Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k_cat/K_m ratio and 7-fold increase in relative activity.     

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Contrasting fates for 6-alpha-methylpenicillin N upon oxidation by deacetoxycephalosporin C synthase (DAOCS) and deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS).

6-alpha-methylpenicillin N was synthesised via known routes from 6-aminopenicillanic acid, and tested as a substrate for recombinant DAOCS and DAOC/DACS. Incubation with DAOCS resulted in conversion of 2-oxoglutarate without oxidation of the penicillin substrate ('uncoupled turnover'). Incubation with DAOC/DACS resulted in oxidation to the cephem aldehyde. This is the first example of substrate-induced 'uncoupled turnover', which has been proposed to be an editing mechanism for these enzymes.     

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium.

Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.     

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.     

Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over that in the untransformed host. Received 5 January 1998/ Accepted in revised form 29 May 1998     

Expression of cefF significantly decreased deacetoxycephalosporin C formation during cephalosporin C production in Acremonium chrysogenum

Deacetoxycephalosporin C (DAOC) is not only the precursor but also one of the by-products during cephalosporin C (CPC) biosynthesis. One enzyme (DAOC/DAC synthase) is responsible for the two-step conversion of penicillin N into deacetylcephalosporin C (DAC) in Acremonium chrysogenum, while two enzymes (DAOC synthase and DAOC hydroxylase) were involved in this reaction in Streptomyces clavuligerus and Amycolatopsis lactamdurans (Nocardia lactamdurans). In this study, the DAOC hydroxylase gene cefF was cloned from Streptomyces clavuligerus and introduced into Acremonium chrysogenum through Agrobacterium tumefaciens-mediated transformation. When cefF was expressed under the promoter of pcbC, the ratio of DAOC/CPC in the fermentation broth significantly decreased. These results suggested that introduction of cefF could function quite well in Acremonium chrysogenum and successfully reduce the content of DAOC in the CPC fermentation broth. This work offered a practical way to improve the CPC purification and reduce its production cost.     

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans

The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.     

Relevant Double Mutations in Bioengineered Streptomyces clavuligerus Deacetoxycephalosporin C Synthase Result in Higher Binding Specificities Which Improve Penicillin Bioconversion

Streptomyces clavuligerus deacetoxycephalosporin C synthase (ScDAOCS) is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid, which is a precursor for cephalosporin synthesis. Single mutations of six amino acid residues, V275, C281, N304, I305, R306, and R307, were previously shown to result in enhanced levels of ampicillin conversion, with activities ranging from 129 to 346% of the wild-type activity. In this study, these mutations were paired to investigate their effects on enzyme catalysis. The bioassay results showed that the C-terminal mutations (N304X [where X is alanine, leucine, methionine, lysine, or arginine], I305M, R306L, and R307L) in combination with C281Y substantially increased the conversion of ampicillin; the activity was up to 491% of the wild-type activity. Similar improvements were observed for converting carbenicillin (up to 1,347% of the wild-type activity) and phenethicillin (up to 1,109% of the wild-type activity). Interestingly, the N304X R306L double mutants exhibited lower activities for penicillin G conversion, and activities that were 40 to 114% of wild-type enzyme activity were detected. Based on kinetic studies using ampicillin, it was clear that the increases in the activities of the double mutants relative to those of the corresponding single mutants were due to enhanced substrate binding affinities. These results also validated the finding that the N304R and I305M mutations are ideal for increasing the substrate binding affinity and turnover rate of the enzyme, respectively. This study provided further insight into the structure-function interaction of ScDAOCS with different penicillin substrates, thus providing a useful platform for further rational modification of its enzymatic properties.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.     

Studies on the cell-free biosynthesis of beta-lactam antibiotics.

Cell walls of Cephalosporium acremonium mycelia were lysed by enzyme preparations from either Helix pomatia (snail) digestive juice or Cytophaga. The yield of protoplasts depended on the lytic-enzyme preparation and the age of the culture, and it increased after the mycelia were pretreated with dithiothreitol. A cell-free preparation, obtained by osmotic lysis of protoplasts, synthesized labelled penicillin N from L-[14C]valine. Approx. 0.03-0.06% of the amino acid was incorporated into penicillin N. Under conditions of penicillin N synthesis, the broken-protoplast preparation failed to produce significant amounts of cephalosporin C or its precursors, deacetylcephalosporin C and deacetoxycephalosporin C.     

Evolving enzyme technology for pharmaceutical applications: case studies

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent. Received 07 March 1997/ Accepted in revised form 15 June 1997     

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.     

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.     

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.     

扩环酶的研究进展及其应用前景

扩环酶,也叫脱乙酰氧基头孢菌素Ｃ合成酶,催化青霉素Ｎ扩环生成脱乙酰氧基头孢菌素Ｃ,是头孢菌素生物合成中的关键酶。在真菌中它是一个双功能酶,同时具有扩环酶和羟化酶的活性;而在细菌中扩环和羟化却是由两个独立的酶来承担的。近年来,扩环酶被纯化成均一蛋白,有关它的性质、分子结构以及基因结构等方面的研究都取得了飞速发展,并不断地应用基因工程的技术探索其在抗生素生产上的应用。     

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.     

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.