Carboxyethylarginine synthase genes show complex cross-regulation in Streptomyces clavuligerus.

Carboxyethylarginine synthase is the first dedicated enzyme of clavam biosynthesis in Streptomyces clavuligerus and is present in two isoforms encoded by two separate genes. When grown on a liquid soy medium, strains with ceaS1 deleted showed only a mild reduction of clavam biosynthesis, while disruption of ceaS2 abolished all clavam biosynthesis. Creation of an in-frame ceaS2 deletion mutant to avoid polarity did not restore clavam production, nor did creation of a site-directed mutant altered only in a single amino acid residue important for activity. Reverse transcriptase PCR analyses of these mutants indicated that the failure to produce clavam metabolites could be traced to reduced or abolished transcription of ceaS1 in the ceaS2 mutants, despite the location of ceaS1 on a replicon completely separate from that of ceaS2. Western analyses further showed that the CeaS1 protein (as well as the CeaS2 protein) was absent from the ceaS2 mutants. Complementation experiments were able to restore clavam production partially, but only by virtue of restoring CeaS2 production. CeaS1 was still absent from the complemented strains. While this dependence of CeaS1 production on the expression of ceaS2 from its native chromosomal location was seen in all of the ceaS2 mutants, the effect was limited to growth in liquid medium. When the same mutants were grown on solid soy medium, clavam production was restored and CeaS1 was produced, albeit at low levels compared to the wild type.

Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation.

The beta-lactam antibiotics have been serving mankind for over 70 years. Despite this old age, they continue to provide health to the world population by virtue of industrial production and discoveries of new secondary metabolite molecules with useful activities. Sales of these remarkable compounds have reached over $20 billion dollars per year. They include penicillins, cephalosporins, cefoxitin, monobactams, clavulanic acid and carbapenems. Strain improvement of the penicillin-producing species of Penicillium has been truly remarkable, with present strains producing about 100,000 times more penicillin that the original Penicillium notatum of Alexander Fleming. A tremendous amount of information has been gathered on the biosynthetic enzymes involved, the pathways of biosynthesis of beta-lactams as well as their regulation, and the genomics and proteomics of the producing organisms. Modern aspects of the processes are discussed in the present review including genetics, molecular biology, metabolic engineering, genomics and proteomics.

Biosynthesis of clavam metabolites.

Naturally occurring clavam metabolites include the valuable ß-lactamase inhibitor, clavulanic acid, as well as stereochemical variants with side-chain modifications, called the 5S clavams. Because of the clinical importance of clavulanic acid, most studies of clavam biosynthesis are based on the industrial producer species Streptomyces clavuligerus. Well-characterized early steps in clavam biosynthesis are outlined, and less well understood late steps in 5S clavam biosynthesis are proposed. The complex genetic organization of the clavam biosynthetic genes in S. clavuligerus is described and, where possible, comparisons with other producer species are presented.

5S clavam biosynthesis is controlled by an atypical two-component regulatory system in Streptomyces clavuligerus.

Streptomyces clavuligerus produces a collection of five clavam metabolites, including the clinically important ß-lactamase inhibitor clavulanic acid, as well as four structurally related metabolites called 5S clavams. The paralogue gene cluster of S. clavuligerus is one of three clusters of genes for the production of these clavam metabolites. A region downstream of the cluster was analyzed, and snk, res1, and res2, encoding elements of an atypical two-component regulatory system, were located. Mutation of any one of the three genes had no effect on clavulanic acid production, but snk and res2 mutants produced no 5S clavams, whereas res1 mutants overproduced 5S clavams. Reverse transcriptase PCR analyses showed that transcription of cvm7p (which encodes a transcriptional activator of 5S clavam biosynthesis) and 5S clavam biosynthetic genes was eliminated in snk and in res2 mutants but that snk and res2 transcription was unaffected in a cvm7p mutant. Both snk and res2 mutants could be complemented by introduction of cvm7p under the control of an independently regulated promoter. In vitro assays showed that Snk can autophosphorylate and transfer its phosphate group to both Res1 and Res2, and Snk-H365, Res1-D52, and Res2-D52 were identified as the phosphorylation sites for the system. Dephosphorylation assays indicated that Res1 stimulates dephosphorylation of Res2∼P. These results suggest a regulatory cascade in which Snk and Res2 form a two-component system controlling cvm7p transcription, with Res1 serving as a checkpoint to modulate phosphorylation levels. Cvm7P then activates transcription of 5S clavam biosynthetic genes.

A comparison of the clavam biosynthetic gene clusters in Streptomyces antibioticus Tü1718 and Streptomyces clavuligerus.

The production of clavam metabolites has been studied previously in Streptomyces clavuligerus , a species that produces clavulanic acid as well as 4 other clavam compounds, but the late steps of the pathway leading to the specific end products are unclear. The present study compared the clavam biosynthetic gene cluster in Streptomyces antibioticus , chosen because it produces only 2 clavam metabolites and no clavulanic acid, with that of S. clavuligerus. A cosmid library of S. antibioticus genomic DNA was screened with a clavaminate synthase-specific probe based on the corresponding genes from S. clavuligerus, and 1 of the hybridizing cosmids was sequenced in full. A clavam gene cluster was identified that shows similarities to that of S. clavuligerus but also contains a number of novel genes. Knock-out mutation of the clavaminate synthase gene abolished clavam production in S. antibioticus, confirming the identity of the gene cluster. Knock-out mutation of a novel gene encoding an apparent oxidoreductase also abolished clavam production. A potential clavam biosynthetic pathway consistent with the genes in the cluster and the metabolites produced by S. antibioticus, and correspondingly different from that of S. clavuligerus, is proposed.

Clavulanic acid biosynthesis and genetic manipulation for its overproduction.

Clavulanic acid, a ß-lactamase inhibitor, is used together with ß-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other ß-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.

A conserved lysine in beta-lactam synthetase assists ring cyclization: Implications for clavam and carbapenem biosynthesis.

beta-Lactam synthetase (beta-LS) is the paradigm of a growing class of enzymes that form the critical beta-lactam ring in the clavam and carbapenem antibiotics. beta-LS catalyzes a two-stage reaction in which N(2)-(2-carboxyethyl)-L-arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to beta-lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. beta-Lactam formation was evaluated on the basis of X-ray crystal structures, site-specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate preorganization, a well-stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four-membered ring formation to the magnitude of a protein conformational change.

Alanylclavam biosynthetic genes are clustered together with one group of clavulanic acid biosynthetic genes in Streptomyces clavuligerus.

Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway.

Enhancement of clavulanic acid by replicative and integrative expression of ccaR and cas2 in Streptomyces clavuligerus NRRL3585.

Clavulanic acid (CA) is an inhibitor of beta-lactamase that is produced from Streptomyces clavuligerus NRRL3585 and is used in combination with other antibiotics in clinical treatments. In order to increase the production of CA, the replicative and integrative expressions of ccaR (encoding for a specific regulator of the CA biosynthetic operon) and cas2 (encoding for the rate-limiting enzyme in the CA biosynthetic pathway) were applied. Six recombinant plasmids were designed for this study. The pIBRHL1, pIBRHL3, and pIBRHL13 were constructed for overexpression, whereas pNQ3, pNQ2, and pNQ1 were constructed for chromosomal integration with ccaR, cas2, and ccaR-cas2, respectively. All of these plasmids were transformed into S. clavuligerus NRRL3585. CA production in transformants resulted in a significantly enhanced amount greater than that of the wild type, a 2.25-fold increase with pIBRHL1, a 9.28-fold increase with pNQ3, a 5.06-fold increase with pIBRHL3, a 2.93-fold increase with pNQ2 integration, a 5.79-fold increase with pIBRHL13, and a 23.8-fold increase with pNQ1. The integrative pNQ1 strain has been successfully applied to enhance production.

5S clavam biosynthetic genes are located in both the clavam and paralog gene clusters in Streptomyces clavuligerus.

The Streptomyces clavuligerus clavam gene cluster was examined to identify genes specifically involved in 5S clavam biosynthesis. A reduction/loss of 5S clavam production was seen in cvm2 and cvm5 gene mutants, and a clavam metabolite not previously observed, 2-carboxymethylideneclavam, accumulated in the cvm5 mutant. Disruption of additional genes from the region of the clavam cluster did not have any effect on 5S clavam production. Examination of the paralog gene cluster region for 5S clavam biosynthetic genes led to the identification of cvm6P and cvm7P, which encode a putative aminotransferase and a transcriptional regulator, respectively. Mutants defective in cvm6P and cvm7P were completely blocked in 5S clavam but not clavulanic acid production. The loss of 5S clavam production in cvm7P mutants suggests that this gene encodes a transcriptional regulator specific for 5S clavam metabolite biosynthesis.

The enzymology of clavam and carbapenem biosynthesis.

The enzyme-catalysed reactions involved in formation of the bicyclic clavam and carbapenem nuclei, including beta-amino acid and beta-lactam formation, are discussed and compared with those involved in penicillin and cephalosporin biosynthesis. The common role of unusual oxidation reactions in the biosynthetic pathways and the lack of synthetic reagents available to effect them are highlighted.

The paralogous pairs of genes involved in clavulanic acid and clavam metabolite biosynthesis are differently regulated in Streptomyces clavuligerus.

Carboxyethylarginine synthase, encoded by the paralogous ceaS1 and ceaS2 genes, catalyzes the first reaction in the shared biosynthetic pathway leading to clavulanic acid and the other clavam metabolites in Streptomyces clavuligerus. The nutritional regulation of ceaS1 and ceaS2 expression was analyzed by reverse transcriptase PCR and by the use of the enhanced green fluorescent protein-encoding gene (egfp) as a reporter. ceaS1 was transcribed in complex soy medium only, whereas ceaS2 was transcribed in both soy and defined starch-asparagine (SA) media. The transcriptional start points of the two genes were also mapped to a C residue 98 bp upstream of ceaS1 and a G residue 51 bp upstream of the ceaS2 start codon by S1 nuclease protection and primer extension analyses. Furthermore, transcriptional mapping of the genes encoding the beta-lactam synthetase (bls1) and proclavaminate amidinohydrolase (pah1) isoenzymes from the paralogue gene cluster indicated that a single polycistronic transcript of approximately 4.9 kb includes ceaS1, bls1, and pah1. The expression of ceaS1 and ceaS2 in a mutant strain defective in the regulatory protein CcaR was also examined. ceaS1 transcription was not affected in the ccaR mutant, whereas that of ceaS2 was greatly reduced compared to the wild-type strain. Overall, our results suggest that different mechanisms are involved in regulating the expression of ceaS1 and ceaS2, and presumably also of other paralogous genes that encode proteins involved in the early stages of clavulanic acid and clavam metabolite biosynthesis.

Two sets of paralogous genes encode the enzymes involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus.

Recently, a second copy of a gene encoding proclavaminate amidinohydrolase (pah1), an enzyme involved in the early stages of clavulanic acid and clavam metabolite biosynthesis in Streptomyces clavuligerus, was identified and isolated. Using Southern analysis, we have now isolated second copies of the genes encoding the carboxyethylarginine synthase (ceaS) and beta-lactam synthetase (bls) enzymes. These new paralogues are given the gene designations ceaS1 and bls1 and are located immediately upstream of pah1 on the chromosome. Furthermore, sequence analysis of the region downstream of pah1 revealed a second copy of a gene encoding ornithine acetyltransferase (oat1), thus indicating the presence of a cluster of paralogue genes. ceaS1, bls1, and oat1 display 73, 60, and 63% identities, respectively, at the nucleotide level to the original ceaS2, bls2, and oat2 genes from the clavulanic acid gene cluster. Single mutants defective in ceaS1, bls1, or oat1 were prepared and characterized and were found to be affected to variable degrees in their ability to produce clavulanic acid and clavam metabolites. Double mutants defective in both copies of the genes were also prepared and tested. The ceaS1/ceaS2 and the bls1/bls2 mutant strains were completely blocked in clavulanic acid and clavam metabolite biosynthesis. On the other hand, oat1/oat2 double mutants still produced some clavulanic acid and clavam metabolites. This may be attributed to the presence of the argJ gene in S. clavuligerus, which encodes yet another ornithine acetyltransferase enzyme that may be able to compensate for the lack of OAT1 and -2 in the double mutants.

Three unlinked gene clusters are involved in clavam metabolite biosynthesis in Streptomyces clavuligerus.

In Streptomyces clavuligerus, three groups of genes are known to be involved in the biosynthesis of the clavam metabolites. Since antibiotic biosynthetic genes are invariably clustered on the chromosome in prokaryotes, chromosome walking was undertaken in an attempt to show that the three groups of clavam genes would resolve into a single super-cluster when analyzed at larger scale. However, no evidence of linkage between the three groups was obtained. Furthermore, Southern analysis of macro-restriction fragments of genomic DNA separated by pulsed-field gel electrophoresis also indicated that the three groups of genes are not linked. Despite the structural and biosynthetic relatedness of the clavam metabolites, our results suggest that the genes involved in their production lie in three unlinked gene clusters. We believe that this represents the first instance in bacteria of genes involved in the biosynthesis of a single family of antibiotics sharing a common biosynthetic pathway and yet residing in three separate locations on the chromosome.

A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds.

A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid.

Analysis of bacterial carbapenem antibiotic production genes reveals a novel beta-lactam biosynthesis pathway.

Carbapenems are beta-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora, makes the simple beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the beta-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing beta-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC, encode proteins which show significant homology with proteins encoded by the Streptomyces clavuligerus gene cluster responsible for the production of the beta-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the beta-lactamase inhibitor. Our observation are consistent with the evolution of a second major biosynthetic route to the production of beta-lactam-ring-containing antibiotics.

Cell density influences antibiotic biosynthesis in Streptomyces clavuligerus.

Production of cephamycin C and clavulanic acid by Streptomyces clavuligerus took place during the exponential phase of growth in a defined medium. Both antibiotic biosynthetic pathways were activated shortly after spore germination, but the timing and kinetics of activation were affected by inoculum density. Rapid activation was favoured by high inoculum density or by growth in medium conditioned by previous incubation of S. clavuligerus spores or mycelium. A heat-resistant conditioning factor able to accelerate the acquisition of antibiotic-biosynthetic capacity when added to low-density cultures was released in suspensions of spores in water. Conditioning factor was also obtained in suspensions of spores from different Streptomyces species or of Bacillus cells, indicating that the signal was not produced specifically by S. clavuligerus. Fractionation of conditioning factor showed that its effect was not due to a single molecule. The fractions contained amino acids (as free amino acids and oligopeptides) in amounts that roughly correlated with their respective conditioning power. Furthermore, the conditioning effect was reproduced by supplementing defined medium with amino acids and peptides in concentration that mimicked those found in conditioning factor. When individually tested at concentrations in the micromolar range, only some amino acids were able to stimulate antibiotic biosynthetic capacity. This stimulation ws also promoted by low concentrations (less than 1 microgram ml-1) of peptide mixtures obtained with different proteolytic enzymes. The results suggest that both amino acids and peptides are responsible for the effects of conditioning factor released by spores. Possible implications of intercellular signalling on activation of secondary metabolism are discussed.

Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations.

Clavulanic acid (CA) was produced by Streptomyces clavuligerus in medium containing glycerol and soy meal or soy meal extract. With regard to growth and CA productivity, the microorganism showed significant differences if solid soy meal as such or its extract were applied as the major nitrogen source. If the extract is used, growth and CA production take place simultaneously and in the stationary phase the CA concentration is stagnant or reduces. If soy meal is used, growth is threefold faster and CA is only generated in the stationary phase. In the case of using the soy meal extract, the decrease of the CA concentration is mainly due to decomposition or re-metabolisation of CA in the presence of the microorganism. This conclusion is supported by in vivo and in vitro data on CA decomposition.

Clavulanic acid biosynthesis in Streptomyces clavuligerus: gene cloning and characterization.

Seven classes of Streptomyces clavuligerus mutants defective in clavulanic acid (CLA) biosynthesis have been identified and used to clone the chromosomal DNA encoding eight CLA biosynthetic genes. The complete sequences of three and the partial sequences of two of these biosynthetic genes are reported, together with their known or predicted functions.