Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine -aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

Time-lapsed confocal microscopy reveals temporal and spatial expression of the lysine epsilon-aminotransferase gene in Streptomyces clavuligerus

To investigate the temporal and spatial expression patterns of the gene (lat ) encoding lysine epsilon-aminotransferase (LAT) for cephamycin C biosynthesis, a mutant form of green fluorescent protein (mut1GFP) was integrated into the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of the recombinant protein paralleled the native LAT enzyme activity profile in wild-type S. clavuligerus, which peaked during exponential growth phase and decreased slowly towards stationary phase. These results indicate that the LAT-Mut1GFP fusion protein retains both LAT and GFP functionality in S. clavuligerus LH369. LH369 produced wild-type levels of cephamycin C in minimal medium culture conditions supplemented with lysine. Time-lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and spatial characteristics of lat gene expression and demonstrated that physiological development of S. clavuligerus colonies leading to cephamycin C biosynthesis is limited to the substrate mycelia.     

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

Cell-free extracts from Streptomyces clavuligerus, purified by elution from heparin-agarose with an ARE-containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE(ccaR)). This sequence is [corrected] located upstream of the ccaR gene, encoding [corrected] the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty-one nucleotides upstream of brp another ARE sequence (ARE(brp)) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp-deleted mutant, S. clavuligerus Deltabrp::neo1, produced 150-300% clavulanic acid and 120-220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Deltabrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a [corrected] slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C [corrected] biosynthesis by its action on ccaR gene expression.     

Mutants of Streptomyces clavuligerus with disruptions in different genes for clavulanic acid biosynthesis produce large amounts of holomycin: possible cross-regulation of two unrelated secondary metabolic pathways

A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a beta-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.     

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.     

Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit

The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin.     

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus

Cell-free extracts from Streptomyces clavuligerus, purified by elution from heparin-agarose with an ARE-containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE_ccaR). This sequence is located upstream of the ccaR gene, encoding the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty-one nucleotides upstream of brp another ARE sequence (ARE_brp) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp-deleted mutant, S. clavuligerus Δbrp::neo1, produced 150–300% clavulanic acid and 120–220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Δbrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C biosynthesis by its action on ccaR gene expression.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites

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.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase (lat) gene in cephamycin C biosynthesis.

Targeted gene insertion methodology was used to study the effect of perturbing alpha-aminoadipic acid precursor flux on the overall production rate of beta-lactam biosynthesis in Streptomyces clavuligerus. A high-copy-number plasmid containing the lysine epsilon-aminotransferase gene (lat) was constructed and used to transform S. clavuligerus. The resulting recombinant strain (LHM100) contained an additional complete copy of lat located adjacent to the corresponding wild-type gene in the chromosome. Biological activity and production levels of beta-lactam antibiotics were two to five times greater than in wild-type S. clavuligerus. Although levels of lysine epsilon-aminotransferase were elevated fourfold in LHM100, the level of ACV synthetase, whose gene is located just downstream of lat, remained unchanged. These data strongly support the notion that direct perturbation of alpha-aminoadipic acid precursor flux resulted in increased antibiotic production. This strategy represents a successful application of metabolic engineering based on theoretical predictions of precursor flux in a secondary metabolic pathway.     

Rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces clavuligerus

Clavulanic acid is a potent beta-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, we genetically engineered the glycolytic pathway in Streptomyces clavuligerus. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild type, suggesting that arginine became limiting for biosynthesis. This is the first reported application of genetic engineering to channel precursor flux to improve clavulanic acid production.     

Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein

The DNA-binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investigated by gel mobility retardation and DNase I footprinting analysis. These results revealed that CcaR specifically binds to the promoter region of the lysine-epsilon-aminotransferase gene (lat). Green fluorescent protein (GFP) was subsequently used as a reporter to analyse in vivo expression of CcaR. The corresponding isogenic strain containing ccaR:gfp in the chromosome produced cephamycin C at levels similar to those of wild-type S. clavuligerus. Confocal laser scanning microscopy revealed that expression of CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. The highly fluorescent seed culture mycelia quickly lost fluorescence upon inoculation into fresh culture medium. The characteristic green colour reappeared in a small portion of mycelia during mid-exponential growth phase. As the culture aged, the population expressing CcaR expanded, and the expression level increased. This was followed by a reduction in the CcaR-expressing population towards the end of the culture period. During peak expression, CcaR was distributed uniformly in mycelia, but became localized distal to the chromosome when the culture entered stationary phase. In solid phase analysis, abundant CcaR expression was evident in the substrate mycelia, but was completely absent in aerial hyphae. These results show regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic production. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrates a distinct pattern of localization over the course of physiological differentiation.     

Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064

The effects of growth rate and nutrient uptake rate on the production of cephamycin C were determined in the parental strain, deltarelA mutant, and deltarsh null mutant of S. clavuligerus. Production of cephamycin C was inversely related to mycelium growth and the phosphate feed rate was more critical for the production of cephamycin C. On the contrary, the production of cephamycin C was completely abolished in the deltarelA mutant, but not in deltarsh mutant. The changes in the cephamycin C production by disruption of the relA and rsh genes are presumably associated with the consequent ability of the mutants to accumulate (p)ppGpp under nutrient starvation. Therefore, it is concluded that the stringent response of S. clavuligerus to starvation for nutrients is governed mainly by RelA rather than Rsh and that the response is more apparently regulated by the limitation of phosphate.     

Two oligopeptide-permease-encoding genes in the clavulanic acid cluster of Streptomyces clavuligerus are essential for production of the beta-lactamase inhibitor

orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical. These proteins show sequence similarity to periplasmic oligopeptide-binding proteins. Mutant S. clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production. Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2. The mutant S. clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5). Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1. Both genes encode oligopeptide permeases with different substrate specificities. The disrupted S. clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes. The null S. clavuligerus oppA1::acc and S. clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins.     

CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus

The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.     

Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine

The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.     

Coordinate production of cephamycin c and clavulanic acid by Streptomyces clavuligerus

Production of cephamycin c and clavulanic acid by Streptomyces clavuligerus was investigated using different media in shake flask condition. Highest cell growth (3.8 g/L) was observed in glycerol, sucrose, proline and glutamic acid (GSPG) medium. Although, GSPG medium supported maximum growth, it was least effective for the synthesis of both cephamycin and clavulanic acid. Yield of cephamycin and clavulanic acid was maximum in dextrin and K medium, respectively. High and low level of constituents of dextrin medium, affected production of both cephamycin and clavulanic acid. Biosynthesis of clavulanic acid was associated with production of cephamycin c.