J. Microbiol. Biotechnol. (2003), 13(6), 906–911 Functional Analysis of Spectinomycin Biosynthetic Genes from Streptomyces spectabilis ATCC 27741 JO, YOU-YOUNG1, SUN-HEE KIM1, YOUNG-YELL YANG1, CHOONG-MIN KANG2, 3 1 JAE-KYUNG SOHNG , AND JOO-WON SUH 1Department of Biological Science, Institute of Bioscience and Biotechnology, Myong Ji University, Yongin 449-728, Korea 2Childrens Hospital, Harvard Medical School, Boston, MA 02467, U.S.A. 3Department of Chemistry, SunMoon University, Asansi, Chungnam 337-840, Korea Received: May 23, 2003 Accepted: July 15, 2003 Abstract The function of genes related to spectinomycin Spectinomycin is an aminoglycoside antibiotic produced biosynthesis (spcD, speA, speB, spcS2) from Streptomyces by several Streptomyces species such as Streptomyces spectabilis ATCC 27741, a spectinomycin producer, was analyzed. flavopersicus [17], Streptomyces hygroscopicus [31], and Each gene was subcloned from a spectinomycin biosynthetic Streptomyces spectabilis [15]. The antibiotic has a unique gene cluster and overexpressed in E. coli BL21 (DE3) using tricyclic structure in which a single sugar component, pET vector. After incubating each purified protein with its actinospectose, is linked to the diaminocyclitol moiety possible substrates, the final products were analyzed using (actinamine) by β-glycosidic and hemiketal bonds [4, 30]. high-performance liquid chromatography (HPLC). From these It blocks the translocation step of protein synthesis by results, spcD, speA, and speB have been identified to be inhibiting the binding of the elongation factor G to the dTDP-glucose synthase, myo-inositol monophosphatase, and ribosome [3]. It possesses a broad spectrum of activities myo-inositol dehydrogenase, respectively. In addition, the against many Gram-positive as well as Gram-negative results suggest that the spcS2 gene product functions downstream bacteria [29]. Specifically, it possesses potent activity of the speB gene product in the biosynthetic pathway of against Neisseria gonorrhoeae, the gonorrhoea bacteria spectinomycin. Taken together, the present study elucidates that have acquired resistance to penicillin and may be an the early steps of the biosynthetic pathway for 6-deoxyhexose alternate drug in such cases or for patients allergic to β- (6-DOH) part (actinospectose) and aminocyclitol part (actinamine) lactams [20]. However, the whole biosynthetic pathway of spectinomycin. for spectinomycin has not yet been elucidated, although Key words: Spectinomycin biosynthetic gene cluster, dTDP- there have been reports on the glycosylation process of glucose synthase, myo-inositol monophosphatase, myo-inositol the two subunits of spectinomycin [16] and the partial dehydrogenase biochemical pathway using cell-free extract and labeling [5, 28]. Therefore, to study the spectinomycin biosynthetic pathways, a 45 kb DNA fragment containing the genes required for Many useful antibiotics from actinomycetes (macrolides, spectinomycin biosynthesis was isolated from Streptomyces β peptide antibiotics, -lactams, etc.) have been studied by spectabilis ATCC 27741, as previously described [7], and cloning their biosynthetic genes, analyzing the regulatory this study focuses on the functional analysis of the genes mechanisms, and creating recombinant antibiotics. However, participating in the early steps of the biosynthetic pathway due to the difficulties in purification and chemical synthesis, of each spectinomycin subunit using biochemical methods, research on biosynthesis and genetic engineering of since a stable transformation system is not available for S. aminoglycoside antibiotics has been very slow, until recently spectabilis. when the biosynthetic gene clusters for streptomycin, Objectives of this study were to investigate whether 1) spectinomycin, bluensomycin, kasugamycin, and butirosin spcD encodes dTDP-glucose synthase, 2) speA encodes were discovered [1,7,8,9,14,18,26]. myo-inositol monophosphatase, and 3) SpeB can convert myo-inositol to scyllo-inosose. Furthermore, it was found *Corresponding author Phone: 82-31-330-6190; Fax: 82-31-335-8249; that spcS2 plays a role following speB in the biosynthetic E-mail: [email protected] pathway of spectinomycin. FUNCTIONAL ANALYSIS OF SPECTINOMYCIN BIOSYNTHETIC GENES 907 MATERIALS AND METHODS pETspcS2 were grown in LB medium supplemented with µ - 1 o 50 g ml ampicillin at 37 C until OD600 of 0.6 was reached, Bacterial Strains then induced with 1 mM isopropyl β-D-thiogalactoside Spectinomycin biosynthetic genes were isolated from (IPTG), and allowed to grow at 28oC for 3 additional S. spectabilis ATCC 27741, a spectinomycin producer. hours. pETspeA and pETspeB were grown in LB medium α - + µ - 1 o E. coli DH5 F' [F'/endA1 hsdR17 (rKmK ) supE44thi-1 supplemented with 50 g ml kanamycin at 37 C until r ∆ φ recA1 gyrA (Nal ) relA1 (lacZYA-argF) U169 ( 80dlac OD600 of 0.6 was reached, then induced with 1 mM IPTG ∆(lacZ)M15)] was used as a host strain for transformation and allowed to grow at 37oC for 3 additional hours [25]. of plasmid harboring spectinomycin biosynthetic genes. E. The cells were harvested by centrifugation and resuspended - - + coli BL21 (DE3) [F , dcm, ompT, hsdS (rBmB ), gal] (ATCC in the binding buffer (5 mM imidazole, 0.5 mM NaCl, 47092) was used to overexpress the transformants. The 20 mM Tris-HCl, pH 7.9). Resuspended cells were lysed pET21a plasmid (Novagen, Germany) was used to express by sonication (Branson sonifer 450 sonicator), and cell debris spcD and spcS2 genes, and pET28a (Novagen, Germany) was removed by centrifugation [10]. Each soluble protein was used for speA and speB. All E. coli cells were grown was purified with a single nickel-affinity chromatography by on LB agar or broth media supplemented with 50 µg ml- 1 using HisBind resin (Novagen, Germany). Protein concentration ampicillin or kanamycin (Sigma-Aldrich corp., U.S.A.). was determined by the Bradford [2] method, and the molecular weights were determined by SDS-PAGE [11]. General Methods General procedures for manipulating DNA were carried Enzymatic Assay for SpcD and SpeA out according to Sambrook et al. [24]. DNA was isolated For the functional analysis of SpcD, a reaction mixture from agarose gels (BentechBio Co., Ltd. Korea) with the (300 µl) containing 50 mM Tris-HCl (pH 8.0), 12 mM Qiagen kit (Chartworth, U.S.A.). Plasmid was purified MgCl2, 6 mM dTTP (deoxythymidyltriphosphate), 24 mM using a Wizard plasmid kit (Promega, U.S.A.) following D-glucose-1-phosphate, 1.8 U of inorganic pyrophosphatase, the manufacturer’s instructions. and 1 µg of purified SpcD was incubated at 37oC for 0, 30, or 60 min, then the reaction was terminated by adding Cloning of spcD and speA Genes 50 mM potassium phosphate (pH 3.0) [12]. The reaction PCR Amplification. For the functional analysis of spcD, product was analyzed by HPLC with a N(CH3)2-1101-N speA, speB, and spcS2 gene products, each gene was column (0.46×10 cm) using a stepwise gradient of 50 to amplified using PCR from the cosmid template containing 600 mM potassium phosphate buffer (pH 4.0) at 30oC. The the spectinomycin biosynthetic cluster [7]. Oligoprimers flow-rate was 1.5 ml min- 1, and the products peaks were included the restriction enzyme sites (underlined) to facilitate detected by a UV detector at 254 nm. Standard reagents, subcloning. The sequences of each primer used in the dTTP, and dTDP-glucose were purchased from Sigma- experiments were as follows; for spcD gene, forward primer Aldrich Corp (U.S.A.). To determine the substrate specificity was 5'-GCCGAATTCATGCG CGGAATCATACTT-3'; and of SpcD, dCTP, dATP, and dGTP were tested as substrates reverse primer was 5'-ATTCTCGAGCAACAGCGCCTC- and the products from the reaction were analyzed by the CTCGTC-3', for speA gene, forward primer was 5'-GCC- method described above. GAATTCATGTCTTTGGCACATGCC-3' and reverse primer For the functional analysis of SpeA, a reaction mixture µ was 5'-TTCTCGAGGGCATGGCCCACCAG-3'. For speB (300 l) containing 20 mM Tris-HCl (pH 7.5), 3 mM MgCl2, gene, forward primer was 5'-GCCGAATTCGTGCAGAA- 0.7 mM myo-inositol-1-phosphate, and 10 mg SpeA cell AGAACGTGTC-3' and reverse primer was 5'-ATTCTC- extract was incubated at 37oC for 0, 30, or 60 min, and the GAGCATTCCTTTGAAGTT-3'. For spcS2 gene, forward reaction was then terminated by freezing at -20oC [13]. As primer was 5'-ATTGAATTCATGTCAGCGGTACGCAGT- a control experiment to eliminate the effect of the E. coli’s 3' and reverse primer was 5'-ATTAAGCTTTGCCCGG- primary metabolism of myo-inositol monophosphatase, the cell CCCCTCTC-3'. PCR reactions were performed using the extract from E. coli cells harboring only the vector plasmid EX Taq DNA polymerase (Takara, Japan). The genes was reacted instead of the SpeA cell extract. The reaction TM amplified by PCR were sequenced to confirm the absence product was analyzed by HPLC with a mBondapak C18 of mutation during PCR reactions. column (0.46×10 cm) using 20% acetonitrile. The flow-rate Construction, Overexpression, and Purification of was 1.0 ml min- 1, and the product peaks were detected by a Recombinant Proteins. Each PCR-amplified spcD and UV detector at 254 nm. The standard reagent used was the spcS2 gene was cloned into the E. coli expression vector commercial myo-inositol (Sigma-Aldrich Corp., U.S.A.). pET21a, and speA and speB genes were cloned into pET28a to give pETspcD, pETspcS2, pETspeA, and pETspeB, Enzymatic Assay for SpeB and SpcS2 respectively. These plasmids were transformed into E. coli For the functional analysis of SpeB, a reaction mixture BL21