Inactivation, Complementation, and Heterologous Expression ofencP, a Novel Bacterial Phenylalanine Ammonia-Lyase Gene*

The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination ofl-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the “Streptomyces maritimus” enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.

The enzyme phenylalanine ammonia-lyase, which catalyzes the nonoxidative deamination of L-phenylalanine to trans-cinnamic acid, is ubiquitously distributed in plants. We now report its characterization for the first time in a bacterium. The phenylalanine ammonia-lyase homologous gene encP from the "Streptomyces maritimus" enterocin biosynthetic gene cluster was functionally characterized and shown to encode the first enzyme in the pathway to the enterocin polyketide synthase starter unit benzoyl-coenzyme A. The disruption of the encP gene completely inhibited the production of cinnamate and enterocin, whereas complementation of the mutant with benzoyl-coenzyme A pathway intermediates or with the wild-type gene encP restored the formation of the benzoate-primed polyketide antibiotic enterocin. Heterologous expression of the encP gene under the control of the ermE* promoter in Streptomyces coelicolor furthermore led to the production of cinnamic acid in the fermented cultures, confirming that the encP gene indeed encodes a novel bacterial phenylalanine ammonia-lyase.
Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) 1 is a ubiquitous higher plant enzyme that catalyzes the nonoxidative deamination of the primary amino acid L-phenylalanine to trans-cinnamic acid. Its product is the precursor of several important classes of plant phenylpropanoids including lignins, flavonoids, and coumarins. These cinnamate-derived natural products are relatively nonexistent in bacteria; however, because of the rarity of PAL in prokaryotes.
Bacteria and animals do carry out a similar enzymatic reaction, the conversion of L-histidine to trans-urocanic acid by the highly homologous histidine ammonia-lyase (HAL, EC 4.3.1.3). The recent crystal structure of HAL from Pseudomonas putida (1) along with numerous biochemical studies on both enzymes (2, 3) established the reaction mechanism of this novel deamination reaction. The structure revealed that the novel prosthetic group 4-methylidene imidazol-5-one (4,5), which is formed autocatalytically from the cyclization and dehydration of the active site tripeptide Ala-Ser-Gly, serves as the essential catalytic electrophile of this Friedel-Crafts-type enzymatic reaction (6). Although HALs and PALs have analogous mechanisms of action, PALs are considerably larger homotetrameric proteins (312 versus 215 kDa), suggesting that these related enzymes evolved independently.
We recently cloned and sequenced the putative PAL gene encP, which is associated with the enterocin biosynthetic gene cluster, from the sediment-derived bacterium "Streptomyces maritimus" (7,8). 2 Although the product of encP is more homologous in sequence and size to bacterial HALs than to plant PALs, circumstantial evidence suggested that EncP functions as a PAL. The bacteriostatic agent enterocin is biosynthesized by a unique type II polyketide synthase system that utilizes a novel cinnamate-derived benzoyl-coenzyme A (CoA) starter unit ( Fig. 1) (9). A sequence analysis of the 20 open reading frame enc cluster and feeding experiments with labeled precursors demonstrated that benzoyl-CoA is produced in this bacterium in a plantlike manner from phenylalanine via cinnamic acid followed by ␤-oxidation (10,11).
To functionally assign encP and its role in benzoyl-CoA biosynthesis in "S. maritimus," we developed a genetics system in this bacterium. Here, we report the inactivation, complementation, and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase-encoding gene.

MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Culture Conditions-All of the strains and plasmids used in this work are listed in Table I. "S. maritimus" strain BD26T was grown as described previously (8). A1 medium was used for sporulation, and R2YE medium was used for isolation of genomic DNA. Mutant strains were grown at 37°C on A1 plates containing 100 g/ml apramycin for ϳ24 -30 h until sporulation, whereas the complemented mutant strains were similarly grown with added thiostrepton (50 g/ml). Escherichia coli XL1-Blue was used for subcloning and grown on LB plates or in LB liquid medium. E. coli S17-1 was used as the host for E. coli-"S. maritimus" conjugation (12).
DNA Manipulations-"S. maritimus" total genomic DNA was isolated as described previously (7). Recombinant DNA procedures were performed by standard techniques (13,14). Biotin labeling and detection of chemiluminescent positives were performed with the DNA-Detector TM HPR Southern blotting kit (KPL, Inc.). Oligonucleotides were obtained from Sigma Genosys. PCR was carried out on a PTC-2000 thermal cycler (MJ Research) with Taq (Invitrogen) or PfuTurbo (Stratagene) DNA polymerase. DNA sequencing by BigDye terminator cycle sequencing reaction using an ABI 377 sequencer was performed at the Laboratory of Molecular Systematics and Evolution at the University of Arizona. The gene sequence of the enterocin biosynthetic gene cluster including encP has been deposited at GenBank under accession number AF254925. * This work was supported by National Institutes of Health AI47818 for generous support. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF254925 and AAF81735.
Construction, Analysis, and Complementation of the encP Mutant KP-The encP disruption vector pBM4 was constructed as follows. A 800-bp internal fragment of encP was PCR-amplified from pJP15F11 with the primers 5Ј-GGTCCACGCGCCGGTTGGCG-3Ј and 5Ј-GCTG-GCGCGCGGGAACTCCG-3Ј and ligated into pCR2.1-TOPO. The EcoRI fragment from the resulting plasmid was cloned into pKC1139 to create pBM4, which was conjugated into "S. maritimus" as described previously (15). The encP single crossover mutant KP was selected after propagating trans-conjugants on SGGP (16) plates at 37°C, and apramycin-resistant colonies were confirmed by Southern hybridization with biotinylated encP.
To facilitate the genetic complementation of the encP mutant, we constructed the conjugative expression plasmid pBM6 by first inserting the 0.8-kb PstI oriT fragment from pOJ446 into a unique PstI site of pWHM3 to create pBM5 followed by ligation of a 0.4-kb EcoRI-XbaI ermE* promoter DNA fragment. The gene encP was PCR-amplified, blunt end-cloned into pCR2.1-Blunt to yield pBM7, and cloned into the BamHI-XbaI cloning site of pBM6 to create pBM8. The sequence of the 1.679-kb encP PCR product was confirmed by sequencing. The primers for the amplification of encP were 5Ј-GTCTAGATGGTGTGTCTCCCT-TCCAG-3Ј (reverse) and 5Ј-GACTTAATTGGCATGGGCAGACG-GTCTG-3Ј (forward). Plasmids pBM6 (negative control) and pBM8 were introduced into "S. maritimus" KP using the plasmid conjugal transfer method. Transformants were directly selected on A1 plates containing 100 g/ml apramycin and 50 g/ml thiostrepton at 37°C.

Production of Cinnamic Acid in Streptomyces coelicolor-Plasmids
pBM6 and pBM8 were introduced into S. coelicolor A3 (2) and S. coelicolor YU105 by protoplast transformation as described previously (14). The nonmethylating E. coli strain ET12567 (17) was used to obtain DNA for transformation of S. coelicolor. The strains were grown on R2YE agar plates containing 20 g/ml thiostrepton at 30°C for up to 7 days. The cultured agar was chopped and extracted with 95:5 EtOAc:MeOH, and the dried crude extract was analyzed by HPLC as described above.

RESULTS
DNA Sequence Analysis of encP-The encP sequence starting with a methionine start codon encodes a 523-amino acid protein and is preceded by a putative ribosome binding site (5Ј-AGGGA-3Ј) at Ϫ10 to Ϫ6. EncP shows greater sequence homology to prokaryotic HALs than to eukaryotic PALs and contains a conserved motif around Ser-143 (Fig. 2), which is the probable precursor of the modified dehydroalanine residue in the 4-methylidone imidazol-5-one prosthetic group (1). Although most HALs and PALs contain either an alanine or cysteine residue adjacent to the active site serine, EncP instead uniquely harbors a threonine residue at this position. Most other HAL active site residues are conserved in EncP. However, a notable exception is Val-83, which in HALs is a conserved histidine residue that coordinates its imidazole group through a hydrogen bond with that of the bound histidine substrate (3). On the other hand, plant PALs carry aliphatic residues such as valine and isoleucine at this position, which is consistent with that of EncP, to provide a hydrophobic environment for the benzene ring of the substrate phenylalanine.
Construction and Complementation of the encP Knock-out Mutant "S. maritimus" KP-To establish the in vivo function of the EncP gene product, we developed a genetics system in "S. maritimus" and disrupted encP by single crossover homologous recombination. The pKC1139-based temperature-sensitive plasmid pBM4 was constructed with an internal 0.8-kb fragment of the targeted encP gene. The conjugal transfer of pBM4 from E. coli to "S. maritimus" and growth of the resulting trans-conjugant under selective conditions resulted in the mutant strain KP (Fig. 3). The single crossover event resulted in the tandem duplication of truncated encP genes with vector containing an apramycin resistance gene between the sequences. Southern blot hybridization of genomic DNA from the wild type and mutant with a biotinylated DNA probe carrying encP verified the gene disruption. Predicted band shifts were detected in SphI digests of the total DNA. "S. maritimus" KP did not exhibit any different phenotypes in comparison with the wild-type strain in A1 medium at 37°C.
HPLC analysis of an organic extract from the encP-inactivated strain "S. maritimus" KP demonstrated that cinnamic acid and the benzoate-primed polyketides enterocin and the  wailupemycins were not produced (Fig. 4). Upon supplementation of cinnamate and benzoate to the culture medium, enterocin production was restored albeit at 5 and 14%, respectively, of wild-type levels. The administration of d 5 -labeled benzoic acid resulted in no dilution of the deuterium label in the resultant enterocin as detected by HPLC mass spectrometry, verifying the complete abolishment of benzoate biosynthesis in the mutant strain. The encP mutant strain KP was next complemented with the wild-type encP gene to measure the level of pathway restoration. The pWHM3-based E. coli streptomycete expression vector pBM8 was constructed with wild-type encP under the strong constitutive ermE* promoter with oriT to allow conjugation from E. coli. The complementation of mutant KP with pBM8 resulted in the biosynthesis of cinnamic acid and the complete restoration of enterocin production to wild-type levels (Fig. 4).
Heterologous Expression of encP and Characterization of Cinnamic Acid Production in S. coelicolor-The pWHM3-based plasmid pBM8 containing the gene encP was heterologously expressed in S. coelicolor wild-type strain A3 (2) and the genetically engineered S. coelicolor host YU105. In each case, organic extracts of the transformants were analyzed for the production of cinnamic acid by HPLC using commercial cinnamic acid as a reference standard. As with "S. maritimus" KP, the expression of the gene encP resulted in the in vivo produc-tion of cinnamic acid in the two S. coelicolor strains. Cinnamic acid biosynthesis was greater in the mutant strain YU105 than in the wild-type strain A3 (2) (100 versus 40 g/20 ml R2YE agar plate). This result verified that the encP gene product is a bona fide phenylalanine ammonia-lyase and demonstrated for the first time the engineered biosynthesis of cinnamic acid in a bacterium.

DISCUSSION
Our data clearly show that the "S. maritimus" gene encP, which is associated with the enterocin biosynthesis gene cluster, encodes a rare bacterial phenylalanine ammonia-lyase whose product cinnamic acid is converted to benzoyl-CoA during the biosynthesis of the polyketide antibiotic enterocin. Although ubiquitous in plants and found in some fungi, PAL activity has only once before been identified in a bacterium where it appears to catalyze the first step in the biosynthesis of cinnamamide in the actinomycete Streptomyces verticillatus (18). A partially purified enzyme with an estimated molecular mass of 226 kDa was shown to behave similarly to PALs isolated from plants and fungi (19). More recently, a bacterial tyrosine ammonia-lyase from Rhodobacter capsulatus was characterized and shown to be 150 times more catalytically efficient toward L-tyrosine than L-phenylalanine as the substrate (20). Like EncP, there are two amino acid substitutions is not drawn to scale. B, Southern analysis of SphI-digested genomic DNA from "S. maritimus" wild-type (wt) and mutant KP with biotinylated 0.8-kb encP fragment from pBM4 as probe.
in the conserved active site residues in the R. capsulatus tyrosine ammonia-lyase when compared with HAL enzymes, His-83 to Leu and Glu-414 to Gln (Fig. 2). A biochemical analysis of all aromatic amino acid ammonia-lyases to date reveals that these proteins are homotetramers and that the prokaryotic enzymes are considerably smaller than their eukaryotic counterparts (Fig. 2).
Downstream of the disrupted encP gene in mutant KP and separated by 26 nucleotides is the methionine start codon of encO. As this gene may be co-transcribed with encP, insertional inactivation of encP via a single crossover may have given rise to polar effects on the expression of encO. The results from the complementation and expression experiments suggest that EncO does not serve a biosynthetic role in the EncP-catalyzed conversion of L-phenylalanine to trans-cinnamic acid. The inability of cinnamate and benzoate supplementation to fully restore wild-type levels in the encP mutant rather suggests that encO may serve a regulatory role in either cinnamic acid or enterocin biosynthesis. Preliminary data on the inactivation and overexpression of encO indicate that cinnamic acid and enterocin production is dramatically reduced. 2 The gene encO may exert feedback control on encP whose product funnels the proteinogenic amino acid L-phenylalanine to the dedicated secondary metabolic pathway to polyketide antibiotic enterocin. Thus, encO may represent a novel regulatory gene as its putative 130 amino acid gene product does not resemble any protein in the databases. We are currently examining the in vivo and in vitro function of this novel gene.
In summary, we have characterized the first phenylalanine ammonia-lyase-encoding gene from a bacterium whose protein product is more homologous in sequence and size to bacterial HALs than to plant PALs. The enzymatic product of the "S. maritimus" PAL EncP is trans-cinnamic acid, which is the first dedicated pathway intermediate to the enc polyketide synthase primer unit benzoyl-CoA.