The δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of β-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Naturally occurring β-lactam compounds fall into four basic structural groups, the penicillins/cephalosporins, the clavams, the carbapenems and the monocyclic β-lactams. Biosynthetic studies have clarified the steps involved in the formation of the β-lactam ring for the first three of these groups, but the corresponding process or processes for the monocyclic β-lactams remains obscure. Isopenicillin N synthase is responsible for formation of the β-lactam ring in all penicillin/cephalosporin compounds, and the reaction catalyzed is completely separate from that of β-lactam synthetase, the enzyme responsible for ring formation in all clavam compounds. Conversely, carbapenam synthetase, the enzyme responsible for β-lactam ring formation for all carbapenem compounds, shows clear relatedness to β-lactam synthetase, despite differences in the substrates and the products for the two enzymes. The mechanism of ring formation has not yet been clarified for any of the monocyclic β-lactams, but a third distinct mechanism of β-lactam ring formation seems likely, and this group includes such a diverse collection of structures that even more new ring-forming reactions may be involved.

Naturally occurring clavam metabolites include the valuable β-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.

ABSTRACT Streptomyces clavuligerus produces a collection of five clavam metabolites, including the clinically important β-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.

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.

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.

Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptide synthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B(1) (1) and B(2) (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues.

Clavulanic acid, a β-lactamase inhibitor, is used together with β-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 β-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.

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 angstrom resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis. (C) 2009 Elsevier Ltd. All rights reserved.

Genetic Engineering To Regulate Production of Secondary Metabolites in Streptomyces clavuligerus Susan E. Jensen, Susan E. JensenSearch for more papers by this author Susan E. Jensen, Susan E. JensenSearch for more papers by this author Book Editor(s):Alan T. Bull, Alan T. Bull School of Biosciences, University of Kent, Canterbury, Kent, United KingdomSearch for more papers by this authorBeth Junker, Beth Junker Bioprocess Research and Development, Merck Research Laboratories, Rahway, New JerseySearch for more papers by this authorLeonard Katz, Leonard Katz SynBERC - Synthetic Biology Engineering Research Center, University of California-Berkeley, Emeryville, CaliforniaSearch for more papers by this authorLee R. Lynd, Lee R. Lynd Thayer School of Engineering, Dartmouth College, Hanover, New HampshireSearch for more papers by this authorPrakash Masurekar, Prakash Masurekar Department of Plant Biology & Plant Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New JerseySearch for more papers by this authorChristopher D. Reeves, Christopher D. Reeves Amyris Biotechnologies, Emeryville, CaliforniaSearch for more papers by this authorHuimin Zhao, Huimin Zhao Departments of Chemical and Biomolecular Engineering, Chemistry, and Bioengineering, Institute for Genomic Biology, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IllinoisSearch for more papers by this author First published: 25 March 2010 https://doi.org/10.1128/9781555816827.ch28 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat Summary Many early studies on penicillin and cephamycin biosynthesis were carried out in Streptomyces clavuligerus, but because of the commercial importance of clavulanic acid, most recent studies have focused on that compound. In particular, genetic engineering to generate improved clavulanic acid producer strains has been a main interest. S. clavuligerus grows and produces secondary metabolites optimally at 28 to 30°C, but it is unusually sensitive to elevated temperature and will not grow above 31 to 32°C. Liquid media employed by research groups studying the production of the various types of β-lactam metabolites produced by S. clavuligerus vary widely. Numerous bioprocess-type studies examining growth medium optimization as a means of improving production of clavulanic acid have appeared in recent years, typically reinforcing the association of soy-based growth medium constituents with high productivity. In S. clavuligerus, biosynthesis begins with the amino acids L-lysine, L-cysteine, and L-valine and proceeds through the synthesis of penicillin and cephalosporin intermediates to give the final cephamycin product. While improvements in productivity can be achieved by increasing production of pathway enzymes, another approach to achieving this is to identify genes encoding critical regulatory proteins and increase their expression levels. More global approaches to improvement of antibiotic production could also be coupled with strategies aimed at the pathway-specific regulators. Effects of greater magnitude are seen when pathway-specific regulators are targeted for overproduction, and combination approaches targeting both global and specific regulators along with individual pathway enzymes for overproduction, together with elimination of competing pathways, will likely yield even greater results. Manual of Industrial Microbiology and Biotechnology, Third Edition RelatedInformation

ABSTRACT Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

Beta-lactamase inhibitory protein (BLIP) binds a variety of beta-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein-protein interactions. Published studies have examined the binding of BLIP to diverse target beta-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme-inhibitor interface has remained unexplored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has beta-lactamase inhibitory activity very similar to that of BLIP; and (2) beta-lactamase-inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra-high-resolution structure), which is unable to inhibit any tested beta-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I.TEM-1 versus those formed at the interface of BLIP.TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein-protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation possible in the subnanomolar binding interaction with TEM-1. The corresponding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone-backbone/backbone-side-chain interactions with a protein partner can be negated by the presence of a few, strongly unfavorable interactions, especially electrostatic repulsions.

The generation of unmarked deletion mutants free from polar effects on downstream genes is typically a lengthy and arduous process in Streptomyces spp. The use of FLP recombinase can greatly facilitate this process when combined with established polymerase chain reaction (PCR)-targeting techniques. In vivo production of FLP within Streptomyces cells would streamline the process further, but expression of flp in Streptomyces spp. has proven difficult to achieve. Two Escherichia coli–Streptomyces shuttle plasmids that constitutively express native flp within Streptomyces cells were constructed and tested within Streptomyces clavuligerus and Streptomyces coelicolor to produce in-frame mutations in genes associated with antibiotic production. Only one of the flp-expressing plasmids was functional in S. clavuligerus, but both functioned in S. coelicolor and both were easily lost from cells. Although a separate study has recently shown successful expression of a synthetic flp gene in Streptomyces, this is the first report of expression of the native flp gene within Streptomyces spp. Through the use of these plasmids to generate unmarked deletion mutants, C7p was shown to be essential for production of 5S clavams in S. clavuligerus, and RedJ was demonstrated to be important for optimal undecylprodigiosin biosynthesis in S. coelicolor but traces of the antibiotic were still produced in a ΔredJ mutant.

The beta-lactamase inhibitory protein, BLIP-II, found in the culture supernatant of Streptomyces exfoliatus SMF19, shows no discernible sequence identity with other beta-lactamase inhibitory proteins identified in Streptomyces spp. A null mutant of the gene encoding BLIP-II (bliB::hygr) showed a bald appearance on solid media. Although BLIP-II was initially isolated from the supernatant of submerged cultures, sites of BLIP-II accumulation were seen in the cell envelope. The mutation of bliB was also associated with changes in the formation of septa and condensation of the chromosomal DNA associated with sporulation. The bliB mutant exhibited infrequent septa, showing dispersed chromosomal DNA throughout the mycelium, whereas the condensed chromosomes of the wild-type were separated by regularly spaced septa giving the appearance of a string of beads. Therefore, on the basis of these results, it is suggested that BLIP-II is a regulator of morphological differentiation in S. exfoliatus SMF19.

The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by increasing dosoge of a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.

ABSTRACT 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.

Paenibacillus polymyxa PKB1 produces fusaricidins, a family of lipopeptide antibiotics that strongly inhibits the growth of many plant pathogenic fungi. The fusaricidin biosynthetic gene cluster was cloned and sequenced, and it spans 32.4 kb, including an open reading frame (fusA) encoding a six-module nonribosomal peptide synthetase. The second, fourth, and fifth modules of fusaricidin synthetase each contain an epimerization domain, consistent with the structure of fusaricidins. However, no epimerization domain is found in the sixth module, corresponding to d-Ala. This sixth adenylation domain was produced at a high level in Escherichia coli and is shown to activate d-Ala specifically, providing evidence for direct activation of a d-amino acid by a prokaryotic peptide synthetase. The fusaricidin gene cluster also includes genes involved in the biosynthesis of the lipid moiety, but no genes for resistance, regulation, or transport functions were encountered.