Isopenicillin N Acyltransferase Research Articles

Comprehensive Profiling of Proteome Changes Provide Insights of Industrial Penicillium chrysogenum During Pilot and Industrial Penicillin G Fermentation.

The intracellular proteomes of the Penicillium chrysogenum throughout pilot and industrial processes were investigated by using 2-DE combined with MALDI-TOF-TOF MS, respectively. We detected a total of 223 spots corresponding to 154 proteins and 231 spots corresponding to 157 proteins throughout pilot and industrial processes, respectively. The levels of glyceraldehyde-3-phosphate dehydrogenase increased (5.1- and 2.5-fold) under the pilot process, while its levels were no significant changes under the industrial process at 140 and 170h when compared with that at 2h. The levels of isocitrate lyase and fumarate hydratase were increased significantly under the industrial process, while their levels had no obvious changes after 20h of fermentation throughout the pilot process. These results indicate that there were remarkable differences in carbohydrate metabolism (including glycolysis, gluconeogenesis, pentose phosphate pathway, and tricarboxylic acid cycle) of P. chrysogenum during the pilot and industrial fermentations, which likely result in alterations of the primary metabolism and penicillin biosynthesis. Moreover, the differences in the levels of proteins involved in amino acid metabolisms (including valine, cysteine, and α-aminoadipic acid biosynthesis) indicated that the pilot and industrial processes influenced the supplies of penicillin precursors. Compared with that at 2h, the maximum levels of superoxide (6.9-fold, at 32h) and catalase (9-fold, at 80h) during the industrial process and the maximum levels of superoxide (1.2-fold, at 20h) and catalase (7.7-fold at 128h) during the pilot process revealed the significant difference in cell redox homeostasis and stress responses during scale-up fermentation. Particularly, 10 spots corresponding to isopenicillin N synthetase and 4 spots corresponding to isopenicillin N (IPN) acyltransferase in pilot and industrial processes were identified, respectively. The levels of IPN acyltransferase (spots 197 and 198) and CoA ligase at 80h during the industrial process were around 2-fold of that during the pilot process, indicating that the industrial process with a higher penicillin production per cell might provide available environments to induce over-expression of IPN acyltransferase and accelerate penicillin formation. These results provide new insights into the globally potential responses of P. chrysogenum to variations of environments in different fermentation scales so as to consequently regulate the penicillin production.

The inducers 1,3-diaminopropane and spermidine cause the reprogramming of metabolism in Penicillium chrysogenum, leading to multiple vesicles and penicillin overproduction

In this article we studied the differential protein abundance of Penicillium chrysogenum in response to either 1,3-diaminopropane (1,3-DAP) or spermidine, which behave as inducers of the penicillin production process. Proteins were resolved in 2-DE gels and identified by tandem MS spectrometry. Both inducers produced largely identical changes in the proteome, suggesting that they may be interconverted and act by the same mechanism. The addition of either 1,3-DAP or spermidine led to the overrepresentation of the last enzyme of the penicillin pathway, isopenicillin N acyltransferase (IAT). A modified form of the IAT protein was newly detected in the polyamine-supplemented cultures. Both inducers produced a rearrangement of the proteome resulting in an overrepresentation of enzymes involved in the biosynthesis of valine and other precursors (e.g. coenzyme A) of penicillin. Interestingly, two enzymes of the homogentisate pathway involved in the degradation of phenylacetic acid (a well-known precursor of benzylpenicillin) were reduced following the addition of either of these two inducers, allowing an increase of the phenylacetic acid availability. Both inducers produced also an increase in the intracellular content of vesicles that derived to vacuoles in late stages and promoted sporulation of P. chrysogenum in solid medium. The analysis of global protein changes produced in response to polyamines 1,3-DAP and spermidine provides a valuable information for the understanding of the molecular mechanisms underlying the production of penicillin. This represents useful information to improve the production of this antibiotic and many other bioactive secondary metabolites not only in P. chrysogenum, but in other filamentous fungi as well.

ChemInform Abstract: Role of Peroxisomes in the Biosynthesis and Secretion of β‐Lactams and Other Secondary Metabolites

AbstractReview: 154 refs.

Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase

Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H(2)O(2) (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillinN acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillinN) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.

Homology modeling of the structure of acyl coA:isopenicillin N-acyltransferase (IAT) from Penicillium chrysogenum. IAT interaction studies with isopenicillin-N, combining molecular dynamics simulations and docking

In the last step of penicillin biosynthesis, acyl-CoA:isopenicillin N acyltransferase (IAT) (E.C. 2.3.1.164) catalyzes the conversion of isopenicillin N (IPN) to penicillin G. IAT substitutes the α-aminoadipic acid side chain of IPN by a phenylacetic acid phenolate group (from phenylacetyl-CoA). Having a three-dimensional (3D) structure of IAT helps to determine the steps involved in side chain exchange by identifying the atomic details of substrate recognition. We predicted the IAT 3-D structure (α- and β-subunits), as well as the manner of IPN and phenylacetyl-CoA bind to the mature enzyme (β-subunit). The 3D IAT prediction was achieved by homology modeling and molecular docking in different snapshots, and refined by molecular dynamic simulations. Our model can reasonably interpret the results of a number of experiments, where key residues for IAT processing as well as strictly conserved residues most probably involved with enzymatic activity were mutated. Based on the results of docking studies, energies associated with the complexes, and binding constants calculated, we identified a site located in the region generated by β1, β2 and β5 strands, which forms part of the central structure of β-subunit, as the potential binding site of IPN. The site comprises the amino acid residues Cys103, Asp121, Phe122, Phe123, Ala168, Leu169, His170, Gln172, Phe212, Arg241, Leu262, Asp264, Arg302, Ser309, and Arg310. Through hydrogen bonds, the IPN binding site establishes interactions with Cys103, Leu169, Gln172, Asp264 and Arg310. Our model is also validated by a recently revealed crystal structure of the mature enzyme.

An Engineered Yeast Efficiently Secreting Penicillin

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's.

Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum.

BackgroundPenicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.ResultsThe IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null ial mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic) and amidohydrolase (6-APA forming) activities in vivo. When the canonical ARL motif (leading to peroxisomal targeting) was added to the C-terminus of the IAL protein (IALARL) by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in Escherichia coli. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive in vitro.ConclusionNo activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the P. chrysogenum IAL, the A. nidulans IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the ial gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in A. nidulans, the ial and penDE genes might have been formed from a single ancestral gene that became duplicated during evolution, although a separate evolutive origin for the ial and penDE genes, is also discussed.

The unprocessed preprotein form IAT C103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE C103S gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT C103S) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE C103S and wild-type penDE genes in P. chrysogenum (Wis54- DE C103S strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (β subunit formation) were observed in the Wis54- DE C103S strain, suggesting a regulatory role of the unprocessed IAT C103S in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT C103S was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing.

Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase

A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

Synthesis of acetyl-CoA:isopenicillin acyltransferase in : First step towards the introduction of a new metabolic pathway

The enzyme acetyl-CoA:isopenicillin N acyltransferase (IAT) is a peroxisomal enzyme that mediates the final step of penicillin biosynthesis in the filamentous fungi Penicillium chrysogenum and Aspergillus nidulans. However, the precise role of peroxisomes in penicillin biosynthesis is still not clear. To be able to use the power of yeast genetics to solve the function of peroxisomes in penicillin biosynthesis, we introduced IAT in the yeast Hansenula polymorpha. To this purpose, the P. chrysogenum penDE gene, encoding IAT, was amplified from a cDNA library to eliminate the three introns and introduced in H. polymorpha. In this organism IAT protein was produced as a 40 kDa pre-protein and, as in P. chrysogenum, processed into an 11 and 29 kDa subunit, although the efficiency of processing seemed to be slightly reduced relative to P. chrysogenum. The P. chrysogenum IAT, produced in H. polymorpha, is normally localized in peroxisomes and in cell-free extracts IAT activity could be detected. This is a first step towards the introduction of the penicillin biosynthesis pathway in H. polymorpha.

Biodegradation of nylon oligomers.

This mini-review is a compendium of the degradation of a man-made compound, 6-aminohexanoate-oligomer, in Flavobacterium strains. The results are summarized as follows: 1. Three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (EI), 6-aminohexanoate-dimer hydrolase (EII), and endotype 6-aminohexanoate-oligomer hydrolase (EIII) were responsible for degradation of the oligomers. 2. The genes coding these enzymes were located on pOAD2, one of three plasmids harbored in Flavobacterium sp. KI72, which comprised 45,519 bp. 3. The gene coding the EII' protein (a protein having 88% homology with EII) and five IS6100 elements were identified on pOAD2. 4. The specific activity of EII was 200-fold higher than that of EII'. However, altering two amino acid residues in the EII' enzyme enhanced the activity of EII' to the same level as that of the EII enzyme. 5. The deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (encoding oligopeptide permease), ftsX (filamentation temperature sensitivity), penDE (isopenicillin N-acyltransferase) and rep (plasmid replication). 6. The EI and EII genes of Pseudomonas sp. NK87 (another nylon oligomer-degrading bacterium) were also located on plasmids. 7. Through selective cultivation using nylon oligomers as a sole source of carbon and nitrogen, two strains which initially had no metabolic activity for nylon oligomers, Flavobacterium sp. KI725 and Pseudomonas aeruginosa PAO1, were given the ability to degrade xenobiotic compounds. A molecular basis for the adaptation of microorganisms toward xenobiotic compounds was described.

Compartmentalization and transport in beta-lactam antibiotic biosynthesis by filamentous fungi.

A proper description of the biosynthesis of fungal beta-lactam antibiotics requires detailed knowledge of the cell biology of the producing organisms. This involves a delineation of the compartmentalization of the biosynthetic pathways, and of the consequential transport steps across the cell-boundary plasma membrane and across organellar membranes. Of the enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum and Aspergillus nidulans, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS) probably have a cytosolic location. Acyl-coenzyme A:isopenicillin N acyltransferase (IAT) is located in microbodies. Of the two enzymes that may be involved in activation of the side chain, acetyl-coenzyme A synthetase (ACS) is located in the cytosol, and phenylacetyl-coenzyme A ligase (PCL) is probably located in the microbody. All enzymes of the cephalosporin biosynthesis pathway in Cephalosporium acremonium probably have a cytosolic location. The vacuole may play an ancillary role in the supply of precursor amino acids, and in the storage of intermediates. The distribution of precursors, intermediates, end- and side-products, the transport of nutrients. precursor, intermediates and products across the plasma membrane, and the transport of small solutes across organellar membranes, is discussed. The relevance of compartmentalization is considered against the background of recent biotechnological innovations of fungal beta-lactam biosynthesis pathways.

New aspects of genes and enzymes for beta-lactam antibiotic biosynthesis.

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of L-alpha-aminoadipic acid, L-cysteine and L-valine to form the tripeptide delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase and isopenicillin N (IPN) synthase respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the alpha-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3' of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P7 and P8) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3'-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a beta-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the beta-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the beta-lactam biosynthetic pathways. Several new beta-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules.

A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2

The entire nucleotide sequence of nylon oligomer degradative plasmid pOAD2 from Flavobacterium sp. KI723T1 was determined. pOAD2 comprises 45519 bp, with a 66.6 mol% G+C content. The precise loci of the four nylon oligomer degradation genes, namely nylA (6-aminohexanoate-cyclic-dimer hydrolase gene), nylB (6-aminohexanoate-dimer hydrolase), nylB' (a gene having 88% homology to nylB) and nylC (endo-type 6-aminohexanoate oligomer hydrolase), and five IS6100 elements were identified on this plasmid. Comparison of the sequence of pOAD2 with those in the GenBank and EMBL databases revealed that the deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (oligopeptide permeases), ftsX (filamentation temperature sensitive), penDE (isopenicillin N-acyltransferase) and rep (plasmid incompatibility). A functional map of pOAD2 is presented.

The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene.

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.

On the production of α,β-heterodimeric acyl-coenzyme A: isopenicillin N-acyltransferase of Penicillium chrysogenum: Studies using a recombinant source

A high level E. coli expression system has been constructed for the Penicillium chrysogenum penDE gene, which encodes the acyl-coenzyme A: isopenicillin N-acyltransferase (AT) enzyme. Induction of overexpression of recombinant AT (recAT) by increasing the growth temperature of the host adversely affected solubility and activity of the AT enzyme. Addition of isopropylthio-β- d-galactopyranoside (IPTG) at decreased growth temperatures (less than 32°C) resulted in the overproduction of soluble, active recAT. When purified to homogeneity, recAT was an α,β-heterodimer, comprised of 11 kDa (α) and 29 kDa (β) subunits, derived from a 40 kDa precursor polypeptide by a posttranslational cleavage. The recAT enzyme contained both the acyl-coenzyme A: isopenicillin N-acyltransferase and the acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase activities. The processing event that generated the two subunits of recAT from the 40 kDa precursor polypeptide occurred between Gly 102/Cys 103. This expression system produced a large amount of soluble, active recAT that is identical to native AT, making it a suitable source of AT enzyme for further characterization.