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 epsilon-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.     

Differential stringent responses of Streptomyces coelicolor M600 to starvation of specific nutrients

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.     

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.     

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.     

Connecting primary and secondary metabolism: AreB, an IclR-like protein, binds the ARE(ccaR) sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production

A protein binding to the autoregulatory element (ARE) upstream of the regulatory ccaR gene of Streptomyces clavuligerus was isolated previously by DNA affinity binding. The areB gene, encoding this protein, is located upstream and in opposite orientation to the leuCD operon of S. clavuligerus; it encodes a 239-amino-acid protein of the IclR family with a helix-turn-helix motif at the N-terminal region. An areB-deleted mutant, S. clavuligerusDeltaareB, has been constructed by gene replacement. This strain requires leucine for optimal growth in defined media. Expression of the leuCD operon is retarded in S. clavuligerusDeltaareB, because AreB binds the areB-leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the DeltaareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the ARE(ccaR) sequence (as shown by EMSA) unless filtered extracts from S. clavuligerus ATCC 27064-containing molecules of Mr lower than 10 kDa are added to the binding reaction. Restoration of binding to the ARE(ccaR) sequence is not observed when filtered extracts are obtained from the DeltaareB mutant, suggesting that biosynthesis of the small-molecular-weight effector is also controlled by AreB.     

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.     

Effect of water activity on production of β-lactam antibiotics by Streptomyces clavuligerus in submerged culture

The amount of available water in the environment of micro-organisms, defined as water activity ( a _W), has been shown to affect growth, respiration, enzyme synthesis, sporulation and other physiological functions. The aim of this study was to examine the impact of a _W on production/excretion of a secondary metabolite. For this purpose, the production of β-lactam antibiotics and biomass of Streptomyces clavuligerus was studied in relation to the a _W-depressing agents glucose, sorbitol and NaCl. These were chosen because NaCl and sorbitol are often used to depress a _W and glucose was not thought to be taken up by S. clavuligerus. The filamentous bacterium S. clavuligerus NRRL 3585 (ATCC 27064) is a prokaryotic producer of penicillin N, cephalosporins including cephamycin C and clavulanic acid. Under water stress conditions, a greater effect upon antibiotic biosynthesis than upon growth was consistently observed. When a _W was decreased to below 0·997, antibiotic production began to decrease. For growth, inhibition was much more gradual and did not become intensive until an a_W of 0·990 was reached.     

Isopenicillin N synthase and desacetoxycephalosporin C synthase activities during defined medium fermentations of Streptomyces clavuligerus: effect of oxygen and iron supplements

When the level of dissolved oxygen was increased to saturation in defined media fermentations of Streptomyces clavuligerus, the total duration of activity of the penicillin ring cyclization enzyme, isopenicillin N synthase (IPNS), was extended by at least 20 h; however, no increase in the stability of the ring expansion enzyme, desacetoxycephalosporin C synthase (DAOCS), was observed. Consequently, the conversion of the excreted intermediate penicillin N to cephamycin C was 15-20% less efficient at this high oxygen concentration. The increased dissolved oxygen level also led to the complete loss of IPNS and DAOCS activities for 4 h during the period of fastest growth, and the rate of specific cephamycin C production fell to zero. A several hundred fold increase in the level of iron in the defined media resulted in a sixfold improvement in the rate of specific cephamycin C production after 60 h fermentation. This increased rate appeared to be due to an elevation in the in vivo activities of a number of the cephamycin biosynthetic enzymes, particularly those catalysing later pathway steps.     

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus

Streptomyces clavuligerus produces a large array of natural compounds with antibiotic, antitumor, beta-lactamase inhibition or inmunomodulating activities. The production of cephamycin C, clavulanic acid and other compounds with a clavam structure has been studied for many years. A network of regulatory mechanisms is present in S. clavuligerus to control the formation of different compounds by pathway-specific regulators or pleiotropic regulators. The possible existence of a gamma-butyrolactone signaling system in this streptomycete is emerging. In addition, S. clavuligerus possesses a stringent control mechanism somehow different from those previously reported in other Streptomyces species.     

Oxygen derepresses deacetoxycephalosporin C synthase and increases the conversion of penicillin N to cephamycin C in Streptomyces clavuligerus.

When dissolved oxygen (DO) was maintained at saturation level during batch fermentations of Streptomyces clavuligerus (NRRL 3585), the accumulation of the intermediate penicillin N was lowered while formation of the end product cephamycin C was increased relative to fermentations without DO control. The specific activity of the penicillin ring-expansion enzyme deacetoxycephalosporin C synthase (DAOCS) was increased 2.3-fold under oxygen saturated conditions, whereas the penicillin ring-cyclizing enzyme isopenicillin N synthase (IPNS) showed only a 1.3-fold increase. Thus oxygen derepression of DAOCS appears to be an important regulatory mechanism in the conversion of penicillin N to cephamycin C in S. clavuligerus. IPNS, an early acting enzyme in cephamycin C biosynthesis, and DAOCS, which acts late in the pathway, both disappeared from cell extracts at 60 h, just prior to cessation of cephamycin production.     

Effect of support materials on cephamycin C production by immobilized Streptomyces clavuligerus

In order to know the effect of supports on cephamycin C production, under similar experimental conditions, S. clavuligerus cells were immobilized with--sponge, 2% agar, 2% and 4% alginate support materials. An experimental set of free cell was also maintained as control. Cephamycin C production by these immobilized and free cells was estimated at 48, 96 and 120 hr of fermentation. In all the cases cephamycin C production was found to be high at 120 hr of fermentation. Sponge was found to be a better support material than other supports used for immobilization.     

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.     

Lysine epsilon-aminotransferase, the initial enzyme of cephalosporin biosynthesis in actinomycetes

Streptomyces clavuligerus, Streptomyces lipmanii and Nocardia (formerly Streptomyces) lactamdurans are Gram-positive mycelial bacteria that produce medically important beta-lactam antibiotics (penicillins and cephalosporins including cephamycins) that are synthesized through a series of reactions starting from lysine, cysteine and valine. L-lysine epsilon-aminotransferase (LAT) is the initial enzyme in the two-step conversion of L-lysine to L-alpha-aminoadipic acid, a specific precursor of all penicillins and cephalosporins. Whereas S. clavuligerus uses LAT for cephalosporin production, it uses the cadaverine pathway for catabolism when lysine is the nitrogen source for growth. Although the cadaverine path is present in all examined streptomycetes, the LAT pathway appears to exist only in beta-lactam-producing strains. Genetically increasing the level of LAT enhances the production of cephamycin. LAT is the key rate-limiting enzyme in cephalosporin biosynthesis in S. clavuligerus strain NRRL 3585. This review will summarize information on this important enzyme.     

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.     

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.     

Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: effects on cephamycin C production

In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the wild strain resulted in a maximum of 3.1- and 3.3-fold increase in specific, 1.7- and 1.9-fold increase in volumetric cephamycin C production when grown in trypticase soy broth (TSB) and a modified chemically defined medium (mCDM), respectively. However, expression of multicopy ask gene in a hom-deleted background significantly decreased cephamycin C yields when the cells were grown in either TSB or mCDM, most probably due to physiological disturbance resulting from enzyme overexpression and high copy number plasmid burden in an auxotrophic host, respectively.     

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.     

Biosynthesis and molecular genetics of cephamycins

Cephamycin C is produced in a nine steps pathway by the actinomycetes S. clavuligerus and N. lactamdurans. The genes encoding the biosynthesis enzymes are clustered in both microorganisms as well as in the cephabacin producer Lysobacter lactamgenus, a Gram negative bacterium. The clusters of genes include genes encoding enzymes common to the biosynthesis of penicillin and cephalosporin C by the eukaryotic producers Penicillium chrysogenum and Cephalosporiun acremonium and genes for steps specific for the formation of the precursor -aminoadipic acid as well as for the enzymes involved in the late modification of the cephalosporin intermediates of the pathway. Present are also genes for proteins involved in the export and/or resistance to cephamycin C. In S. clavuligerus a gene encoding a regulatory protein controlling the formation of cephamycin C and clavulanic acid is also present in the cluster.