δ-(<Emphasis Type="SmallCaps">l</Emphasis>-α-aminoadipyl)-<Emphasis Type="SmallCaps">l</Emphasis>-cysteinyl-<Emphasis Type="SmallCaps">d</Emphasis>-valine synthetase (ACVS): discovery and perspectives

The δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of β-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.     

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase: production of dipeptides containing valine residue at its C-terminus

L-delta-(alpha-Aminoadipoyl)-L-cysteine-D-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of alpha-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [(14)C]Cysteine was incorporated into the[(14)C]cysteinyl-valine analogue dipeptides. Notably, [(14)C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The alpha-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.     

Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the lld-ACV:bisACV ratio on the flux control

An extended kinetic model for the first two steps of the penicillin biosynthetic pathway in Penicillium chrysogenum is set up. It includes the formation and reduction of the dimer bis--(l--aminoadipyl)-l-cysteinyl-d-valine (bisACV) from the first pathway intermediate lld-ACV and their parallel inhibition of the enzyme ACV synthetase (ACVS). The kinetic model is based on Michaelis-Menten type kinetics, with non-competitive inhibition of the ACVS by both lld-ACV and bisACV, and competitive inhibition of the isopenicillin N synthetase (IPNS) by glutathione. The inhibition constant of lld-ACV, K_ACV is determined to be 0.54 mm. With the kinetic model metabolic control analysis is performed to identify the distribution of rate-control in the pathway at all ratios of lld-ACV:bisACV. It is concluded that the flux control totally resides at the IPNS. This is a result of the regulation of the ACVS by both the lld-ACV and bisACV demanding a higher flux through the IPNS enzyme to alleviate their inhibition. The measurement of an intracellular ratio of lld-ACV:bisACV to be in the range of 1–2 moles per moles emphasises the importance of a fast conversion of lld-ACV to IPN, and accumulation of lld-ACV above the K_m-value of the IPNS should therefore be avoided.     

Purification and partial characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) was purified from Streptomyces clavuligerus by a combination of salt precipitation, ultrafiltration, and anion-exchange chromatography. The final purified material gave two protein bands with molecular weights of 283,000 and 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis in nondenaturing gels gave a single protein band with an estimated molecular weight of 560,000. These results suggest that ACVS is a multimer composed of nonidentical subunits.     

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.     

Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes.

A cosmid clone containing closely linked beta-lactam antibiotic biosynthetic genes was isolated from a gene library of Flavobacterium sp. SC 12,154. The location within the cluster of the DNA thought to contain the gene for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first step in the beta-lactam antibiotic biosynthetic pathway, was identified by a novel method. This DNA facilitated the isolation, by cross-hybridization, of the corresponding DNA from Streptomyces clavuligerus ATCC 27064, Penicillium chrysogenum Oli13 and Aspergillus nidulans R153. Evidence was obtained which confirmed that the cross-hybridizing sequences contained the ACVS gene. In each case the ACVS gene was found to be closely linked to other beta-lactam biosynthetic genes and constituted part of a gene cluster.     

Nonribosomal peptide synthetases-evidence for a second ATP-binding site

δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP–[³²P]PP_i exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP^2?, l-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5′)tetraphospho(5′)adenosine (Ap₄A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.     

Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations.     

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.     

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.     

ATPase activity of non-ribosomal peptide synthetases

Adenylation domains of non-ribosomal peptide synthetases (NRPS) catalyse the formation of aminoacyl adenylates, and in addition synthesize mono- and dinucleoside polyphosphates. Here, we show that NRPS systems furthermore contain an ATPase activity in the range of up to 2 P(i)/min. The hydrolysis rate by apo-tyrocidine synthetase 1 (apo-TY1) is enhanced in the presence of non-cognate amino acid substrates, correlating well with their structural features and the diminishing adenylation efficiency. A comparative analysis of the functional relevance of an analogous sequence motif in P-type ATPases and adenylate kinases (AK) allowed a putative assignment of the invariant aspartate residue from the TGDLA(V)R(K) core sequence in NRPS as the Mg(2+) binding site. Less pronounced variations in ATPase activity are observed in domains with relaxed amino acid specificity of gramicidin S synthetase 2 (GS2) and delta-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), known to produce a set of substitutional variants of the respective peptide product. These results disclose new perspectives about the mode of substrate selection by NRPS.     

Analysis of the regulation of penicillin biosynthesis in Aspergillus nidulans by targeted disruption of the acvA gene

To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.     

Analysis of the regulation of penicillin biosynthesis in Aspergillus nidulans by targeted disruption of the acvA gene

To analyse the regulation of the biosynthesis of the secondary metabolite penicillin in Aspergillus nidulans, a strain with an inactivated acvA gene produced by targeted disruption was used. acvA encodes -(l--aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), which catalyses the first step in the penicillin biosynthetic pathway. To study the effect of the inactivated acvA gene on the expression of acvA and the second gene, ipnA, which encodes isopenicillin N synthase (IPNS), A. nidulans strain XEPD, with the acvA disruption, was crossed with strain AXB4A carrying acvA-uidA and ipnA-lacZ fusion genes. Ascospores with the predicted non-penicillin producing phenotype and a hybridization pattern indicating the presence of the disrupted acvA gene, and the fusion genes integrated in single copy at the chromosomal argB locus were identified. Both fusion genes were expressed at the same level as in the non-disrupted strain. Western blot analysis (immunoblotting) revealed that similar amounts of IPNS enzyme were present in both strains from 24 to 68 h of a fermentation run. In the acvA disrupted strain, IPNS and acyl-CoA: 6-aminopenicillanic acid acyltransferase (ACT) specific activities were detected, excluding a sequential induction mechanism of regulation of the penicillin biosynthesis gene ipnA and the third gene aat.     

Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae

Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using δ-(l-α-aminoadipyl)–l-cysteinyl–d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5′ end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 °C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast.