Cephamycin Biosynthesis Research Articles

Streptomyces clavuligerus: The Omics Era.

The Streptomyces clavuligerus genome consists in a linear chromosome of about 6.7 Mb and four plasmids (pSCL1 to pSCL4), the latter one of 1.8 Mb. Deletion of pSCL4, results in viable mutants with high instability in the chromosome arms, which may lead to chromosome circularisation. Transcriptomic and proteomic studies comparing different mutants with the wild-type strain improved our knowledge on the biosynthesis and regulation of clavulanic acid, cephamycin C and holomycin. Additional knowledge has been obtained on the SARP-type CcaR activator and the network of connections with other regulators (Brp, AreB, AdpA, BldG, RelA) controlling ccaR expression. The transcriptional pattern of the cephamycin and clavulanic acid clusters is supported by the binding of CcaR to different promoters and confirmed that ClaR is a CcaR-dependent activator that controls the late steps of clavulanic biosynthesis. Metabolomic studies allowed the detection of new metabolites produced by S. clavuligerus such as naringenin, desferroxamines, several N-acyl tunicamycins, the terpenes carveol and cuminyl alcohol or bafilomycin J. Heterologous expression of S. clavuligerus terpene synthases resulted in the formation of no less than 15 different terpenes, although none of them was detected in S. clavuligerus culture broth. In summary, application of the Omic tools results in a better understanding of the molecular biology of S. clavuligerus, that allows the use of this strain as an industrial actinobacterial platform and helps to improve CA production.

Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster

The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC–MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains.

Role of the cmcH–ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus

The effect of the CcaR regulatory protein on expression of the cephamycin C gene cluster is studied. Quantitative reverse transcription PCR (qRT-PCR) expression analysis of the cephamycin biosynthesis genes in the ccaR-disrupted strain, S. clavuligerus ccaR::aph, revealed that in the absence of CcaR, the lat and cmcI genes expression was reduced 2,200- and 1,087-fold compared with the wild type. Expression of pcbAB-pcbC-cefD-cefE-cmcJ-cmcH and blp was 225- to 359-fold lower, while expression of pcbR-pbpA-bla and orf10 was only slightly affected if at all, indicating that resistance and regulatory genes are not under CcaR control as opposed to pathway biosynthetic genes. In the intergenic cmcH-ccaR region, a small messenger RNA (mRNA) overlaps with the cmcH transcription terminator. Deletion of 688 bp of the intergenic region results in a strain, S. clavuligerus ΔRI, still able to produce cephamycin C and clavulanic acid but at levels 30-40% lower than the parental strain. Therefore, specific sequences in the intergenic region upstream of ccaR enhance the expression of ccaR but are not essential for its expression. Strains containing an additional ccaR gene integrated in the chromosome, S. clavuligerus pSET-PC, or multiple copies of ccaR expressed from the PglpF promoter, S. clavuligerus pAK23, were constructed. Fermentations of the pAK23 strain resulted in a 6.1-fold increase in specific cephamycin C production relative to the wild type. In the same experiments, qRT-PCR analysis of the cephamycin biosynthesis genes showed a 5.1-fold increase in ccaR expression and similar increases in expression of lat and cmcI, while expression of other cluster genes were increased in the order of 2- to 3-fold.

Characterization of DNA‐binding sequences for CcaR in the cephamycin–clavulanic acid supercluster of Streptomyces clavuligerus

RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

Pcd Mutants of Streptomyces clavuligerus Still Produce Cephamycin C

Biosynthesis of cephamycin C in Streptomyces clavuligerus involves the initial conversion of lysine to alpha-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the lat gene causes complete loss of cephamycin production, pcd mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by pcd mutants could be restored to wild-type levels either by supplementation of the growth medium with alpha-aminoadipic acid or by complementation of the mutation with an intact copy of the pcd gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second pcd gene. Furthermore, cell extracts from pcd mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that S. clavuligerus must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis.

Insights into Cephamycin Biosynthesis: the Crystal Structure of CmcI from Streptomyces clavuligerus

Cephamycin C-producing microorganisms use two enzymes to convert cephalosporins to their 7alpha-methoxy derivatives. Here we report the X-ray structure of one of these enzymes, CmcI, from Streptomyces clavuligerus. The polypeptide chain of the enzyme folds into a C-terminal Rossmann domain and a smaller N-terminal domain, and the molecule packs as a hexamer in the crystal. The Rossmann domain binds S-adenosyl-L-methionine (SAM) and the demethylated product, S-adenosyl-L-homocysteine, in a fashion similar to the common binding mode of this cofactor in SAM-dependent methyltransferases. There is a magnesium-binding site in the vicinity of the SAM site with a bound magnesium ion ligated by residues Asp160, Glu186 and Asp187. The expected cephalosporin binding site near the magnesium ion is occupied by polyethyleneglycol (PEG) from the crystallisation medium. The geometry of the SAM and the magnesium binding sites is similar to that found in cathechol O-methyltransferase. The results suggest CmcI is a methyltransferase, and its most likely function is to catalyse the transfer of a methyl group from SAM to the 7alpha-hydroxy cephalosporin in the second catalytic reaction of cephamycin formation. Based on the docking of the putative substrate, 7alpha-hydroxy-O-carbamoyldeacetylcephalosporin C, to the structure of the ternary CmcI-Mg2+-SAM complex, we propose a model for substrate binding and catalysis. In this model, the 7-hydroxy group of the beta-lactam ring ligates the Mg2+ with its alpha-side facing the methyl group of SAM at a distance that would allow methylation of the hydroxyl-group.

Enhancement of clavulanic acid production in Streptomyces clavuligerus with ornithine feeding

The addition of glycerol, ornithine, and arginine had significant effects on the biosynthesis of clavulanic acid in Streptomyces clavuligerus. Adding ornithine in batch culture could enhance clavulanic acid production but arginine showed little effect. With an addition of 5 mM ornithine to the culture, the CA production (200 mg l −1) was about 2-fold of that without any addition of amino acids (115 mg l −1). In the fed-batch experiments, feeding glycerol could raise clavulanic acid production to 220 mg l −1. In the absence of glycerol, feeding ornithine or arginine only could not enhance the clavulanic acid production. We suggested that glycerol (C 3) supply rather than amino acid (C 5) supply is rate-limiting. Feeding glycerol with ornithine resulted in further increase in clavulanic acid production to 311 mg l −1, but feeding glycerol with arginine showed little enhancing effect. Feeding ornithine not only provided an enough supply of arginine for clavulanic acid production, but also inhibited the glycerol-utilizing cephamycin biosynthesis. Thus, it could increase the flux of C 3 precursor into clavulanic acid. The results reveal that ornithine rather than arginine could enhance effectively clavulanic acid production if the amount of C 3 precursor was sufficient.

The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the alpha-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.

Heterogeneous distribution of lysine 6-aminotransferase during cephamycin C biosynthesis in Streptomyces clavuligerus demonstrated using green fluorescent protein as a reporter.

The cellular distribution of the cephamycin biosynthetic enzyme lysine 6-aminotransferase (LAT) has been studied in Streptomyces clavuligerus hyphae by confocal microscopy using the S65T mutant of green fluorescent protein (GFP) as a reporter. LAT mediates the first committed step in the biosynthesis of the secondary metabolite cephamycin C by S. clavuligerus. The enzymic activity of LAT varies with time during the growth of S. clavuligerus in liquid medium. To investigate if this temporal variation occurs uniformly amongst all hyphae, S. clavuligerus was transformed with a plasmid containing the LAT-encoding gene translationally fused to the GFP-encoding gene. The LAT-GFP fusion product displayed fluorescence spectral characteristics of GFP, and showed similar temporal characteristics of LAT activity compared to the wild-type strain of S. clavuligerus. The transformed strain exhibited a heterogeneous distribution of fluorescence in mycelia grown in liquid cultures. This distribution varied significantly as the batch progressed: only a fraction of the mycelia fluoresced in the early growth phase, whereas nearly all hyphae fluoresced by the late growth phase. Thereafter, a non-uniform distribution of fluorescence was again observed in the declining growth phase. A large fraction of the non-fluorescent cells in the declining growth phase were found to be non-viable. Observations of S. clavuligerus colonies grown on solid agar also showed variation of LAT-GFP expression at different stages of growth. These observations in the solid phase can be explained in terms of nutrient deprivation and signalling molecules. The results suggest that physiological differentiation of S. clavuligerus mycelia leading to cephamycin C biosynthesis is both temporally and spatially distributed. The findings also revealed that the observed heterogeneity was independent of the position of individual cell compartments within the hypha. The potential of GFP as a reporter for the quantitative study of cephamycin biosynthesis at the cellular level has also been demonstrated.

Overexpression of the lat gene in Nocardia lactamdurans from strong heterologous promoters results in very high levels of lysine-6-aminotransferase and up to two-fold increase in cephamycin C production.

The level of lysine-6-aminotransferase (encoded by the lat gene), an enzyme that commits lysine to the cephamycin biosynthesis pathway, is very low in wild type Nocardia lactamdurans. Two lat overexpression systems (pAMEXlat and pSAFlat) were constructed to express the promoterless lat gene of N. lactamdurans from the strong promoters amyP (of the alpha-amylase gene) and safP (of the secretion activating factor gene) of Streptomyces griseus. Both constructions led to very high levels of lysine-6-aminotransferase (between 8- and 15-fold) in the cells. Expression of lat from the amy promoter was optimal in glycerol-containing medium and was negatively regulated by glucose. The high levels of lysine-6-aminotransferase resulted in a 50-200% increase in cephamycin C production in the standard fermentation conditions. Onset of cephamycin C biosynthesis occurred at the same time in control and in lat-overexpressing strains, but the cephamycin production rate was clearly higher in transformants overexpressing the lat gene. Furthermore, HPLC analysis of cephamycin C in the culture broths revealed an early depletion of biosynthetic intermediates and an accumulation of cephamycin C when the lat gene was overexpressed. These results indicate that lysine-6-aminotransferase activity is limiting for cephamycin C biosynthesis under some culture conditions.

Enzymes catalyzing the early steps of clavulanic acid biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus.

Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a "supercluster" in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.

Precursor and cofactor as a check valve for cephamycin biosynthesis in Streptomyces clavuligerus.

The biosynthesis of secondary metabolites is closely linked to primary metabolism via the supply of precursors, cofactors, and cellular energy. The availability of these precursors and cofactors can potentially be rate-limiting for secondary metabolism. A combined experimental and kinetic modeling approach was used to examine the regulation of flux in the cephamycin biosynthetic pathway in Streptomyces clavuligerus. The kinetic parameters of lysine 6-aminotransferase (LAT), the first enzyme leading to cephamycin biosynthesis and one which was previously identified as being a rate-limiting enzyme, were characterized. LAT converts lysine to alpha-aminoadipic acid using alpha-ketoglutarate as a cosubstrate. The K(m) values for lysine and alpha-ketoglutarate were substantially higher than those for their intracellular concentrations, suggesting that lysine and alpha-ketoglutarate may play a key role in regulating the flux of cephamycin biosynthesis. The important role of this precursor/cosubstrate was supported by simulated results using a kinetic model. When the intracellular concentrations and high K(m) values were taken into account, the predicted intermediate concentration was similar to the experimental measurements. The results demonstrate the controlling roles that precursors and cofactors may play in the biosynthesis of secondary metabolites.

Inducing effect of diamines on transcription of the cephamycin C genes from the lat and pcbAB promoters in Nocardia lactamdurans.

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the alpha-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbAB promoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

Investigation of the Streptomyces clavuligerus cephamycin C gene cluster and its regulation by the CcaR protein.

As part of a search for transcriptional regulatory genes, sequence analysis of several previously unsequenced gaps in the cephamycin biosynthetic cluster has revealed the presence in Streptomyces clavuligerus of seven genes not previously described. These include genes encoding an apparent penicillin binding protein and a transport or efflux protein, as well as the CmcI and CmcJ proteins, which catalyze late reactions in the cephamycin biosynthetic pathway. In addition, we discovered a gene, designated pcd, which displays significant homology to genes encoding semialdehyde dehydrogenases and may represent the gene encoding the long-sought-after dehydrogenase involved in the conversion of lysine to alpha-aminoadipate. Finally, two genes, sclU and rhsA, with no obvious function in cephamycin biosynthesis may define the end of the cluster. The previously described CcaR protein displays homology to a number of Streptomyces pathway-specific transcriptional activators. The ccaR gene was shown to be essential for the biosynthesis of cephamycin, clavulanic acid, and non-clavulanic acid clavams. Complementation of a deletion mutant lacking ccaR and the adjacent orf11 and blp genes showed that only ccaR was essential for the biosynthesis of cephamycin, clavulanic acid, and clavams and that mutations in orf11 or blp had no discernible effects. The lack of cephamycin production in ccaR mutants was directly attributable to the absence of biosynthetic enzymes responsible for the early and middle steps of the cephamycin biosynthetic pathway. Complementation of the ccaR deletion mutant resulted in the return of these biosynthetic enzymes and the restoration of cephamycin production.

The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene

Two genes, claR and car, encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb BglII-EcoRI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the cephamycin and clavulanic acid biosynthesis gene cluster. claR encoded a protein of 431 amino acids (deduced Mr 47080), that showed a significant degree of homology with several transcriptional activators of the LysR family. The ClaR protein contained two helix-turn-helix (HTH) motifs in the amino and carboxyl terminal regions. The second gene, car, encoded a protein of 247 amino acids (Mr 26629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus. Amplification of the claR gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas cephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or cephamycin production. Both claR and car are expressed as monocistronic transcripts; the level of transcript declined rapidly after 48h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96h. claR and car were not significantly expressed in mutants disrupted in the ccaR gene, a regulatory gene that controls positively clavulanic acid and cephamycin biosynthesis. These results indicate that clavulanic acid and cephamycin biosynthesis in S. clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.

Improved oxygen transfer in cultures of Nocardia lactamdurans maintains the cephamycin biosynthetic proteins for prolonged time and enhances the conversion of deacetylcephalosporin into cephamycin C

Batch cultures of Nocardia lactamdurans MA4213 in stirred tank fermentors (400 rpm) became oxygen limited after 45 h of incubation. When the stirrer speed was increased at 36 h from 400 to 600 rpm there was a 5-fold increase in the dissolved oxygen (DO) in the broth what resulted in a 2-fold stimulation of cephamycin biosynthesis. Immunoblotting studies using antibodies against the α-aminoadipyl-cysteinyl-valine synthetase and the protein P7 (cephem-7-hydroxylase) component of the methoxylation system showed that cultures with increased DO concentrations maintained high levels of these proteins for an extended period of time. Under these conditions there was an enhanced conversion of cephamycin intermediates into cephamycin C. Allophane and alumina exerted a strong stimulatory effect on cephamycin biosynthesis that correlated with high DO levels in allophane- or alumina-supplemented cultures at low agitation speeds. Allophane or alumina supplementation of N. lactamdurans cultures resulted in a delayed decay of the cephem-7-hydroxylase protein in the cells favoring conversion of the cephamycin intermediates into cephamycin C.

Δ-1-Piperideine-6-carboxylate dehydrogenase, a new enzyme that forms α-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes

Delta-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into alpha-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 microM) and NAD+ (apparent Km 115 microM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6 amino-transferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but no in actinomycetes that do no produce beta-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to alpha-aminoadipic acid.

Allophane increases the protein levels of several cephamycin biosynthetic enzymes in Nocardia lactamdurans

Summary: Addition of allophane, a phosphate-trapping agent, to two different strains of Nocardia lactamdurans exerted a large stimulatory effect on cephamycin biosynthesis. The biosynthesis of cephamycin is inhibited by inorganic phosphate at concentrations above 5 mM and allophane reversed this inhibitory effect. Allophane-supplemented cultures showed increased activities and/or an extended life in the cell of four cephamycin biosynthetic enzymes: isopenicillin N synthase, the two-protein-component 7α-cephem methoxylase (7α-cephem hydroxylase and 7-hydroxycephem methyltransferase) and 3′-hydroxymethylcephem O-carbamoyltransferase. However, the first enzyme of the pathway, lysine 6-aminotransferase, was not stimulated by allophane. Allophane-supplemented cultures showed increased protein levels of (i) α-aminoadipyl-cysteinyl-valine synthetase (the condensing multienzyme that forms the tripeptide intermediate), and (ii) the two proteins involved in the 7α-cephem methoxylase, as shown by immunoblotting with antibodies against each of these proteins. Phosphate repressed the de novo synthesis of these proteins but did not increase their degradation. These results indicated that allophane stimulates expression of the cluster of genes extending from the pcbAB gene (encoding α-aminoadipyl-cysteinyl-valine synthetase) to cmcl-cmcJ (encoding the two-protein methoxylase) and cmcH (encoding the O-carbamoyltransferase).

Effects of enhanced lysine ε-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus

Effects of enhanced lysine ε-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus

Effects of enhanced lysine ?-aminotransferase activity on cephamycin biosynthesis in Streptomyces clavuligerus

A recombinant strain of S. clavuligerus (LHM100) that contains an additional copy of the gene (lat) encoding lysine epsilon-aminotransferase (LAT) was analyzed and compared to the wild-type for intracellular concentrations of primary metabolites involved in cephamycin C biosynthesis. This strain had been shown previously to produce higher levels of the antibiotic because of increased levels of LAT, a rate-limiting enzyme involved in the production of alpha-aminoadipic acid. The results showed that the overall growth kinetics of the two strains were comparable, including the intracellular concentrations of cysteine, valine and lysine. In contrast, 60% higher antibiotic production was observed in LHM100, which reflected a significant temporal variation in specific metabolite production rate. The time profile of LAT activity was consistently higher in LHM100; however, alpha-aminoadipic acid levels showed unexpected variation during the growth cycle. These results support the proposal that rate-limiting enzymes in cephamycin C biosynthesis are temporally controlled, and indicate that optimization of metabolite production will require differential overexpression of several biosynthetic genes.