ABSTRACT
Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the kcat/Km ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.
Cephalosporins, which constitute one of the major β-lactam antibiotic classes, are produced by several microorganisms, such as filamentous fungi, actinomycetes, and gram-negative bacteria. The biosynthesis of cephalosporins in Streptomyces clavuligerus and Acremonium chrysogenum has been studied extensively (19). In S. clavuligerus, deacetoxycephalosporin C synthase (DAOCS) (expandase), encoded by cefE, catalyzes the oxidative expansion of the five-membered thiazolidine ring of penicillin N into the six-membered dihydrothiazine ring of deacetoxycephalosporin C (DAOC) (13) (Fig. 1). DAOC is sequentially converted to deacetylcephalosporin C (DAC) by deacetylcephalosporin C synthase (DACS) (hydroxylase), encoded by cefF (12). Both expandase and hydroxylase are α-ketoglutarate and Fe(II) dependent oxygenases and share 55 to 60% amino acid identity. Interestingly, the enzyme from A. chrysogenum can catalyze the direct conversion of penicillin N to DAC due to possession of both expandase and hydroxylase activity; hence the encoded gene is termed cefEF (24).
The biosynthesis of cephalosporins from penicillins catalyzed by the expandase (DAOCS) and hydroxylase (DACS).
7-Aminodeacetoxycephalosporanic acid (7-ADCA) is one of the most important semisynthetic cephalosporins, and its current production includes the ring expansion of penicillin G into phenylacetyl-7-ADCA and the well-documented enzymatic removal of the phenylacetyl group. Due to potential pollution by the toxic chemicals used for the ring expansion, Penicillium chrysogenum and A. chrysogenum have been engineered metabolically to produce 7-ADCA derivatives (7, 27). However, the derivatives from these strains still need further complicated enzymatic processing. Therefore, the production of a penicillin G-specific expandase would be a more straightforward solution to the problems of 7-ADCA production.
Several expandases have been screened from actinomycetes, and the S. clavuligerus enzyme has been well characterized. These enzymes have broad substrate specificity and can catalyze ring expansion in penicillins with various side chains. However, they prefer their physiological substrate, penicillin N, rather than penicillin G (4). Random mutagenesis and structure-based mutation analysis have improved the substrate specificity for penicillin G but with only a limited degree of enhancement (2, 3, 17, 18, 28). For example, Wei and colleagues have obtained a more active S. clavuligerus expandase mutant but only with an increase in the kcat/Km value of 32-fold (28). In this study, we first screened several new expandase genes from actinomycetes and enhanced the substrate specificity for penicillin G using DNA family shuffling. After two rounds, we have generated a chimeric expandase with a significantly better kcat/Km value, 118-fold higher than that of the S. clavuligerus enzyme.
MATERIALS AND METHODS
Screening of new expandase genes from soil actinomycetes.Various soil samples were collected from Fu-Shan Research Station, Ilan; Chatienshan, Taoyuan; and Smangus, Hsinchu, and Green Island, Taitung, in Taiwan. Soil suspensions were pretreated with sodium dodecyl sulfate (SDS)-yeast extract solution (6% yeast extract and 0.05% SDS) at 40°C for 30 min (9) or with 1.5% phenol solution at 30°C for 30 min (10) before being plated on humic acid-vitamin agar (8) and actinomycete isolation agar (Difco) with 20 μg of nalidixic acid/ml and 200 μg of cycloheximide/ml. The plates were incubated at 30°C for 7 to 14 days. The actinomycete isolates were then streaked on ISP (International Streptomyces Project) no. 4 medium plates and stored.
For the Escherichia coli ESS assay (4), a piece of agar (∼0.5 cm3) containing each actinomycete isolate was placed on Luria broth-penicillinase plates seeded with the E. coli ESS strain, a β-lactam-supersensitive mutant, and the plates were incubated overnight at 37°C. Formation of a clear zone indicates that the actinomycete isolate secretes non-penicillin-type antibiotics, causing bacterial lysis. Expandase-homologous genes from those non-penicillin-type strains were then identified by screening with two radiolabeled probes (nucleotides 157 to 305 and 487 to 611 of S. clavuligerus cefE, generated by incorporation of [α-32P]dCTP, using PCR amplification).
Construction and screening of genomic DNA.The chromosomal DNA from the selected isolates were purified and partially digested with BamHI or Sau3AI. The recovered 2- to 7-kb DNA fragments were ligated into the BamHI site of the λZAP Express vector (Stratagene), and the recombinant phagemids were packaged in vitro by the Gigapack III gold packaging extract (Stratagene) to construct a genomic library. Plaque hybridization was performed, and phagemids were excised from positive phage clones as recommended by the manufacturer.
DNA electrophoresis, Southern blotting, and general recombinant DNA techniques were carried out by standard procedures. Plasmid DNA was isolated from E. coli strains using a plasmid miniprep kit (Viogene). All restriction enzymes were purchased from Promega, and digests were purified by using a QIAquick PCR purification kit (QIAGEN). DNA fragments from agarose gels were recovered by using the QIAquick gel extraction kit (QIAGEN). Genomic DNA of actinomycetes was prepared as described by Kutchma et al. (15). The oligonucleotides were synthesized by Mission Biotech, Taipei, Taiwan, on an ABI 3900 high-throughput DNA synthesizer. DNA sequencing was performed using an ABI PRISM dye terminator cycle sequence kit and an ABI 377 DNA sequencer by Mission Biotech.
Identification and phylogenetic analysis of isolates. Taxonomic identification of actinomycete isolates was deputized to the Bioresources Collection and Research Center (Hsinchu, Taiwan). Together with the cefE genes from S. clavuligerus (accession number P18548 ), Streptomyces jumonjinensis (AAL09460 ), and Nocardia lactamdurans (Q03047 ), and the cefEF gene from A. chrysogenum (P11935 ), phylogenetic analysis of our newly identified genes was performed by the PHYLIP package using the neighbor-joining method (23). Robustness of the tree (100 pseudo-replications) was assessed by the bootstrap and interior branch tests, and the tree was drawn by using TreeView software (21).
Enzyme-directed evolution.The previously identified cefE genes from S. clavuligerus (ATCC 27064), N. lactamdurans (ATCC 27382), and S. jumonjinensis (ATCC 29864), the cefF gene from S. clavuligerus, the cefEF gene from A. chrysogenum (ATCC 11550), and our newly cloned genes were isolated by PCR and cloned into the NdeI/HindIII-treated expression vector, pET24a (Novagen). After amplification of the homologous genes by PCR, DNase I was used to fragment the genes randomly, and fragments of around 100 bp in size were collected. The 100-bp fragments were added to a primerless polymerase reaction for reassembly, and external primers were added to amplify the recombinant expandase genes (6, 25), which were then expressed in Tuner(DE3) (Novagen). Conditions for primerless and amplification PCR were as follows: 5 min at 96°C and 40 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C, followed by 10 min at 72°C; 5 min at 98°C and 20 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C, followed by 10 min at 72°C. Transformants were cultured in 96-well plates followed by an E. coli ESS bioassay as described previously (28), except for use of a substrate concentration of 10 mM penicillin G. The Tuner(DE3)-based transformant YS16, containing the S. clavuligerus cefE gene, was used as a positive control. For the second shuffling round, the clones with a higher activity than that of YS16 were used, and one of the clones, F42, was used as the positive control. Finally, the family-shuffled expandases with significantly enhanced activities were purified by a fast protein liquid chromatography system, and their kinetic parameters were analyzed by a high-performance liquid chromatography system using 20 μg of enzyme and 0.1 to 10 mM penicillin G in a total volume of 200 μl of assay mixture as reported previously (28).
RESULTS
Screening of new expandase genes from soil actinomycetes.From 181 soil samples, we isolated 1,835 actinomycetes-like colonies, and most of the isolates were obtained by using the soil pretreatment of SDS-yeast extract method. Subsequently, 133 isolates producing non-penicillin-type antibiotics, as assayed by E. coli ESS bioassay, were selected. Based on Southern blotting analysis, 14 actinomycete isolates were found to carry expandase-homologous genes, and these were distributed into four groups. One isolate from each group, 102SH3, 29SA4, 29SA13, and 65PH1, was selected for library construction. Isolates 102SH3 and 29SA4 were identified as Streptomyces ambofaciens and Streptomyces chartreusis, respectively.
Four homologous expandase genes, including three cefE genes and one cefF gene, were finally obtained by screening. The cefE genes from the S. ambofaciens and S. chartreusis isolates contain 936 nucleotides encoding 311 amino acids and have a G+C content of 63.2 and 66.9%, respectively. The deduced protein sequences of these two cefEs share 79% identity to each other and show 81 and 76% identity with the S. clavuligerus enzyme, respectively (Table 1). Both Streptomyces strains have never been reported to have the cefE gene. A phylogenetic tree of the cefE genes from S. ambofaciens, S. chartreusis, S. clavuligerus, N. lactamdurans, and S. jumonjinensis and the cefEF gene from A. chrysogenum was then constructed (Fig. 2). Because the evolutionary split between cefE and cefEF is probably ancestral to the evolution of cefE in the Actinomycetales, we use the fungal cefEF as the tree root. This distance tree was compared to trees produced by PROTPARS and PROML from PHYLIP by using CONSENSE, and the consensus tree was identical to the distance tree originally obtained. The cefE genes share 67 to 79% protein sequence identity to each other (Table 1). Furthermore, the presence of the conserved residues involved in iron ligation and substrate and cosubstrate recognition suggests that these two newly cloned cefE genes possess the expandase activity (Fig. 3).
A consensus tree constructed using the protein sequences of the five DAOCSs and one DAOCS/DACS, with the latter used as the root. Numbers at the nodes indicate statistical support for each branch as a percentile value. Scale bar, 0.1 substitution per site.
Protein sequences of the six evolved clones and their parents. The shaded residues show the identity to the parents (S. clavuligerus, red; S. ambofaciens, yellow; S. chartreusis, blue), point mutations (green), and the regions of crossover events (gray). The conserved residues in several Fe(II)-dependent oxygenases (5, 22, 29) for iron ligation, H183, D185, and H243, and for interaction with the carboxylate group of the (co)substrate, R258 and S260, are shaded in magenta, while residues involved in substrate binding are in cyan.
Percentages of sequence identities between shuffled genes
The deduced amino acid sequences of the isolates 29SA4 and 29SA13 show 99.6% identity to each other, strongly suggesting that they come from the same species. The new cefF-homologous gene from the isolate 65PH1 has 939 nucleotides and 82% identity to the cefF gene from S. clavuligerus.
Family shuffling and screening.For the first round of family shuffling, 6,578 clones were screened, and most of them had low or even no detectable activity. Only four clones, F42, F58, F53, and F56, showed a higher activity than the S. clavuligerus expandase, YS16. The sequences revealed that F56 and F58 originated by one crossover event, F42 by two such events, and F53 by three events (Fig. 3). Sequence elements were contributed by only three parental genes, namely, genes from S. clavuligerus, S. ambofaciens, and S. chartreusis, even though eight genes were used for the family shuffling. The construction elements in F42, F53, and F56 were derived mainly from S. clavuligerus, with only 10 to 30% from S. ambofaciens. However, 80% of F58 is from S. ambofaciens, and 20% is from S. chartreusis.
The four clones were reshuffled and rescreened to generate progeny with an activity higher than that of clone F42. During the second round, 3,612 clones were screened and 20 clones with enhanced activity were obtained. Two clones, FF1 and FF8, showed no substrate inhibition and were subjected to kinetic and sequence analyses. FF1 was derived mainly from F42 with some sequence elements from S. ambofaciens and S. chartreusis, while FF8 was derived mainly from F58 and F42 (Fig. 3). In addition, both FF1 and FF8 have five amino acid substitutions.
Kinetic parameters of selected expandases.We purified the expandases from F42, F58, FF1, FF8, S. ambofaciens, and S. chartreusis and measured their kinetic parameters for penicillin G expansion (Table 2). The recombinant proteins of our two newly identified cefE genes showed weak activity towards penicillin G. With comparison to the S. clavuligerus expandase when assayed with 1 mM penicillin G, the S. ambofaciens enzyme displayed only 14% activity, the S. chartreusis counterpart had a fivefold higher Km value, while F42 and F58 from the first shuffling round showed 3.4-fold and a 5.7-fold increases, respectively in penicillin G binding. After the second round of shuffling, the kinetic parameters of clones FF1 and FF8 were improved further. FF1 and FF8 had an enhancement in the Km value by 8.6- and 184-fold, respectively. To the best of our knowledge, the two-round-shuffled FF8 had the highest known kcat/Km value, 2,121 (M−1 s−1), for penicillin G, which is 118-fold higher than that for the S. clavuligerus expandase.
Kinetic parameters for penicillin G conversiona
DISCUSSION
After screening of 1,835 actinomycetes from soil, two isolates containing new cefE genes were found and identified as S. ambofaciens and S. chartreusis. S. ambofaciens has been shown to produce the macrolide antibiotic spiramycin (1), but no expandase-related gene has been reported. The production of cephamycins by S. chartreusis (11) implies the existence of an expandase gene in this species. We could not identify promoter regions for the newly discovered cefE genes; hence these genes might be expressed as part of an operon, or they might exist as a silent gene within the chromosome. Although the two cefE genes share high homology to S. clavuligerus cefE, their physiological functions in the original strains need to be addressed further.
Although five cefE genes, two cefF genes, and one cefEF gene were used for DNA family shuffling, the most-active clones from the first round were constructed from the S. clavuligerus, S. ambofaciens, and S. chartreusis genes. The sequences of some randomly selected clones, such as FS1 to FS9 in Fig. 4, demonstrated parts of cefEs from two other sources, indicating that the shuffling pool indeed consisted of five cefE sources. The first-round clones F42 and F58 showed only a slight increase, less than threefold, in the kcat/Km value, relative to the S. clavuligerus expandase. Nevertheless, the two clones, FF1 and FF8, obtained in the second shuffling carried out with the most active four clones from the first round showed a significant improvement for penicillin G expansion. Therefore, the evolution of penicillin G-specific expandase has been successfully directed. These results also indicate the remarkable plasticity of this enzyme, and this suggests that more-active chimeras might be achievable with further rounds.
Distinct residues among the most active clone, FF8, and the clones FS1 to FS9 with little or even no detectable activity. The secondary structure elements (αA to αE and β1 to β15) for the expandase are labeled. The solvent-accessible area for each residue is calculated based on the crystal structure of the S. clavuligerus expandase by using PROCHECK (16) and colored according to the degree of exposure from burial (blue) to exposure (white). The blanks indicate the disordered regions in the crystal structures. Obviously, the structural core is mainly maintained by the hydrophobic packing of β-strands, and this hydrophobic core is conserved in this Fe(II)-dependent oxygenase superfamily (5, 22, 29).
During screening, most evolved expandases with higher activities showed severe substrate inhibition, which would be a major disadvantage for industrial applications. However, there is no significant correlation between the enzyme activity and the substrate inhibition, because some of the more-active evolved enzymes have no substrate inhibition. This implies that there are at least two penicillin G-binding sites: one is responsible for the expansion activity, whereas the other has the inhibition effect. Our observation of substrate inhibition and the low activities of some evolved clones might be due to penicillin G preferring the inhibitory site and thus attenuating activity. Therefore, the penicillin G concentration was increased to 10 mM in the screening mixture to allow evolution of a higher affinity to the substrate-binding site instead of to the inhibitory site. Consistent with our observation, crystallographic studies indeed reveal two penicillin-binding sites, which overlap with each other and clash with the cosubstrate α-ketoglutarate (26) (Fig. 5).
Stereo view of the penicillin G-binding sites. The substrate penicillin G (penGS) is colored in black, whereas penicillin G in the inhibitory site (penGI) is in orange. The iron is shown as an orange sphere, the conserved residues in several oxygenases are shown in magenta, and some other protein residues are shown in cyan. This figure was prepared using MOLSCRIPT (14).
Many inactive clones may be due to mutations at the essential active-site residues. For example, replacement of the conserved R162 with glutamine in FS1 results in no detectable activity because the R162Q mutant has been demonstrated to show a loss of penicillin binding (18) (Fig. 4 and 5). However, we do not know why the chimera FF8 has such a high penicillin G-binding affinity, whereas most other evolved clones, such as FS2 to FS9 in Fig. 4, contain little or even no detectable activity. In order to identify some possible clues, we mapped the distinct residues among expandases onto the enzyme structures. These include the naturally occurring substitutions between different species and shuffled replacements in the crossover events and random mutations during family shuffling (Fig. 3 and 4). Most of them are exposed at the protein surface, have no close contacts with other parts of the protein structure, and are far away from the active-site cavity and hence might not significantly affect structural integrity and enzyme activity.
In contrast, several residues are buried (Fig. 4) and may involve compactable side chains for the hydrophobic packing, contributing to part of the penicillin G-binding sites. The hydrophobic edges of the substrate penicillin G face several hydrophobic regions, and these hydrophobic contacts contribute the major interactions between penicillin G and the expandase (Fig. 5). The phenylacetyl ring makes close contacts with M73, L158, and F264, the β-lactam ring makes close contacts with M180, I305, and V262, and the thiazolidine ring makes close contacts with I192, L204, and V245. The I305L and I305M mutants, which show 3.9-fold and 3.4-fold reductions in Km for penicillin G, respectively (28), suggest that replacement with a leucine or methionine residue enhances the hydrophobic contacts. The N304K mutant with an 11.7-fold reduction in Km (28) and the N304L and R306L mutants with significantly enhanced activity (3) also suggest that the C-terminal tail is an attractive candidate for enhancement of penicillin G binding as well as residues around other hydrophobic regions. An example of the latter is the residue M188, near the iron ligands H183 and D185, which is buried in a hydrophobic pocket made up of A134, V137, L138, F152, L153, V189, I229, A230, V233, and L265. Our M188V and M188I mutants contain a 3.4-fold and 1.3-fold reduction in Km for penicillin G, respectively (Wei et al., unpublished data). In addition, the residue A246, as well as several surrounding hydrophobic residues, L178, F201, V202, L204, and A247, is close to the iron ligand H243, which constitutes part of the active-site cavity. Replacement of this residue with valine in FF8 might result in better steric complementarity and hydrophobic packing (Fig. 6) and therefore might enhance binding of penicillin G to the substrate site in a manner similar to that for the R306L mutant. In short, it should be possible to use these predictions in the future as a structural basis for further mutational analysis.
Predicted hydrophobic contacts surrounding V246 in FF8. Based on the enzyme structure, residue A246 (A) and the modeled V246 (B) are colored blue and cyan, respectively, while the surrounding hydrophobic residues are shown in gray. Replacement of A246 with valine might result in better hydrophobic packing. This figure was displayed by using GRASP (20).
In summary, we have obtained two new natural expandase genes from actinomycetes by screening and the most active penicillin G-specific expandase known to date by using family shuffling. In the next stage, we will attempt to further improve the enzyme activity through a combination of DNA shuffling and mutagenesis based on structural analysis.
ACKNOWLEDGMENTS
We thank A. L. Demain for kindly providing the E. coli ESS strain and R. Kirby for critical reading of the manuscript.
This work was supported by a grant from the Ministry of Economic Affairs of the Republic of China (89-EC-2-A-17-0285-035) and by the Synmax Biochemical Co., Ltd., Hsinchu, Taiwan.
FOOTNOTES
- Received 25 February 2004.
- Accepted 16 June 2004.
- Copyright © 2004 American Society for Microbiology
