Expression of Novel Salicylate Oxygenase

1
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 
2
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-spectrometr
ic 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.
3
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. 46565-601.)
ISP (ox)
Ferredoxin (red)
Reductase (ox)
NADH H
O2
ISP (red)
Ferredoxin (ox)
Reductase (red)
NAD
4
? 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. 46565-601.)

• 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. 23294-302.)

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Construction of an Expression System for an ISP
Gene(bphA1cA2c) from S. yanoikuyae B1
ATGTCGAACAAATTGCGC
AAGCCCTGGCGTGCTACT
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
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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) ?The culture of E.coli BL21(DE3)
Spectra of 1-ml cleaved samples were recorded
immediately after Inoculation and then after
3,6 and 9 h.

7
Schematic Description of (methyl)Salicylate
Transformation by BphA1CA2C from B1
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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.