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Journal of Bacteriology, January 2003, p . 399-404, Vol . 185, No . 2

Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium "Streptomyces maritimus"

Longkuan Xiang1 and Bradley S . Moore1,2*

College of Pharmacy the,1 Department of Chemistry, University of Arizona, Tucson, Arizona 857212

Received 26 June 2002/ Accepted 6 October 2002


   ABSTRACT

 
The novel benzoyl coenzyme A (benzoyl-CoA) biosynthesis pathway in "Streptomyces maritimus" was investigated through a series of target-directed mutations . Genes involved in benzoyl-CoA formation were disrupted through single-crossover homologous recombination, and the resulting mutants were analyzed for their ability to biosynthesize the benzoyl-CoA-primed polyketide antibiotic enterocin . Inactivation of the unique phenylalanine ammonia-lyase-encoding gene encP was previously shown to be absolutely required for benzoyl-CoA formation in "S . maritimus" . The fatty acid ß-oxidation-related genes encH, -I, and -J, on the other hand, are necessary but not required . In each case, the yield of benzoyl-CoA-primed enterocin dropped below wild-type levels . We attribute the reduced benzoyl-CoA formation in these specific mutants to functional substitution and cross-talk between the products of genes encH, -I, and -J and the enzyme homologues of primary metabolism . Disruption of the benzoate-CoA ligase encN gene did not perturb enterocin production, however, demonstrating that encN is extraneous and that benzoic acid is not a pathway intermediate . EncN rather serves as a substitute pathway for utilizing exogenous benzoic acid . These experiments provide further support that benzoyl-CoA is formed in a novel bacterial pathway that resembles the eukaryotic assembly of benzoyl-CoA from phenylalanine via a ß-oxidative path .


   INTRODUCTION

 
Benzoyl coenzyme A (benzoyl-CoA) is a rare bacterial metabolite that serves as a starter unit for the biosynthesis of the polyketides enterocin and soraphen (11) and as a central intermediate of anaerobic aromatic metabolism (3) . Its biosynthesis involves at least two oxidative pathways from the amino acid phenylalanine . Feeding experiments with 2H- and 13C-labeled intermediates (5, 6) and sequence analysis of the enterocin biosynthetic gene cluster (enc) (12) revealed that benzoyl-CoA is biosynthesized in a plant-like manner in the sediment-derived bacterium "Streptomyces maritimus" (Fig . 1) . In plants where benzoyl-CoA is a common metabolite and a component of numerous natural products, phenylalanine is nonoxidatively deaminated to cinnamic acid and then converted in a CoA-dependent manner to benzoyl-CoA via two proposed routes involving ß-oxidation (5) and reverse aldol (1) reactions . The denitrifying bacterium Thauera aromatica, conversely, metabolizes phenylalanine to benzoyl-CoA, which is then reductively degraded (15) . This anaerobic conversion involves the transamination of phenylalanine to phenylpyruvate, followed by two successive {alpha}-oxidative decarboxylations .


FIG . 1 . Proposed biosynthetic pathway for benzoyl-CoA, the starter unit of the polyketide enterocin . EncP, PAL; EncH, cinnamate-CoA ligase; EncI, cinnamoyl-CoA hydratase; EncJ, 3-keto-3-phenylpropionyl-CoA thiolase; EncN, benzoate-CoA ligase.

 
The biosynthesis of benzoyl-CoA in "S . maritimus," which parallels common eukaryotic pathways in plants and fungi, is a novel bacterial pathway . Analysis of the 20-open-reading-frame (ORF) enc gene cluster revealed five ORFs (Fig . 2) putatively involved in the biosynthesis of the benzoyl-CoA starter unit of the bacteriostatic agent enterocin (12) . These genes are arranged on four transcripts neighboring the enterocin polyketide synthase (PKS) genes encA, encB, and encC . We previously characterized the unique phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) gene encP and showed that its inactivation resulted in the abolishment of de novo cinnamic acid and enterocin synthesis (20) . Enterocin biosynthesis could be restored in the encP-inactivated mutant "S . maritimus" KP through supplementation with cinnamic or benzoic acid as well as complementation with plasmid-borne encP . Subsequent biosynthetic reactions are thought to involve the activation of cinnamic acid to its CoA thioester, presumably by the encH gene product cinnamate-CoA ligase, followed by one round of ß-oxidation . Two ß-oxidation homologous encoding genes, encI and encJ, which encode an enoyl-CoA hydratase and a ß-oxoacyl-CoA thiolase, respectively, are clustered in the enc gene set . The third gene product necessary for a complete ß-oxidation series, a hydroxyacyl-CoA dehydrogenase, was, however, not identified in the cluster . The fifth gene putatively involved in the formation of benzoyl-CoA in "S . maritimus" that was identified in the gene cluster is encN, a putative benzoate-CoA ligase .


FIG . 2 . Partial organization of the enterocin biosynthetic gene cluster (enc) . The orientation of the arrows indicates the direction of each ORF, and those that are shaded are involved in benzoyl-CoA biosynthesis . Suicide plasmids pBM10, pBM12, pBM14, and pBM16 contain internal fragments of encH, encI, encJ, and encN, respectively, and were used for gene inactivation by single recombination . Nc, NcoI . B . subtilis, Bacillus subtilis; R . palustris, Rhodopseudomonas palustris; T . subterraneum, Trifolium subterraneum.

 
In the present study, we examined the genes encH, -I, -J, and -N in benzoyl-CoA formation . Each gene was inactivated, and the resulting mutants were biochemically examined . Our results describe for the first time the benzoyl-CoA biosynthesis genes in a bacterium whose products catalyze a plant-like ß-oxidative pathway .


   MATERIALS AND METHODS

 
Bacterial strains, plasmids, and culture conditions. All strains and plasmids used in this work are listed in Table 1 . "S . maritimus" strain BD26T (GenBank accession number AF233338) was grown as previously described (12) . A1 medium was used for sporulation, and R2YE medium was used for the isolation of genomic DNA . Escherichia coli XL1-Blue was used for subcloning and grown on Luria-Bertani plates or in Luria-Bertani liquid medium . E . coli S17-1 was used as the host for E . coli-"S . maritimus" conjugation .


TABLE 1 . Bacterial strains and plasmids

 
DNA manipulations. "S . maritimus" total genomic DNA was isolated as described previously (12) . Recombinant DNA procedures were performed by standard techniques (8, 14) . Biotin-labeling and detection of chemiluminescent positives were performed with the DNADetector 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 (GIBCO) DNA polymerase . DNA sequencing by a BigDye terminator cycle sequencing reaction with an ABI 377 sequencer was performed at the Laboratory of Molecular Systematics and Evolution at the University of Arizona .

Gene disruptions. DNA fragments containing the targeted genes were PCR amplified (Table 2), cloned into the E . coli-streptomycete conjugal transfer vector pKC1139, and conjugated into "S . maritimus" as previously described for the construction of the related encP mutant "S . maritimus" KP (20) . The single-crossover mutants were selected after propagating transconjugants on SGGP (21) plates at 37°C, and apramycin-resistant colonies were confirmed by Southern hybridization with biotinylated gene probes . Mutant strains were grown at 37°C on A1 plates containing 100 µg of apramycin/ml for approximately 24 to 30 h until sporulation .


TABLE 2 . Oligonucleotide primers used to amplify enc fragments for gene disruption experiments

 
HPLC-MS analysis. Cinnamic acid and polyketide production in "S . maritimus" mutants was analyzed by high-performance liquid chromatography (HPLC) and electrospray-mass spectrometry (MS) as previously described for the encP mutant "S . maritimus" KP (12) . Feeding experiments involved overlaying ring-2H5-labeled intermediates (0.5 to 1 mg/plate) .

Nucleotide sequence accession number. The gene sequence of the enterocin biosynthetic gene cluster has been deposited at GenBank under accession number AF254925 .


   RESULTS

 
Disruption of the benzoyl-CoA formation genes encH-encJ and encN. To examine which of the previously cloned and sequenced enc genes are involved in benzoyl-CoA biosynthesis in the thermotolerant marine bacterium "S . maritimus," we developed a genetics system and disrupted several candidate genes by single-crossover homologous recombination . The PAL-encoding gene encP was previously inactivated (20) and verified to be involved in the initial reaction of the benzoyl-CoA pathway (Fig . 1) . We employed a similar approach involving the E . coli-to-"S . maritimus" conjugal transfer of pKC1139-based temperature-sensitive plasmids carrying the internal 0.5- to 0.8-kb fragments of target enc genes . We constructed plasmids pBM10, pBM12, pBM14, and pBM16 to inactivate the genes encH, encI, encJ, and encN, respectively (Fig . 2) . A single crossover between chromosomal and pKC1139-based enc sequences generated tandemly duplicated enc sequences with the vector containing an apramycin resistance gene between the sequences . In each plasmid, the enc genes were truncated at both termini . As a consequence, the mutant upstream- and downstream-targeted enc genes lacked the carboxyl and amino termini, respectively, thus resulting in nonfunctional enc gene products . In disrupting encH, the downstream gene encI may have been additionally inactivated due to polar effects . Transconjugants were grown under selective conditions, providing the "S . maritimus" mutant strains KH, KI, KJ, and KN in which encH, encI, encJ, and encN were respectively disrupted . Southern blot hybridization of genomic DNA from the wild-type and apramycin-resistant transconjugants with biotinylated enc gene fragment DNA probes verified all gene disruptions (Fig . 3) . Predicted band shifts of approximately 7 kb were detected in NcoI digests of the total DNA . The four mutants did not exhibit any different phenotypes in comparison to the wild-type strain in A1 media when fermented at 37°C in the presence of apramycin .


FIG . 3 . Southern analysis of NcoI-digested genomic DNA from "S . maritimus" wild type (wt) and mutants KH, KI, KJ, and KN by using the following biotinylated DNA probes: 0.8-kb encH fragment from pBM9 (A); 0.5-kb encI fragment from pBM11 (B); 0.7-kb encJ fragment from pBM13 (C); 0.7-kb encN fragment from pBM15 (D) . {lambda}, bacteriophage {lambda} DNA-HindIII digestion marker.

 
Analysis of the mutants and supplementation with deuterium-labeled intermediates. To differentiate the roles of the two putative CoA ligases, the 535-amino-acid EncH and the 522-amino-acid EncN, cinnamic acid and enterocin production in the mutant strains KH and KN was compared . Although the encH mutant KH produced cinnamic acid and enterocin, HPLC analysis showed that their yields decreased to 15 to 20% of that of the wild-type strain (Table 3) . Upon supplementation with d5-benzoate, enterocin production was fully recovered whereas cinnamic acid levels remained unchanged . MS analysis furthermore showed an 84% incorporation of the d5 label into enterocin . No significant difference, however, was observed by HPLC between the encN mutant KN and the wild-type strain, as wild-type levels of cinnamic acid and enterocin were detected . Supplementation with d5-benzoate provided no further increase in enterocin productivity and no notable incorporation of the deuterium label . Control feeding experiments with [ring-d5]phenylalanine gave similar incorporation percentages of approximately 9% in enterocin produced by the two enc CoA ligase mutants as well as the wild-type strain . These experiments support the DNA sequence analyses in assigning functional roles of the encH and encN gene products as cinnamate-CoA and benzoate-CoA ligases, respectively .


TABLE 3 . Characteristics of "S . maritimus" BD-26T-derived enc mutants

 
The functional roles of the two ß-oxidation genes encI and encJ in benzoyl-CoA biosynthesis were similarly analyzed . The encI gene encodes a 258-amino-acid protein homologous to enoyl-CoA hydratases, and its initiation ATG codon overlaps the TGA stop codon of encH . The encI mutant KI behaved similarly to the encH mutant KH in that the production of cinnamic acid and enterocin significantly dropped 20 to 25% and that supplementation with d5-benzoic acid fully restored enterocin biosynthesis to wild-type levels (Table 3) . The stop codon of the convergently transcribed encJ gene, which encodes a 400-amino-acid protein homologous to ß-oxoacyl-CoA thiolase, is located 146 bases downstream of the encI gene . HPLC analysis of the encJ mutant KJ showed elevated levels of cinnamic acid but a 25% loss in enterocin yield . Supplementation with d5-benzoic acid restored enterocin biosynthesis to wild-type levels and resulted in a 43% incorporation of label .

It was previously demonstrated that ß-hydroxy- and ß-ketophenylpropionates were assimilated into the benzoate-derived unit of enterocin and concluded that their respective CoA thioester analogues were pathway intermediates (6) . In order to determine to what degree the CoA ligases EncH and EncN may additionally serve in activating phenylpropanoid intermediates, we fed (3R)-[ring-2H5]-3-hydroxy-3-phenylpropionic acid (5) to the KH and KN mutants (Table 3) . Similar incorporation levels of 3 to 4% were measured in enterocin produced by the wild type and mutant KN, suggesting that EncN does not appreciably activate the phenylpropanoid intermediate . Enterocin from the encH-encI mutant KH, on the other hand, was enriched at a much higher percentage of 38% . This mutant carries not only a disrupted cinnamate-CoA ligase encH but also an inactivated cinnamoyl-CoA hydratase encI gene due to polar effects . Thus, the enrichment from this experiment was compared not against wild-type levels but rather against the encI mutant KI in which a slightly higher 46% incorporation was previously documented (5) . Consequently, 3-hydroxy-3-phenylpropionate may be activated in vivo by the action of more than one CoA ligase, including the encH gene product .


   DISCUSSION

 
The biosynthesis of benzoyl-CoA in "S . maritimus" BD-26T was examined by mutating specific genes associated with the enterocin biosynthetic gene cluster . These experiments provide further support that benzoyl-CoA is formed in a novel bacterial pathway that resembles the eukaryotic assembly of benzoyl-CoA from phenylalanine via a ß-oxidative route (5, 9) . Inactivation of the unique PAL-encoding gene encP was previously shown to be absolutely required for benzoyl-CoA formation in "S . maritimus" (20) . The fatty acid ß-oxidation related genes encH, -I, and -J, on the other hand, were shown by target-directed mutagenesis in this study to be necessary but not required . In each case, the yield of benzoyl-CoA-primed enterocin significantly dropped below wild-type levels . We attribute the reduced benzoyl-CoA formation in these specific mutants to functional substitution and cross-talk between the products of genes encH, -I, and -J and their corresponding fatty acid ß-oxidation homologues .

ß-Oxidation of cinnamoyl-CoA and of fatty acids proceeds with the same absolute configuration of the 3-hydroxyacyl-CoA intermediate (5), thus supporting the evolutionary relationship between these pathways . Furthermore, there is considerable amino acid sequence similarity between the cinnamoyl-CoA hydratase EncI and fatty acid enoyl-CoA hydratases (12) . In particular, the EncI amino acid residues Gly-116, Glu-119, and Glu-139, which are essential for catalytic activity in homologous-fatty-acid-type hydratases (4), suggest that the two enzyme systems operate by a common mechanism .

Inactivation of the encI gene in mutant KI resulted in the largest drop in cinnamic acid and enterocin productivity among the enc ß-oxidation mutants, suggesting that EncI is the most pathway specific of the enterocin ß-oxidation enzymes . While a similar loss was measured in the encH mutant KH, this mutant may effectively be an encI mutant and the overlapping encI gene may be translationally coupled . If this is indeed the case, the role of the encH CoA ligase gene product becomes less clear from these gene inactivation experiments . To this end, we have recently expressed and purified EncH as an octahistidyl-tagged recombinant protein and verified that this protein indeed catalyzes the in vitro CoA activation of trans-cinnamic acid (M . Izumikawa and B . S . Moore, unpublished observations) .

Missing from the sequenced enc cluster is a homologue of ß-hydroxyacyl-CoA dehydrogenase, the ß-oxidation enzyme needed to catalyze the dehydrogenation of (3R)-3-hydroxy-3-phenylpropionoyl-CoA and complete the oxidative cycle . Homologues were found neither 2 kb upstream nor downstream of the 21.3-kb enc cluster (12) . Heterologous expression of the enc-containing E . coli-streptomycete shuttle cosmid pJP15F11, nonetheless, was sufficient to produce enterocin (13), suggesting that either a dedicated dehydrogenase gene is located elsewhere on the cosmid clone or a dehydrogenase isoenzyme is provided by the streptomycete host . This second scenario is entirely plausible, as inactivation of the enterocin ketothiolase encJ gene in mutant KJ resulted in only a 25% loss in enterocin production, thereby suggesting strong complementation by the corresponding ketothiolase associated with fatty acid ß-oxidation . The benzoyl-CoA pathway dehydrogenase may thus be entirely supplied through primary metabolism .

As products of ketothiolases are acyl-CoAs, we predicted that the homologous enc-encoded thiolase EncJ catalyzes the conversion of 3-keto-3-phenylpropionyl-CoA directly to benzoyl-CoA (Fig . 1) . This conversion, however, bypasses the need to directly activate benzoic acid by a dedicated benzoate-CoA ligase such as EncN . Inactivation of the encN gene did not perturb the production of benzoate-primed enterocin, thereby confirming that encN is extraneous and that the product of the thiolase EncJ must be benzoyl-CoA and not free benzoic acid . Supplemental benzoic acid can efficiently enter the enterocin biosynthetic pathway in the wild-type strain but not in the corresponding encN mutant KN . This observation is consistent with our recent analysis of the encM mutant KM, in which the oxygenase encM as well as the coupled encN were disrupted (19) . Although benzoate-primed polyketides were synthesized in this mutant strain, they were not enriched with administered d5-benzoic acid . These observations confirm that, although EncN is a functional benzoate-CoA ligase, EncN does not catalyze a reaction directly along the phenylalanine-to-benzoyl-CoA biosynthetic pathway . Rather, EncN serves as a substitute pathway for utilizing exogenous benzoic acid .

The redundancy of encN in the enterocin biosynthetic gene cluster may explain why benzoic acid is not accepted as a precursor in the biosynthesis of the macrolide soraphen A in the myxobacterium Sorangium cellulosum (7), even though the soraphen A PKS-loading domain harbors a benzoyl-CoA-specific acyltransferase (18) . If benzoyl-CoA is synthesized in a similar manner in S . cellulosum, the bacterium may harbor homologues to just the encH-encJ and encP genes and not encN . Although the soraphen A biosynthetic gene cluster has been cloned and sequenced (10), genes encoding the benzoyl-CoA biosynthetic pathway are not clustered with the soraphen PKS genes and have not been reported to date .

In summary, we have assigned the enzymatic roles of the products of the novel benzoyl-CoA biosynthesis genes encH-encJ, encN, and encP through target-based mutagenesis . This pathway to benzoyl-CoA involves a plant-like conversion of phenylalanine to cinnamic acid, followed by a single round of ß-oxidation, and is reported at the genetic level here for the first time in a bacterium .

 


   ACKNOWLEDGMENTS

 
This work was generously supported by grant AI47818 from the National Institutes of Health .

We thank Paul R . Shipley for assistance with MS experiments .


   FOOTNOTES

 
* Corresponding author . Mailing address: College of Pharmacy, 1703 E . Mabel St., University of Arizona, Tucson, AZ 85721-0207 . Phone: (520) 626-6931 . Fax: (520) 626-2466 . E-mail: moore{at}pharmacy.arizona.edu .


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