Biotechnology and Bioprocess Engineering 2008, 13: 752-757 DOI/10.1007/s12257-008-0128-y  Functional Characterization of orf6 and orf9 Genes Involved in the Biosynthesis of L -Oleandrose from Streptomyces antibioticus Tü99 Binod Babu Pageni 1 , Tae-Jin Oh 1 , Jin Cheol Yoo 2 , and Jae Kyung Sohng 1 * 1 Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, Sun Moon University, Asan 336-708, Korea 2 Department of Pharmacy, College of Pharmacy, Chosun University, Gwangju 501-759, Korea Abstract Two genes, orf6 and orf9 located in the L-oleandrose sugar biosynthetic gene cluster of Streptomyces antibioticus Tü99. NovU has been characterized as C-5 methyltrnaferase involved in noviose biosynthetic pathway. We have cloned and het- erologously expressed the orf6, orf9, and novU genes in S. venezuelae YJ003-OTBP1. This established the function of orf6 and orf9 as 4-ketoreductase and 3-epimerase, respectively. All of analytical data of the noviosylated 10-deoxymethynolide also is in support of proving their functions. Furthermore biosynthetic pathway 5,5-gem-dimethyl-6-deoxyglucose (TDP-L- noviose) has been proposed. © KSBB Keywords: deoxysugar biosynthesis, 3-epimerase, 4-ketoreductase, L-noviose, Streptomyces antibioticus Tü99 INTRODUCTION A number of natural products, such as antibiotics, anti- tumors, or immunosuppressive agents, have useful biological activities with wide clinical applications. Bioactive secondary metabolites produced by microorganisms have deoxysugars as one of the essential component which play the key roles for their activity [1] like as in pikromycin [2], oleandomycin [3], and erythromycin [4]. A number of deoxysugar moieties par- ticipate in the molecular recognition of a drug target site and often play crucial roles in determining the biological activity of the parent natural products in secondary metabolites [1,5]. During the past few years, there has been a great increase in the knowledge of biosynthetic pathways of secondary metabo- lites by altering the structure of natural products in polyketide backbone and in deoxysugars through combinatorial biosyn- thesis technique. The aminocoumarin antibiotic novobiocin produced by Streptomyces spheroides NCIB 11891, a potent inhibitor of bacterial gyrase, contains an unusual branched deoxysugar with 5,5-gem-dimethyl structure (noviose sugar), where novWUS sugar genes has been well characterized [6,7]. S. venezuelae ATCC 15439 produces group of macrolactones *Corresponding author Tel: +82-41-530-2246 Fax: +82-41-544-2919 e-mail: sohng@sunmoon@ac.kr including the 12-membered 10-deoxymethynolide obtained from single multifunctional polyketide synthase (pikA). In addition, the pikromycin glycosyltransferase DesVII/DesVIII system, which replaces the native partner with a heterologous activator protein, affects the efficiency of glycosylation of deoxysugar [8] and shows flexibility in accepting different deoxysugars generating novel macrolide derivatives [9-11]. These unique features in generating structural variability can be applied for designing strategies to accomplish hybrid mac- rolide antibiotics by combinatorial biosynthesis. The orf6 (GenBank accession no. AAF59936) and orf9 (GenBank ac- cession no. AAF59931) are located in L-oleandrose biosyn- thetic gene cluster of S. antibioticus Tü99 (Fig. 1A) [12], and their function has been annotated by sequence similarities. However the definitive assignment of their functions has not been verified yet; this is largely (but not exclusively) caused by the unavailability of biosynthetic intermediates and sugar donor cofactors [13]. Therefore, we have cloned and heterolo- gously expressed these genes by in vivo process. In this study, S. venezuelae YJ003-OTBP1 [10] was used as the host bacteria. An expression plasmid pOTBP6 (Fig. 1B) was constructed including L-oleandrose sugar genes, orf6 and orf9, from S. antibioticus Tü99 and novU from S. spheroides [14] which is introduced into S. venezuelae YJ003-OTBP1 to show the definitive assignment of orf6 and orf9. Electrospray ionization-mass spectrometry (ESI-MS), liquid chromatography-mass spectrometry (LC-MS), and