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Clavulanic Acid Production by Streptomyces Clavuligerus: Biogenesis, Regulation and Strain Improvement

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Clavulanic Acid Production by Streptomyces Clavuligerus: Biogenesis, Regulation and Strain Improvement The Journal of Antibiotics (2013) 66, 411–420 & 2013 Japan Antibiotics Research Association All rights reserved 0021-8820/13 www.nature.com/ja REVIEW ARTICLE Clavulanic acid production by Streptomyces clavuligerus: biogenesis, regulation and strain improvement Ashish Paradkar Clavulanic acid (CA) is a potent b-lactamase inhibitor produced by Streptomyces clavuligerus and has been successfully used in combination with b-lactam antibiotics (for example, Augmentin) to treat infections caused by b-lactamase-producing pathogens. Since the discovery of CA in the late 1970s, significant information has accumulated on its biosynthesis, and regarding molecular mechanisms involved in the regulation of its production. Notably, the genes directing CA biosynthesis are clustered along with the genes responsible for the biosynthesis of the b-lactam antibiotic, cephamycin C, and co-regulated, which makes this organism unique in that the production of an antibiotic and production of a small molecule to protect the antibiotic from its enzymatic degradation are controlled by shared mechanisms. Traditionally, the industrial strain improvement programs have relied significantly on random mutagenesis and selection approach. However, the recent availability of the genome sequence of S. clavuligerus along with the capability to build metabolic models, and ability to engineer the organism by directed approaches, has created exciting opportunities to improve strain productivity more efficiently. This review will include focus mainly on the gene organization of the CA biosynthetic genes, regulatory mechanisms that affect its production, and will include perspectives on improving strain productivity. The Journal of Antibiotics (2013) 66, 411–420; doi:10.1038/ja.2013.26; published online 24 April 2013 Keywords: Actinomycetes; b-lactams; clavulanic acid; secondary metabolism; Streptomyces INTRODUCTION inhibitory property is due to the characteristic 3R, 5R stereochemistry Clavulanic acid (CA) is a potent inhibitor of a wide range of of the strained bicyclic nucleus structure (Figure 1). This b-lactamase b-lactamases from pathogenic organisms.1,2 It is used in combination inhibitor was discovered in the fermentation broth of Streptomyces with b-lactam antibiotics against those organisms that are known or clavuligerus in a screening program aimed at identifying novel suspected to be b-lactamase producers, to protect activity of b-lactamase inhibitors.8 It is notable that S. clavuligerus was previously the b-lactam antibiotics and prevent infections. The mechanism of isolated for its ability to produce the b-lactam antibiotic, cephamycin C b-lactamase inhibition is because of the structural similarity of CA with and related b-lactam antibiotics (Figure 1), and subsequently it was the natural substrate of the b-lactamase enzyme, that is, b-lactam found that it also produces CA. The b-lactam antibiotics penicillin N antibiotic, which results in CA binding to the serine-hydroxyl group in and cephalosporin occur as intermediates in the production of the active site of the enzyme and causing irreversible enzyme cephamycin C. Production of both b-lactam antibiotics and a b- inhibition.3,4 CA is active against group 2 class A or D b-lactamases, lactamase inhibitor by one organism, and as described later, the including both penicillinases and cephalosporinases.5 The combination contiguous chromosomal arrangement of the genes responsible for therapy of CA þ b-lactam antibiotic has been very successful in prevent- the production of these two classes of compounds, and their coregula- ing infections due to Gram-positive (Staphylococcus sp.) and Gram- tion, makes S. clavuligerus a unique case, in which both the antibiotic negative (Klebsiella sp. Hemophilus sp. Proteus, Shigella, Pseudomonas.) and a molecule to protect the antibiotic from degradation seem to have b-lactamase-producing pathogens. Augmentin, a combination of CA coevolved. Thus, the idea and development of Augmentin as a and the b-lactam antibiotic, amoxicillin, is a very successful combina- combination therapy seems to have been inspired by the mother nature. tion therapy with reported global sales of 4$2.1 billion (Glaxo Subsequently, several other Streptomyces sp., including S. jumonjinensis SmithKline).6,7 and S. katsurahamanus, have been shown to produce both cephamycin CA belongs to the clavam group of b-lactams, characterized by the CandCA.9 Among these, S. clavuligerus has been studied the most as a bicyclic b-lactam/oxazolidine ring system, in which its b-lactamase model system, and is also used for producing CA commercially. DuPont Knowledge Center, E.I., DuPont India Ltd., ICICI Knowledge Park, Genome Valley, Hyderabad, India Correspondence: Dr A Paradkar, DuPont Knowledge Center, E.I., DuPont India Ltd., ICICI Knowledge Park, Genome Valley, Shamirpet, Hyderabad 500065, Andhra Pradesh, India. Email: [email protected] Received 22 January 2013; revised 25 February 2013; accepted 11 March 2013; published online 24 April 2013 Clavulanic acid production by Streptomyces clavuligerus AParadkar 412 Clavams -lactam antibiotics + NH4 HN S - COO O N O Penicillin N COOH Clavulanic acid (3R, 5R stereochemistry) Cephalosporin Other clavams (3S, 5S stereochemistry) Cephamycin C Figure 1 b-Lactam metabolites produced by S. clavuligerus. Apart from cephamycin C and CA, S. clavuligerus also produces THE ‘EARLY’ STEPS non-CA clavam type of compounds all of which contain the antipodal The CA pathway begins with the condensation of the 3-carbon 3S, 5S stereochemistry in their b-lactam ring system, as compared glyceraldehyde 3-phosphate with the 5-carbon L-arginine to form with the 3R, 5R stereochemistry of CA, which is required for the N2-(2-carboxyethyl) arginine, catalyzed by carboxyethylarginine b-lactamase inhibitory property10 (Figure 1). Therefore, the 3S, 5S synthase (CeaS2).25 In the next reaction, N2-(2-carboxyethyl) clavams do not possess any b-lactamase inhibitory property, but do arginine is cyclized to form a monocyclic b-lactam compound, possess activities ranging from antibacterial and antifungal in nature. deoxyguanidinoproclavaminate, catalyzed by b-lactam synthase Owing to structural similarity between CA and 3S, 5S clavams, there (Bls2).26–28 Deoxyguanidinoproclavaminate is then hydroxylated to are shared mechanisms involved in their biosynthesis.11,12 However, form guanidinoproclavaminate, catalyzed by clavaminate synthase this review will focus primarily on the molecular aspects of CA (Cas2).29–31 Guanidinoproclavaminic acid undergoes a hydrolysis biogenesis in S. clavuligerus, with special emphasis on the strategies reaction catalyzed by proclavaminate amidino-hydrolase (Pah2) to used for strain development. The biosynthesis of 3S, 5S clavams and form proclavaminate.21,32 The next two consecutive reactions are relevant genetics have been reviewed elsewhere.13,14 again catalyzed by Cas2. In the first reaction, proclavaminate is cyclized to form the first bicyclic ring structure, dihydroclavaminic acid, which then undergoes subsequent desaturation to produce BIOGENESIS clavaminic acid.33 The enzymology involved in the ‘early’ reactions The knowledge on CA biosynthesis has been obtained from several leading to the formation of clavaminic acid is well characterized and lines of investigation including classical approaches of feeding cells reviewed elsewhere.13 The genes encoding the ‘early’ steps, ceasS2, bls, with labeled precursors and tracing the incorporation of the label in pah and cas2, are coexpressed and coregulated. The gene immediately the CA molecule,10,15–17 isolating or constructing non-producing downstream from cas-2 is oat2, which codes for an ornithine mutants13,18,19 and characterizing the accumulated intermediates, and acetyltransferase-like activity.18,34 On the basis of mutational taking molecular approaches including, sequence and transcriptional analysis and similarity of its transcriptional regulation to that of analysis of the biosynthetic and regulatory genes.13 The biosynthetic ‘early’ genes, it is considered a part of the ‘early’ gene cluster. pathway for CA is presented in Figure 2, and the description of However, oat2 is transcribed separately.34,35 The oat2 mutant enzymes and genes involved in the pathway and their functions is showed decreased CA production, however, this phenotype could summarized in Table 1. The CA biosynthetic gene cluster is located be reversed by addition of arginine. Therefore, Oat2 is thought immediately downstream from the cephamycin C gene cluster,20,21 to contribute toward building arginine pools required for CA and stretches from carboxyethylarginine synthase ceaS2 to gcaS;13 biosynthesis. (Figure 3). Beyond ceaS2 on one end of the CA cluster is pcbR,a It is striking to note that in S. clavuligerus the entire ‘early’ gene penicillin-binding protein involved in b-lactam resistance and part of cluster is duplicated. This knowledge came from several independent the cephamycin C gene cluster.22 Beyond gcaS on the other end are studies. The initial enzymology and gene cloning had demonstrated orf-18 and orf-19, encoding another set of penicillin-binding proteins, the existence of duplicate sets (paralogs) of Cas genes, cas1 and cas2, which are not involved in CA biosynthesis. These are followed by located on two different regions of the chromosome.30 cas2 is now orf-20, orf-21 and orf-22, encoding potential regulatory genes, which assigned to the CA gene cluster downstream from the cephamycin C seem to have some effects on CA production.
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