Expression of Novel Salicylate Oxygenase Genes from a Sphingomonas Strain in Escherichia coli Ok-young Cho1, Si Wouk Kim2, and Eungbin Kim1 1Department of Biology, Yonsei University 2Department of Environmental Engineering, Chosun University 

Abstract Bacterial aromatic-ring dioxygenase is a normally three-component enzyme system, which consists of a flavoprotein reductase, a ferredoxin and an iron sulfur protein (terminal oxygenase). Sphingomonas yanoikuyae B1 is unique in that it possesses least six different sets of an iron sulfur protein component, which are apparently associated with a single ferredoxin (BphA3) and a reductase (BphA4) components. Previous studies suggested that the gene for one of the multiple oxygenases (bphA1cA2c) is responsible for metabolizing salicylate and 1-hydroxy-2-naphthoate during the degradation of naphthalene and phenanthrene (Kim et al., Abstr. Am. Soc. Microbiol., Q299, p. 470. 1998). To investigate the function of the bphA1cA2c gene in more detail, the genes for bphA1cA2c and bphA3 were amplified by PCR, and cloned together into an expression vector. The Escherichia coli strain harboring the recombinant plasmid converted salicylate, 3‑methylsalicylate, 4‑methylsalicylate, or 5‑methylsalicylate to catechol, 3‑methylcatechol, 4‑methylcatechol, or 4‑methylcatechol, respectively, as confirmed by UV-visible spectral, HPLC, and GC/mass-spectrometric analyses. Furthermore, this expression system was also able to oxidize benzoate, m‑toluate, or 1‑hydroxy‑2‑naphthoate although the metabolite structures have not been identified yet.

Introduction  Aromatic ring dioxygenase - normally a three-component enzyme system :a flavoprotein reductase, ferredoxin , iron sulfur protein (oxygenase) (Harayama and Kok. 1992. Ann. Rev. Microbiol. 46:565-601.) ISP (ox) Reductase (ox) Ferredoxin (red) NADH + H+ O2 ISP (red) NAD Reductase (red) Ferredoxin (ox)

 Salicylate hydroxylase - a member of aromatic ring oxygenase transforming salicylate into catechol - generally, a monomeric flavoprotein with the molecular weight of c.a. 50 kd (Harayama and Kok. 1992. Ann. Rev. Microbiol. 46:565-601.) H2O NADH + H+ NAD+ O2 Prescence of multiple genes for ISPs, but single genes for ferredoxin and reductase, respectively, in Shingomonas yanoikuyae B1. (Kim and Zylstra.1999. J. Indust. Microbiol. Biotechnol. 23:294-302.)

Construction of an Expression System for an ISP Gene(bphA1cA2c) from S. yanoikuyae B1 ATGTCGAACAAATTGCGC AAGCCCTGGCGTGCTACT A3 A2c A1c 1.PCR Amplication 94C for 5 min 94C for 30 sec 55C for 30 sec 72C for 1 min 72C for 7 min 2. Ligation into a pCR T7/CT-TOPO vector 3. Transformation into E.coli BL21 (DE3) 25cycles pKEB1501

Transformation of Salicylate into Catechol by the Expression System The culture of E.coli BL21(DE3) (pKEB 1501)(MSB containging 2.5 mM salicylate, 20 mM glucose, 100 g of ampicillin per ml and 1 mM IPTG) was incubated at 30C with shaking 200rpm) Spectra of 1-ml cleaved samples were recorded immediately after Inoculation and then after 3,6 and 9 h.

Schematic Description of (methyl)Salicylate Transformation by BphA1CA2C from B1  catechol

Conclusion  The genes for one of the multiple ISPs (bphA1cA2c) and a ferredoxin was cloned from S. yanoikuyae B1 and coordinately expressed in E. coli  Metabolites formed from (methyl)salicylates were identified as corresponding catechols by UV-visible spectral, HPLC, and GC/mass-spectrometric analyses.  To our best knowledge, BphA1cA2c is the first three-component oxygenase possessing aromatic-ring monooxygenase activities.