 8 days in A. hibisca SP1, SP2 and SP3, respectively, compared to that of parental strain. The higher transcriptional level of pdmR1 and pdmR2 genes in the pdmR1 and pdmR2 harboring strains compared to the wild type was consistent with the enhanced production. The plasmids pSP1, pSP2, and pSP3 were expressed in Strepto- myces venezulae and S. lividians, which are sensitive to pradimicin. The transformants acquired higher resistance to pradimicin than the wild-type Streptomyces strains suggest- ing that pdmR1 and pdmR2 acts as a set of resistance genes in A. hibisca to pump out pradimicin produced throughout the cell. Keywords: Actinomadura hibisca, ABC transporter genes, pradimicin 1. Introduction Actinomycetes are producers of approximately three-quarters of all known antibiotics. This prolific group of Gram positive, mycelial, sporulating bacteria has developed specific resistance mechanisms to facilitate its survival during production of the potentially toxic compounds [1]. Self-resistance is an important requirement for antibiotic- producing microorganisms and is mediated by (i) drug inactivation, (ii) target site modification, and (iii) reduction of the intracellular concentration via pumping-out and sequestration of drug by the formation of a protein-drug complex [2]. The last resistance mechanism is designated “membrane associated system” consisting of two classes. In one class, resistance is mediated by membrane proteins that are believed to energize export of antibiotic molecules by proton-dependent transmembrane electrochemical gradi- ents. The other class belongs to the ABC transporter super- family that comprises many membrane-associated export and import systems, which are present in both prokaryotic and eukaryotic cells [3]. ABC transporters share a common domain organization: transmembrane domains (TMDs) and nucleotide binding domains (NBDs, also called ABC ATP-binding cassette) [4]. Most prokaryotic ABC transporters consist of TMD and NBD polypeptides, which form a half transporter that then dimerizes to become a functional transporter [5,6]. Presence of highly conserved ATP-binding cassette is the most important characteristic feature of the ABC trans- porters superfamily. They participate in the secretion of Sarita Paudel, Je Won Park, Joo Ho Lee, Jae Kyung Sohng * Institute of Biomolecule Reconstruction (iBR), Department of Pharm- aceutical Engineering, SunMoon University, Asan 336-708, Korea Tel: +82-41-530-2246; Fax: +82-41-544-2919 E-mail: sohng@sunmoon.ac.kr Yeo Joon Yoon Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea RESEARCH PAPER