Biosynthesis of clavam metabolites

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.     

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.     

Biosynthesis and Molecular Genetics of Clavulanic Acid

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic -lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modfication as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the -lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack -lactamase inhibitory activity.     

Induction of holomycin production and complex metabolic changes by the argR mutation in Streptomyces clavuligerus NP1

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus.     

Clavulanic acid biosynthesis and genetic manipulation for its overproduction

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.     

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.     

Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56

Myxobacteria belonging to the genus Sorangium are known to produce a variety of biologically active secondary metabolites. Chivosazol is a macrocyclic antibiotic active against yeast, filamentous fungi and especially against mammalian cells. The compound specifically destroys the actin skeleton of eucaryotic cells and does not show activity against bacteria. Chivosazol contains an oxazole ring and a glycosidically bound 6-deoxyglucose (except for chivosazol F). In this paper we describe the biosynthetic gene cluster that directs chivosazol biosynthesis in the model strain Sorangium cellulosum So ce56. This biosynthetic gene cluster spans 92 kbp on the chromosome and contains four polyketide synthase genes and one hybrid polyketide synthase/nonribosomal peptide synthetase gene. An additional gene encoding a protein with similarity to different methyltransferases and presumably involved in post-polyketide modification was identified downstream of the core biosynthetic gene cluster. The chivosazol biosynthetic gene locus belongs to the recently identified and rapidly growing class of trans-acyltransferase polyketide synthases, which do not contain acyltransferase domains integrated into the multimodular megasynthetases.     

Agrobacterium tumefaciens TR-DNA encodes a pathway for agropine biosynthesis

Summary A pathway for biosynthesis of the crown-gall opine, agropine is proposed and three potential new precursors characterised. The location of genes involved in the three steps of this pathway was determined by site directed insertions and deletions in the T_R region of the octopine Ti plasmid, pTiB6Tra^c. The proposed biosynthetic pathway for agropine which involves three T-DNA genes is in contrast to the biosynthesis of octopine and nopaline where single T-DNA genes are involved.     

Biosynthesis of deoxyaminosugars in antibiotic-producing bacteria

Deoxyaminosugars comprise an important class of deoxysugars synthesized by a variety of different microorganisms; they can be structural components of lipopolysaccharides, extracellular polysaccharides, and secondary metabolites such as antibiotics. Genes involved in the biosynthesis of the deoxyaminosugars are often clustered and are located in the vicinity of other genes required for the synthesis of the final compound. Most of the gene clusters for aminosugar biosynthesis have common features, as they contain genes encoding dehydratases, isomerases, aminotransferases, methyltransferases, and glycosyltransferases. In the present mini-review, the proposed biosynthetic pathways for deoxyaminosugar components of both macrolide and non-macrolide antibiotics are highlighted. The possibilities for genetic manipulations of the deoxyaminosugar biosynthetic pathways aimed at production of novel secondary metabolites are discussed.     

A comprehensive and engaging overview of the type III family of polyketide synthases

Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds.     

Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.     

Molecular biology of mycotoxin biosynthesis

Mycotoxins are secondary metabolites produced by many important phytopathogenic and food spoilage fungi including Aspergillus, Fusarium and Penicillium species. The toxicity of four of the most agriculturally important mycotoxins (the trichothecenes, and the polyketide-derived mycotoxins; aflatoxins, fumonisins and sterigmatocystin) are discussed and their chemical structure described. The steps involved in the biosynthesis of aflatoxin and sterigmatocystin and the experimental techniques used in the cloning and molecular characterisation of the genes involved in the pathway are described in detail. The biosynthetic genes involved in the fumonisin and trichothecene biosynthetic pathways are also outlined. The potential benefits gained from an increased knowledge of the molecular organisation of these pathways together with the mechanisms involved in their regulation are also discussed.