Mutation of N304 to leucine in Streptomyces clavuligerus deacetoxycephalosporin C synthase creates an enzyme with increased penicillin analogue conversion

Superimposition of deacetoxycephalosporin C synthase (DAOCS) and isopenicillin N synthase (IPNS) structures revealed that R74, R160, R266 and N304 are strategically located in the catalytic cavity of Streptomyces clavuligerus DAOCS (scDAOCS) and are crucial for orchestrating different substrates. Substitutions at these sites to a hydrophobic leucine residue were expected to stabilize the hydrophobic substrate bound state. Substantial improvements in the biotransformation of penicillin G, ampicillin and amoxicillin to their respective cephalosporin moieties were observed using the N304L mutant scDAOCS. Thus, our results have demonstrated the enhancement of scDAOCS activity via critical computational analysis and site-directed mutagenesis of endogenous ligands.     

Directed evolution of Streptomyces clavuligerus deacetoxycephalosporin C synthase for enhancement of penicillin G expansion

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the kcat/Km ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a kcat/Km ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

A complete library of amino acid alterations at N304 in Streptomyces clavuligerus deacetoxycephalosporin C synthase elucidates the basis for enhanced penicillin analogue conversion

N304 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic ability. Given that N304A, N304K, N304L, and N304R mutant enzymes exhibited significant improvements in penicillin analogue conversions, we advocate that replacement of N304 with residues with aliphatic or basic side chains is preferable for engineering of a hypercatalytic enzyme.     

Purification and initial characterization of deacetoxycephalosporin C synthase from Streptomyces clavuligerus

Deacetoxycephalosporin C synthase, the penicillin N ring expansion enzyme from Streptomyces clavuligerus, was purified to near homogeneity, as judged by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The synthase was monofunctional and could be completely separated from deacetoxycephalosporin C hydroxylase activity early in the purification sequence. Synthase specific activity was increased 97-fold over crude cell-free extracts, and the purified enzyme appeared to be a monomer with a molecular weight of 36,000 and a Km for the penicillin N substrate of 50 microM. Deacetoxycephalosporin C synthase activity required alpha-ketoglutarate, Fe2+, and oxygen and was specifically stimulated by ascorbate and dithiothreitol. The enzyme was sensitive to thiol-specific inhibitors, the most effective of which was N-ethylmaleimide.     

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.

We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.     

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.     

C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity

Here we investigated the effect of pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, on early-phase hepatic fibrogenesis in vivo caused by acute carbon tetrachloride (CCl(4)) administration in the rat. Pioglitazone (1 mg/kg BW) prevented pericentral fibrosis and induction of alpha-smooth muscle actin (SMA) 72 h after CCl(4) administration (1 ml/kg BW). CCl(4) induction of alpha1(I)procollagen mRNA in the liver was blunted by pioglitazone to the levels almost 2/3 of CCl(4) alone. Pioglitazone also prevented CCl(4)-induced hepatic inflammation and necrosis, as well as increases in serum tumor necrosis factor-alpha levels. Further, pioglitazone inhibited the induction of alphaSMA and type I collagen in primary cultured hepatic stellate cells in a dose-dependent manner. In conclusion, pioglitazone inhibits both hepatic inflammation and activation of hepatic stellate cells, thereby ameliorating early-phase fibrogenesis in the liver following acute CCl(4).     

Glucose regulation of cephamycin biosynthesis in Streptomyces lactamdurans is exerted on the formation of alpha-aminoadipyl-cysteinyl-valine and deacetoxycephalosporin C synthase

Glucose exerted a concentration-dependent negative regulation on the biosynthesis of cephamycin C by Streptomyces lactamdurans. Formation of the cephamycin precursor delta(alpha-aminoadipyl)-cysteinyl-valine was greatly decreased by excess glucose. The ring-expanding enzyme deacetoxycephalosporin C synthase was strongly repressed by glucose in vivo. Isopenicillin N synthase (cyclase) and isopenicillin N epimerase were not repressed by glucose. However, the activity of isopenicillin N synthase was inhibited in vitro by glucose 6-phosphate, and the activity of deacetoxycephalosporin C synthase was inhibited by inorganic phosphate, glucose 6-phosphate, fructose 2,6-diphosphate and fructose 1,6-diphosphate. The intracellular cAMP content decreased as growth proceeded and remained lower in glucose-supplemented cells than in control cultures. cAMP did not seem to be involved in glucose control of cephamycin biosynthesis.     

Solid state cultivation of Streptomyces clavuligerus for cephamycin C production

Solid state cultivation of Streptomyces clavuligerus for cephamycin C production was carried out in a system consisting of wheat rawa 5 g; cotton seed deoiled cake 5 g; sunflower cake 0·5 g; corn steep liquor 1 g; MgSO₄.7H₂O 0·06 g; CaCO₃ 0·1 g; K₂HPO₄ 4·4 g; with initial moisture content of 80%, initial pH 6·5 and a fermentation temperature in the range 28–30°C. The fermentation cycle was about 5 days. Streptomyces clavuligerus growth was observed on the 2nd day and production of cephamycin C was initiated on 3rd day. Abundant mycelial growth was observed from the 3rd day and reached stationary phase by the 5th day. Cephamycin C was produced maximally at a rate of 15 mg/g substrate on the 5th day and was stable until the 30th day with only marginal decrease in titre.     

Carbon catabolite regulation of cephamycin C and expandase biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produces cephamycin C while growing on chemically defined basal medium. Cephamycin C production takes place during the exponential growth phase and is accompanied by vigorous activity of the cephamycin C synthetase system and of expandase. An excessive amount of glycerol decreases cephamycin C production. Its negative effect appears to be greatest when it is added in the first phase of fermentation either alone or in the presence of starch. Starch excess also reduces cephamycin C production, but its effect is slight compared with glycerol. Glycerol hinders cephamycin C production by the repression of the cephamycin C synthetase system and particularly expandase biosynthesis. Starch and glycerol inhibit neither cephamycin C synthetase nor expandase activities. However, the phosphorylated intermediates of the glycolytic pathway, glucose 6-phosphate and fructose 1,6-phosphate, strongly inhibit expandase activity.     

Production of clavulanic acid and cephamycin C by Streptomyces clavuligerus in palm-oil medium

Palm and palm-kernel oils and their olein and stearin fractions were suitable as the main carbon sources for growth and production of clavulanic acid by Streptomyces clavuligerus. However, oleic and lauric acids were not utilized for growth. A spontaneous mutant, which was selected for higher cephamycin C production, also produced more clavulanic acid with these oils in the medium.     

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.     

Genetic recombination in the cephamycin producer,Streptomyces clavuligerus

Summary In order to study the mechanism of cephamycin production by streptomycetes and to use genetic recombination in strain development, we undertook genetic studies inStreptomyces lipmanii andS. clavuligerus. S. lipmanii crosses gave 0.005–1.3% prototroph-like colonies, but all segregated back to parental genotypes. Crosses ofS. clavuligerus resulted in lower frequencies of prototroph-like colonies, i.e., 0.00002–0.9%. In ade x ura and ade x his crosses, the recombinant progeny did not segregate back. In arg x ade and arg x his crosses, segregation occurred in about 50% of the progeny. These data demonstrate that true haploid recombinants occur in crosses ofS. clavuligerus. S. lipmanii yielded only heterokaryons and, therefore, is less suitable thanS. clavuligerus for further genetic study.     

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.     

The effect of cysteine mutations on recombinant deacetoxycephalosporin C synthase from S. clavuligerus

Cysteines 100, 155, and 197 of recombinant deacetoxycephalosporin C synthase were mutated to alanine residues. The C100A mutant had properties similar to those of the wild-type enzyme, but mutation of Cys-155 and Cys-197 reduced enzyme activity with penicillin N and penicillin G to different extents.     

Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus

A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D., and Miller, J. R. (1989) J. Bacteriol. 171, 754-760). The synthase protein was detected mainly from granules of recombinant E. coli. The recombinant synthase was solubilized from the granules by urea, and for the first time a highly active synthase was purified to near homogeneity. The synthase was a monomer with a Mr of 34,600 and exhibited two isoelectric points of 6.1 and 5.3. Its catalytic activity required alpha-ketoglutarate, Fe2+, and O2, was stimulated by dithiothreitol or ascorbate but not by ATP, and was optimal at pH 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and at 36 degrees C. The Fe2+ requirement was specific, and at least one sulfhydryl group in the purified enzyme was apparently essential for the ring expansion. The Km values of the enzyme for penicillin N and alpha-ketoglutarate were 29 and 18 microM, respectively, and the Ka for Fe2+ was 8 microM. The recombinant synthase was indistinguishable from the native synthase of S. clavuligerus by those biochemical properties. In addition to the enzymic ring expansion of penicillin N to deacetoxycephalosporin C, the recombinant synthase catalyzed a novel hydroxylation of 3-exomethylenecephalosporin C to deacetylcephalosporin C.     

Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus

The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for β-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M _r of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protein was purified. The cloned gene was used to construct a plasmid containing a hom disruption cassette which was then transformed into S. clavuligerus. A hom mutant of S. clavuligerus was obtained by insertional inactivation via double crossover, and the effect of hom gene disruption on cephamycin C yield was investigated by comparing antibiotic levels in culture broths of this mutant and in the parental strain. Disruption of hom gene resulted in up to 4.3-fold and twofold increases in intracellular free l-lysine concentration and specific cephamycin C production, respectively, during stationary phase in chemically defined medium.     

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase.

Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.