Production of L-Dihydroxyphenylalanine in Escherichia coli

AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1993, p. 3070-3075 0099-2240/93/093070-06$02.00/0 Copyright © 1993, American Society for Microbiology

APPLIED

Vol. 59, No. 9

Production of L-Dihydroxyphenylalanine in Escherichia coli with the Tyrosine Phenol-Lyase Gene Cloned from Erwinia herbicola FORREST FOOR,* NANCY MORIN, AND KEITH A. BOSTIANt Merck Research Laboratories, Rahway, New Jersey 07065 Received 4 January 1993/Accepted 23 June 1993

The gene (tut4) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed. The enzyme was expressed at high levels in E. coli in the presence of isopropyl-1-D-thiogalactopyranoside or lactose as an inducer. L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells.

L-Dihydroxyphenylalanine (L-DOPA) is used in the treatment of Parkinson's disease. Although most of the world's supply is synthesized chemically, an efficient microbiological method using the enzyme tyrosine phenol-lyase (TPL; EC 4.1.99.2) from Erwinia herbicola has also been developed (1). TPL normally catalyzes the degradation of tyrosine to pyruvate, phenol, and ammonia. However, this reaction is reversible, and if catechol is substituted for phenol, L-DOPA is produced. Induction of high levels of TPL requires up to 2 g of tyrosine per liter in the growth medium (1, 11). This amount of tyrosine is expensive in large-scale production, and carryover of tyrosine into the reaction mixture complicates the purification of the final product because of the similarity of tyrosine to L-DOPA. We therefore sought a method of inducing high levels of TPL in the absence of

tyrosine. MATERUILS AND METHODS Media. Nitrogen-free glucose minimal salts (NF-GMS) contained 4 g of glucose, 10.5 g of K2HPO4, 4.5 g of KH2PO4, and 50 mg of MgSO4- 7H20 per liter. GN medium was NF-GMS supplemented with 0.2% ammonium sulfate. TPL production medium (PM) has been described previously (5). PM is an enriched medium that contains 0.2% additional tyrosine as an inducer of TPL. In some instances, the additional tyrosine was omitted. Ampicillin (75 jig/ml) or kanamycin (50 ,ug/ml) was routinely included in all media used for the growth of strains containing plasmids carrying a resistance gene for either of these two drugs. Molecular cloning. Chromosomal DNA from E. herbicola ATCC 21434 was isolated as described previously (12), partially digested with Sau3AI, and fractionated by sucrose gradient centrifugation (8). Fragments of 2 to 12 kb were ligated with pUC18 (Bethesda Research Laboratories) which had been linearized with BamHI and treated with calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals). The ligated DNA was transformed into Escherichia coli NM522 (International Biotechnologies, Inc.), and cells from 5 x 104 colonies were pooled. Transformants capable of utilizing tyrosine as the nitrogen source were enriched by three rounds of growth in NF-GMS supple*

Corresponding author.

t Present address: Microcide Pharmaceuticals, Menlo Park, CA

94025.

3070

mented with 0.1% tyrosine. The plasmid pTPL1 was isolated from one of these transformants. Detection of TPL activity. Cells were grown to the stationary phase by inoculating 0.05 ml of a saturated culture into 20 ml of PM and incubating with shaking at 28°C for 24 h. In some instances, cells were harvested during exponential growth in GN. The cell density was monitored with a Klett-Summerson colorimeter fitted with a no. 54 (green) filter. TPL was assayed at 30°C by monitoring the release of pyruvate from tyrosine by measuring the oxidation of NADH in the presence of lactate dehydrogenase (10). CTAB (hexadecyltrimethylammonium bromide, 0.1 mg/ml) was included in the assay mix to permeabilize the cells. The specific activity is expressed as the rate of decrease in NADH (nanomoles per minute) divided by the volume (milliliters) of cell suspension multiplied by the optical density of the suspension at 600 nm. Cells were diluted in growth medium to an optical density of 0.1 to 0.5 to determine the value for the undiluted suspension. With E. coli, the oxidation of NADH was dependent upon the presence of tyrosine and lactate dehydrogenase in the assay mix. With E. herbicola, there was some oxidation in the absence of these components, so the values for oxidation rates without tyrosine were subtracted to derive the TPL activity. Plasmid mutagenesis and sequencing. A 5.8-kb fragment conferring the ability to produce TPL was excised from pTPL1 by digestion withXbaI and SstI and inserted between the sites for the same enzymes in the polylinker of pIBI30 (International Biotechnologies, Inc.), giving pTPL30. The new plasmid was mutagenized with a TnJO-lacZ-kanR gene fusion transposon, mini-TnlO-LK (3) (Fig. 1). pTPL30 and its mutagenized derivatives were used for sequencing. Single-stranded template was prepared by superinfecting E. coli NM522 containing various derivatives of pIBI30 with the kanamycin-resistant helper phage M13K07 (17) and sequenced by the dideoxy chain termination method (14) with Sequenase (U.S. Biochemicals) and [a-35S]thio-dATP. Plasmids containing inserts LK7, LK23, and LK52 were first modified to remove one end of the transposon and inactivate its kanamycin resistance gene by deleting the DNA between the XhoI site in the polylinker of the vector and the XhoI site in the transposon. An oligonucleotide, 5'-CGGATCATAT GACAAGATGTGT-3', the sequence of which was derived from that deduced for nucleotides 51 to 72 from the left end of mini-TnlO-LK, was synthesized with an Applied Biosys-

PRODUCTION OF L-DOPA IN E. COLI

VOL. 59, 1993

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region sequenced FIG. 1. Map of the tut genes in pTPL30 and pTPL31. DNA from E. herbicola is indicated by the long open bar. The locations and orientations (direction of lacZ transcription) of the mini-TnlO-LK inserts are indicated by the flags above the map. Distances are measured from the BamHI site. The MluI, XbaI, BamHI, KpnI, and SstI sites are part of the polylinker-lacZa region of the vector. The polylinker is in opposite orientations relative to the lacZ promoter in pTPL30 and pTPL31. The locations and direction of transcription of tutA and tutB are indicated by the arrows below the map. The 3' end of tutA is located somewhere between the insertion sites of LK102 (tutA) and LK72 (tutA +), as indicated by the dotted portion of the arrow. The region which was sequenced is indicated at the bottom.

tems oligonucleotide synthesizer and used to prime synthesis of labelled DNA starting from the end of the remaining part of each transposon and proceeding into the adjoining region of cloned DNA. Since the primer sequence is located in the inverted repeat of the transposon, the primer could be used with inserts in either orientation. To obtain singlestranded template from the opposite strand, the 5.8-kb fragment from pTPL1 was inserted into pIBI31 (identical to pIBI30 except that the polylinker is reversed), giving pTPL31. Primer M (complementary to bases 314 to 333, Fig. 2) was synthesized for sequencing the opposite strand. The sequence of promoter fusions was confirmed with doublestranded templates and primer M. Synthesis and detection of L-DOPA. The L-DOPA reaction mix (1, 18) contained 330 mM sodium pyruvate, 230 mM catechol, 12 mM ammonium acetate, 240 mM boric acid, 30 mM sodium sulfite, 7 mM disodium EDTA, and 330 mM ammonium chloride and was adjusted to pH 8.5 with ammonium hydroxide. Samples (1.5 ml) were removed at various times, acidified with 15 ,ul of H2SO4, and stored at 4°C. L-DOPA content was determined by high-pressure liquid chromatography (HPLC). Samples were diluted 100-fold with 0.02 N HCI, and 10 1.l was injected. A YMC AQ-302 102 A ODS S-5 column (4.6 [inner diameter] by 150 mm) was LK23 1