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.     

The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum

A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in lambda EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB-pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase with a deduced Mr of 404,134. This enzyme contains three repeated domains and a consensus thioesterase active-site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37,469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB-pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.     

The absolute configuration of the amino acids in delta-(alpha-aminoadipyl)cysteinylvaline from Penicillium chrysogenum.

Radioactive carbon-14 L-alpha-aminoadipic acid, L-cysteine, or L-valine were readily incorporated into the intracellular tripeptide, delta-(alpha-aminoadipyl)cysteinylvaline (ACV), by washed starved cells of Penicillium chrysogenum. The labeled ACV in each case was oxidized with performic acid and isolated as its corresponding sulfonic acid derivative. After acid hydrolysis, the configuration of the component acids was determined by L- and D-amino acid oxidases, which showed the tripeptide (ACV) from P. chrysogenum to be delta-(L-aminoadipyl)-L-cysteinyl-D-valine.     

Generality of peptide cyclization catalyzed by isolated thioesterase domains of nonribosomal peptide synthetases

The C-terminal thioesterase (TE) domains from nonribosomal peptide synthetases (NRPSs) catalyze the final step in the biosynthesis of diverse biologically active molecules. In many systems, the thioesterase domain is involved in macrocyclization of a linear precursor presented as an acyl-S-enzyme intermediate. The excised thioesterase domain from the tyrocidine NRPS has been shown to catalyze the cyclization of a peptide thioester substrate which mimics its natural acyl-S-enzyme substrate. In this work we explore the generality of cyclization catalyzed by isolated TE domains. Using synthetic peptide thioester substrates from 6 to 14 residues in length, we show that the excised TE domain from the tyrocidine NRPS can be used to generate an array of sizes of cyclic peptides with comparable kinetic efficiency. We also studied the excised TE domains from the NRPSs which biosynthesize the symmetric cyclic decapeptide gramicidin S and the cyclic lipoheptapeptide surfactin A. Both TE domains exhibit expected cyclization activity: the TE domain from the gramicidin S NRPS catalyzes head-to-tail cyclization of a decapeptide thioester to form gramicidin S, and the TE domain from the surfactin NRPS catalyzes stereospecific cyclization to form a macrolactone analogue of surfactin. With an eye toward generating libraries of cyclic molecules by TE catalysis, we report the solid-phase synthesis and TE-mediated cyclization of a small pool of linear peptide thioesters. These studies provide evidence for the general utility of TE catalysis as a means to synthesize a wide range of macrocyclic compounds.     

Regulation of δ-(L-α-aminoadipyl)-l-cysteinyl-d-valine and isopenicillin N biosynthesis in Penicillium chrysogenum by the α-aminoadipate pool size

The effect of changes in the intracellular concentration of alpha-aminoadipate on the formation of alpha-aminoadipyl-cysteinyl-valine (ACV) and isopenicillin N (IPN)--two intermediates of penicillin biosynthesis--by strains of Penicillium chrysogenum has been investigated by measuring the incorporation of radioactivity from (6-14C)-alpha-aminoadipate into cellular 14C-ACV and 14C-IPN. No ACV or IPN were found in any strain during cultivation on glucose, but were clearly detected in all three strains during growth on lactose, displaying increased formation in strains exhibiting increased penicillin productivity and increased intracellular alpha-aminoadipate pools. ACV and IPN formation was affected by subjected P. chrysogenum mycelia to either general amino acid control (by addition of amitrol) or by exogenous addition of 5 mM L-lysine. In all cases, the changes observed paralleled the changes in the intracellular alpha-aminoadipate pool. These results are consistent with the alpha-aminoadipate pool limiting the biosynthesis of ACV and IPN and hence penicillin biosynthesis in the present strains of P. chrysogenum.     

The thioesterase domain from the pimaricin and erythromycin biosynthetic pathways can catalyze hydrolysis of simple thioester substrates

The recombinant polyketide synthase thioesterase domains from the pimaricin and 6-deoxyerythronolide B biosynthetic pathways catalyze hydrolysis of a number of simple N-acetylcysteamine thioester derivatives. This study demonstrates that thioesterases are not highly substrate selective in formation of the acyl-enzyme intermediate, in contrast to non-ribosomal peptide synthase thioesterase domains that show very high specificity for substrate loading. This observation has important implications for the engineering of biosynthetic pathways to produce polyketide products.     

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.     

Penicillium chrysogenum var. halophenolicum, a new halotolerant strain with potential in the remediation of aromatic compounds in high salt environments

A halotolerant phenylacetate-degrading fungus Penicillium CLONA2, previously isolated from a salt mine at Algarve (Portugal), was identified as a variant of P. chrysogenum using the ITS-5,8S rDNA and the D1/D2 domain of 28S rDNA sequences. The metabolic features and genetic characteristics suggest that this strain belongs to a subgroup of P. chrysogenum, named var. halophenolicum. The presence of the penicillin biosynthetic cluster was proven by Southern hybridizations using probes internal to the pcbAB and penDE genes and sequencing of the pcbAB-pcbC intergenic region. However the pcbAB-pcbC divergent promoter region contained 20 point modifications with respect to that of the wild type P. chrysogenum NRRL1951. The CLONA2 strain produced non-aromatic natural penicillins rather than benzylpenicillin in a medium containing potassium phenylacetate (the precursor of benzylpenicillin) and was able to grow well on phenylacetatic acid using it as sole carbon source. Due to the ability of P. chrysogenum CLONA2 to degrade aromatic compounds, this strain may be an interesting organism for aromatic compounds remediation in high salinity environments.     

Timing of epimerization and condensation reactions in nonribosomal peptide assembly lines: kinetic analysis of phenylalanine activating elongation modules of tyrocidine synthetase B

The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.     

Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase

A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.     

Macrolactonization to 10-deoxymethynolide catalyzed by the recombinant thioesterase of the picromycin/methymycin polyketide synthase

The recombinant thioesterase (TE) domain of the picromycin/methymycin synthase (PICS) catalyzes the macrolactonization of 3, the N-acetylcysteamine thioester of seco-10-deoxymethynolide to generate 10-deoxymethynolide (1) with high efficiency. By contrast, 4, the 7-dihydro derivative of seco-thioester 3, undergoes exclusive hydrolysis by PICS TE to seco-acid 5. The recombinant TE domain of 6-deoxyerythronolide B synthase (DEBS TE) shows the same reaction specificity as PICS TE, but with significantly lower activity.     

Synthesis of long chain acyl-enzyme thioesters by modified fatty acid synthetases and their hydrolysis by a mammary gland thioesterase.

The fatty acid synthetase multienzyme from lactating rat mammary gland was modified either by removal of the two thioesterase I domains with trypsin or by inhibiting the thioesterase I activity with phenylmethanesulfonyl fluoride. The modified multienzymes are able to convert acetyl-CoA, malonyl-CoA, and NADPH to long chain acyl moieties (C₁₆C₂₂), which are covalently bound to the enzyme through thioester linkage, but they are unable to release the acyl groups as free fatty acids. A single enzyme-bound, long chain acyl thioester is formed by each molecule of modified multienzyme. Kinetic studies showed that the modified multienzymes rapidly elongate the acetyl primer moiety to a C₁₆ thioester and that further elongation to C₁₈, C₂₀, and C₂₂ is progressively slower. Thioesterase II, a mammary gland enzyme which is not part of the fatty acid synthetase multienzyme, can release the acyl moiety from its thioester linkage to either modified multienzyme. Kinetic data are consistent with the formation of an enzyme—substrate complex between thioesterase II and the acylated modified multienzymes. The present study demonstrates that the ability of thioesterase II to modify the product specificity of normal fatty acid synthetase is most likely attributable to the capacity of thioesterase II for hydrolysis of acyl moieties from thioester linkage to the multienzyme.     

Thioesterase domain of delta-(l-alpha-Aminoadipyl)-l-cysteinyl-d-valine synthetase: alteration of stereospecificity by site-directed mutagenesis

The carboxy-terminal thioesterase domain of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase catalyzes the hydrolytic release of the tripeptide product (LLD-ACV). By site-directed mutagenesis an S3599A change was introduced into the highly conserved GXSXG motif, resulting in a more than 95 % decrease of penicillin production. Purification of the modified multienzyme showed surprisingly only a 50 % reduction of the peptide formation rate, with the stereoisomer delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine (LLL-ACV) as the dominating product. Thioesterases of ACV synthetases differ from other thioesterases integrated in non-ribosomal peptide synthetases in their direct association with an epimerase domain, and their respective GXSXG-seryl residue is apparently not essential in acyl transfer leading to peptide release. Instead, this motif may be involved in the control of tripeptide epimerization by selection of the isomer to be released, and the construct supports the presence of LLL-ACV as an intermediate in penicillin biosynthesis.     

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.     

The global regulator LaeA controls penicillin biosynthesis, pigmentation and sporulation, but not roquefortine C synthesis in Penicillium chrysogenum

The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.