Appl Microbiol Biotechnol (1991) 35:606-610

Applied Microbiology Biotechnology

017575989100189L

© Springer-Verlag 1991

Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from KlebsieUa aerogenes Hiroyuki Sugino, Kaname Ishibashi, Masashi Sakaue, Mitsuo Yamashita, and Yoshikatsu Murooka Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama 1, Higashi-Hiroshima 724, Japan Received 6 March 1991/Accepted 2 May 1991

Summary. We cloned the structural gene for monoamine oxidase (maoA) from Klebsiella aerogenes into a pKI212 vector in an maoA mutant strain of K. aerogenes. Deletion analysis and complementation tests o f the recombinant plasmid showed that the maoA gene was located entirely within a 4.1-kb segment. In an maoA mutant strain harbouring the cloned maoA gene, synthesis of monoamine oxidase was induced by addition of tyramine and related compounds. Transfer of a plasmid containing the maoA gene into a monoamine oxidase-producing strain of K. aerogenes W70 resulted in about a 30- to 40-fold increase in total production of the enzyme. When cells of K. aerogenes carrying the plasmid containing the maoA gene were grown with tyramine, more than 85% of the monoamine oxidase was produced in soluble form, whereas the parent strain W70 produced most monoamine oxidase as the membrane-bound form.

However, tyramine oxidase from K. aerogenes could not be characterized, since the enzyme bound tightly to the cell membrane and failed to solubilize in an active form (Okamura et al. 1976). Cloning of the gene for tyramine oxidase from K. aerogenes resulted in overproduction of the enzyme, thus making its purification and characterization possible (Sakaue et al. 1990). We found that the enzyme was highly specific for tryptamine and histamine in addition to tyramine and catecholamines. Thus, we classified the Klebsiella enzyme as a monoamine oxidase instead of tyramine oxidase, and changed the genotype name of the corresponding gene from tynA to maoA In this paper, we report on the cloning o f the chromosomal fragment containing the maoA gene into a multicopy plasmid, and demonstrate the high productivity o f the soluble form of monoamine oxidase in K. aerogenes.

Materials and methods Introduction In bacteria, monoamine oxidase (EC 1.4.3.4), which is found in the cell membrane, is induced by tyramine and catecholamines and is specific for tyramine, octopamine, dopamine, and norepinephrine (Murooka et al. 1979; Okamura et al. 1976). Thus, the enzyme has been classified as a tyramine oxidase (EC 1.4.3.9) (Yamada et al. 1971). Tyramine oxidase oxidizes tyramine and catecholamines to hydroxyphenylacetaldehyde compounds and ammonium ions. Tyramine oxidase in Klebsiella aerogenes is specified by the tynA gene and is subject to catabolite and ammonium repression (Oka et al. 1980; Okamura et al. 1976). Expression of the tynA gene resulted in derepression of the ats operon, which is closely linked to tynA (Murooka et al. 1977a, 1978) and is composed of the arylsulphatase (atsA) gene and the positive regulator, atsB gene (Murooka et al. 1990).

Offprint requests to: Y. Murooka

Bacterial strains and plasmids. The bacterial strains used in this study were K. aerogenes W70 (prototroph, MacPhee et al. 1969), a strain producing monoamine oxidase (Okamura et al. 1976) and its derivative strains MKN63 (Okamura et al. 1977) and MKN329 (Murooka et al. 1983), deficient in monoamine oxidase (maoA) and in maoA ats recA, respectively. Plasmid pKI212, conferring resistance to kanamycin (Kmr), was constructed previously (Murooka et al. 1990). Other recombinant plasmids were derivatives of pKI212. Enzymes and chemicals. Restriction endonucleases, T4 DNA ligase, and Escherichia coli alkaline phosphatase were purchased from Takarashuzo (Kyoto, Japan) or Toyobo (Osaka, Japan). [3H]-Tyramine hydrochloride (20 Ci/mmol) was purchased from New England Nuclear Research/Du Pont, (Boston, Mass., USA). The other compounds used were standard commercial preparations. Transformation ofK. aerogenes. A modification of the method described by Davis et al. (1980) was used for transformation of K. aerogenes Mao - (Tyn-) cells. The cells were treated with 100 mM CaC12, 50 mM RbC1, and 25 mM LiC1 in 100 mM 3-(N-morpholi-

607 no)propanesulphonate (pH 6.5). After incubation for 1 h at 0 ° C, the cells were treated at 42°C for 3 min and then washed twice with saline to remove excess metal ions.

0 I

1 I

2 I

B

E

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Cloning of the mao gene. Preparation of plasmid and DNA, restriction endonuclease digestion, ligation, and agarose and polyacrylamide gel electrophoresis were performed according to Maniatis et al. (1982). Chromosomal DNA prepared from K. aerogenes W70 by the method of Marmur (1961) was partially digested with BamHI or EcoRI. Fragments of between 7 kb and 20 kb of chromosomal DNA were isolated by sucrose density gradient centrifugation and ligated to BamHI-cleaved or EcoRIcleaved pKI212 with T4 DNA ligase, after treatment with alkaline phosphatase. Transformants were selected on LB (1% polypeptone, 0.5% yeast extract, 0.5% NaCi) agar plates containing 50 Ixg kanamycin/ml. Km r colonies were replicated onto K medium (Murooka et al. 1981) with 0.5% xylose and 10 mM tyramine as carbon and nitrogen sources, respectively. Mao + transformants, which utilize tyramine as a nitrogen source, were selected.

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4 I

5 I

6 I

7 I

8 I

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KB Phenotype

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Fig. 1. Deletion analysis of the maoA gene. Deletion plasmids were transferred to K. aerogenes MKN63 and assayed for monoamine oxidase activity. The thick line indicates the region essential for expressing the Mao + phenotype; dotted lines show the pKI212 sequence; B, BamHI; C, ClaI; E, EcoRI; S, SalI; Sm,

Sinai

Assay of monoamine oxidase activity. Bacterial cells were grown aerobically at 28°C in K medium. Unless otherwise stated, 0.5% xylose for K. aerogenes (Adachi et al. 1974) and NI-hC1 were used as sources of carbon and nitrogen, respectively. Tyramine (3 mM) was used as an inducer of monoamine oxidase. Growth was followed in a Klett-Summerson colorimeter. Monoamine oxidase activity was assayed by a radiometric procedure with [3H]tyramine as described previously (Okamura et al. 1976) or a colorimetric assay coupled with peroxidase and o-phenylenediamine (lwata et al. 1990). One unit (U) of monoamine oxidase activity was defined as the amount producing 1 Ixmol hydrogen peroxide per minute at 30 ° C.

1. Level of tyramine oxidase in strains of Klebsiella aerogenes, with or without plasmid pTO58, induced by various mon-

Table

oamine compounds a Strain (genotype)

W70 (wild-type)

Plasmid (pTO58)

-

-

Preparation of subcellularfractions. Subcellular fractionation was

-

performed as described previously (Murooka et al. 1978).

-

Results

Cloning o f the monoamine oxidase gene from a chromosome o f K. aerogenes C h r o m o s o m a l D N A f r o m K. aerogenes W70 was partially digested with B a m H I or EcoRI. The fragments were ligated with BamHI-digested or EcoRI-digested p K I 2 1 2 . T h e mixture o f hybrid D N A molecules was used to t r a n s f o r m K. aerogenes M K N 3 2 9 (maoA atsA recA). The K m r t r a n s f o r m a n t s were replicated o n t o xylose-tyramine agar plates. A m o n g a b o u t 5000 colonies tested, o n e M a o + c o l o n y was o b t a i n e d b y using BarnH I - d i g e s t e d fragments, but n o n e were o b t a i n e d f r o m the EcoRI-digested fragments. The M a o + c o l o n y grew o n t y r a m i n e or c a t e c h o l a m i n e s as the sole source o f nitrogen, but the recipient strain M K N 3 2 9 did not. T h e p l a s m i d D N A p r e p a r e d f r o m the M a o + strain h a d an extra B a m H I f r a g m e n t a n d was estimated to be a b o u t 14 kb in length. The p l a s m i d was n a m e d pTO58. A restriction m a p o f the c l o n e d c h r o m o s o m a l fragm e n t was c o n s t r u c t e d (Fig. 1). The size o f the c h r o m o s o m a l f r a g m e n t was estimated to be a b o u t 8.7 kb. T h e t r a n s f o r m a t i o n o f K. aerogenes M K N 3 2 9 (maoA343) or M K N 6 3 (maoA63) with p T O 5 8 yielded 100% K m r M a o ÷ colonies, but those strains t r a n s f o r m e d with p K I 2 1 2 did n o t yield any, indicating that the ability to p r o d u c e m o n o a m i n e oxidase was associated with the r e c o m b i n a n t plasmid.

MKN63 (maoA63) + + + + +

Monoamine compound (3 mM)

-

Tyramine Octopamine Dopamine Norepinephrine Tyramine Tyramine Octopamine Dopamine Norepinephrine

Monoamine oxidase activity (mU/mg of cells)