Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum.

The inhibitory effect of L-lysine on penicillin biosynthesis by Penicillium chrysogenum has been compared in a low-producing strain (Wis. 54-1255) and a high-producing strain (ASP-78). Lysine inhibited total penicillin synthesis to a similar extent in both strains. However, in the high-producing strain the onset of penicillin synthesis occurred even at a high lysine concentration, whereas in the low-producing strain lysine had to be depleted before penicillin production commenced.     

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.     

Formation of bulges associated with penicillin production in high-producing strains ofPenicillium chrysogenum

Bulges were formed in the hyphae of a high penicillin-producing strain ofPenicillium chrysogenum AS-P-78 when penicillin was accumulated in the broth. The mycelium of cultures grown in the presence of 50 mM lysine, which specifically inhibits penicillin formation, showed a greatly reduced number of bulges. The size and number of bulges increased during the fermentation in parallel with penicillin accumulation. A smaller number of bulges was formed in the mycelium of the low-producing strain Wis 54-1255 than in the high-producing mutant. Three mutants blocked in penicillin biosynthesis did not form these globose structures. Bulges were not osmotically sensitive, although some of them burst out. They may be formed by weakening of the cell wall of hyphae following accumulation of high concentrations of penicillin.     

Phosphopantetheinyl transferase CfwA/NpgA is required for Aspergillus nidulans secondary metabolism and asexual development

Polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs) are central components of secondary metabolism in bacteria, plants, and fungi. In filamentous fungi, diverse PKSs and NRPSs participate in the biosynthesis of secondary metabolites such as pigments, antibiotics, siderophores, and mycotoxins. However, many secondary metabolites as well as the enzymes involved in their production are yet to be discovered. Both PKSs and NRPSs require activation by enzyme members of the 4'-phosphopantetheinyl transferase (PPTase) family. Here, we report the isolation and characterization of Aspergillus nidulans strains carrying conditional (cfwA2) and null (DeltacfwA) mutant alleles of the cfwA gene, encoding an essential PPTase. We identify the polyketides shamixanthone, emericellin, and dehydroaustinol as well as the sterols ergosterol, peroxiergosterol, and cerevisterol in extracts from A. nidulans large-scale cultures. The PPTase CfwA/NpgA was required for the production of these polyketide compounds but dispensable for ergosterol and cerevisterol and for fatty acid biosynthesis. The asexual sporulation defects of cfwA, DeltafluG, and DeltatmpA mutants were not rescued by the cfwA-dependent compounds identified here. However, a cfwA2 mutation enhanced the sporulation defects of both DeltatmpA and DeltafluG single mutants, suggesting that unidentified CfwA-dependent PKSs and/or NRPSs are involved in the production of hitherto-unknown compounds required for sporulation. Our results expand the number of known and predicted secondary metabolites requiring CfwA/NpgA for their biosynthesis and, together with the phylogenetic analysis of fungal PPTases, suggest that a single PPTase is responsible for the activation of all PKSs and NRPSs in A. nidulans.     

Sfp-type 4'-phosphopantetheinyl transferase is required for lysine synthesis, tolerance to oxidative stress and virulence in the plant pathogenic fungus Cochliobolus sativus

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are the major enzymes involved in the biosynthesis of secondary metabolites, which have diverse activities, including roles as pathogenicity/virulence factors in plant pathogenic fungi. These enzymes are activated by 4'-phosphopantetheinylation at the conserved serine residues, which is catalysed by 4'-phosphopantetheinyl transferase (PPTase). PPTase is also required for primary metabolism (α-aminoadipate reductase, AAR). In the genome sequence of the cereal fungal pathogen Cochliobolus sativus, we identified a gene (PPT1) orthologous to the PPTase-encoding genes found in other filamentous ascomycetes. The deletion of PPT1 in C. sativus generated mutants (Δppt1) that were auxotrophic for lysine, unable to synthesize melanin, hypersensitive to oxidative stress and significantly reduced in virulence to barley cv. Bowman. To analyse the pleiotropic effects of PPT1, we also characterized deletion mutants for PKS1 (involved in melanin synthesis), AAR1 (for AAR) and NPS6 (involved in siderophore-mediated iron metabolism). The melanin-deficient strain (Δpks1) showed no differences in pathogenicity and virulence compared with the wild-type strain. Lysine-auxotrophic mutants (Δaar1) induced spot blotch symptoms, as produced by the wild-type strain, when inoculated on wounded barley leaves or when lysine was supplemented. The Δnps6 strain showed a slightly reduced virulence compared with the wild-type strain, but exhibited significantly higher virulence than the Δppt1 strain. Our results suggest that an unknown virulence factor, presumably synthesized by PKSs or NRPSs which are activated by PPTase, is directly responsible for high virulence of C. sativus on barley cv. Bowman.     

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.     

A dedicated phosphopantetheinyl transferase for the fredericamycin polyketide synthase from Streptomyces griseus

Polyketide synthases cannot be functional unless their apo-acyl carrier proteins (apo-ACPs) are post-translationally modified by covalent attachment of the 4'-phosphopantetheine group to the highly conserved serine residue, and this reaction is catalyzed by phosphopantetheinyl transferases (PPTases). Cloning and sequence analysis of the 33-kb fredericamycin (FDM) biosynthetic gene cluster from Streptomyces griseus revealed fdmW, whose deduced gene product showed significant sequence homology to known PPTases. Biochemical characterization of FdmW in vitro confirmed that it is a PPTase. Inactivation of fdmW resulted in approximately 93% reduction of FDM production, and complementation of the fdmW::aac (3)IV mutant by expressing fdmW in trans restored FDM production to a level comparable with that of the wild-type strain. Although FdmW can phosphopantetheinylate various ACPs, it prefers its cognate substrate, the FdmH ACP, with a K(m) of 5.8 microM and a k(cat)/K(m) of 8.1 microM(-1) x min(-1), to heterologous ACPs, such as the TcmM ACP with a K(m) of 1.0 x 10(2) microM and a k(cat) /K(m) of 0.6 microM(-1) x min(-1). These findings suggest that FdmW is specific for FDM biosynthesis. FdmW therefore represents the first holo-ACP synthase-type PPTase identified from an aromatic polyketide biosynthetic gene cluster.     

Strain improvement of Penicillium chrysogenum by recombinant DNA techniques.

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.     

An EntD-like phosphopantetheinyl transferase gene from Photobacterium profundum SS9 complements pfa genes of Moritella marina strain MP-1 involved in biosynthesis of docosahexaenoic acid

The EntD-like phosphopantetheinyl transferase (PPTase) gene, cloned from the eicosapentaenoic acid-producing bacterium Photobacterium profundum strain SS9, has an ORF of 690 bp encoding a 230-amino acid protein. When this PPTase gene was expressed in Escherichia coli with pfaA, pfaB, pfaC and pfaD derived from Moritella marina MP-1, which were four of five essential genes for biosynthesis of docosahexaenoic acid (DHA), the DHA production of the recombinant was 2% (w/w) of total fatty acids. This is the first report showing that the EntD-like PPTase is involved in producing n-3 polyunsaturated fatty acids. Shinji Sugihara and Yoshitake Orikasa contributed equally to this work.     

Effects of lysine analogs on Penicillium chrysogenum.

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.     

Effects of lysine analogs on Penicillium chrysogenum.

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

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.     

Improvement of penicillin yields in solid-state and submerged fermentation of Penicillium chrysogenum by amplification of the penicillin biosynthetic gene cluster

Penicillium chrysogenum low and high penicillin producing strains were transformed with a cosmid containing the whole penicillin biosynthetic gene cluster. The cosmid library was constructed in a newly developed cosmid vector, IztapaCos, which allows cloning and direct introduction of large DNA fragments in fungal recipients using phleomycin resistance as selection marker. The effect of increased gene dosage on penicillin production was evaluated both in submerged (SmF) and solid-state fermentation (SSF). Transformants from the low-producing strain Wis 54-1255, showed a 67.3 and 28.3% increased penicillin titer in SSF and SmF, respectively. In transformants from the high-producing strain P2-32 the increase was 92.9 and 158.4% respectively. Strain P2-32 already contains originally about 14 copies of the penicillin biosynthetic cluster, which shows that the strategy of increasing the gene dosage is still valid for high copy-number strains. The different behavior of the two strains in each type of culture is discussed, along with the practical implications for industrial penicillin production.     

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.     

Gene Targeting in Penicillium chrysogenum: Disruption of the lys2 Gene Leads to Penicillin Overproduction

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked α-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The α-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the α-aminoadipate reductase level.     

Albicidin antibiotic and phytotoxin biosynthesis in Xanthomonas albilineans requires a phosphopantetheinyl transferase gene

Xanthomonas albilineans produces a family of highly potent antibiotics and phytotoxins called albicidins, which are key pathogenesis factors in the systemic development of leaf scald disease of sugarcane. A gene (xabA) required for albicidin biosynthesis encodes a peptide of 278 amino acids, including the signature sequence motifs for phosphopantetheinyl transferases (PPTases) that activate polyketide and non-ribosomal peptide synthetases. The Escherichia coli entD gene, which encodes a PPTase involved in biosynthesis of enterobactin (a siderophore), restored biosynthesis of albicidin (a DNA replication inhibitor) in X. albilineans Tox- LS156 (xabA::Tn5). We conclude that XabA is a PPTase required for post-translational activation of synthetases in the albicidin biosynthetic pathway. This is the first antibiotic or toxin biosynthesis gene characterized from the genus Xanthomonas, and the first demonstration that antibiotic synthetases in the Pseudomonadaceae, like those in the Enterobacteriaceae and in Gram-positive bacteria, can require activation by a PPTase. Coupled with the recent demonstration of a separate albicidin biosynthetic gene cluster, the results indicate the possibility for overproduction of albicidins,which allows better understanding and application of these potent inhibitors of prokaryote DNA replication.     

Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. strain SCRC-2738.

The 38 kb eicosapentaenoic acid (EPA) biosynthesis gene cluster of Shewanella sp. strain SCRC-2738 was cloned into the cosmid vector (pEPA). A 27 kb nucleotide sequence of the XhoI to SpeI region of pEPA showed EPA production (6.3%) in E. coli JM109. Among the nine open reading frames (ORFs) in this sequence, only five (ORFs 2 and 5-8) were essential for EPA production. High levels of production (16%-22%) were found in E. coli JM109 transformed with a multicopy pNEB vector carrying only the five essential ORFs and in that transformed with a pNEB vector that integrated ORFs 3, 5, 6, 7 and 8, and vector pSTV28 that integrated the ORF2 encoding phosphopantetheinyl transferase (PPTase). Thus, production of EPA appears to be regulated by the presence of all the biosynthesis gene products and by the ratio of PPTase to the other gene products. The temperature -EPA production relationship in E. coli strain DH5alpha varied between constructs, suggesting that it is controlled not only by EPA biosynthesis enzymes but also by other factors in vivo. There was a strict upper temperature limit for EPA biosynthesis: no EPA was synthesized at 30 degrees C in E. coli transformants carrying any gene construct for EPA biosynthesis.     

Novel Posttranslational Activation of the LYS2-Encoded α-Aminoadipate Reductase for Biosynthesis of Lysine and Site-Directed Mutational Analysis of Conserved Amino Acid Residues in the Activation Domain of Candida albicans

The alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi. The alpha-aminoadipate reductase (AAR) of this pathway catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde by a complex mechanism involving two gene products, Lys2p and Lys5p. The LYS2 and LYS5 genes encode, respectively, a 155-kDa inactive AAR and a 30-kDa phosphopantetheinyl transferase (PPTase) which transfers a phosphopantetheinyl group from coenzyme A (CoA) to Lys2p for the activation of Lys2p and AAR activity. In the present investigation, we have confirmed the posttranslational activation of the 150-kDa Lys2p of Candida albicans, a pathogenic yeast, in the presence of CoA and C. albicans lys2 mutant (CLD2) extract as a source of PPTase (Lys5p). The recombinant Lys2p or CLD2 mutant extract exhibited no AAR activity with or without CoA. However, the recombinant 150-kDa Lys2p, when incubated with CLD2 extract and CoA, exhibited significant AAR activity compared to that of wild-type C. albicans CAI4 extract. The PPTase in the CLD2 extract was required only for the activation of Lys2p and not for AAR reaction. Site-directed mutational analysis of G882 and S884 of the Lys2p activation domain (LGGHSI) revealed no AAR activity, indicating that these two amino acids are essential for the activation. Replacement of other amino acid residues in the domain resulted in partial or full AAR activity. These results demonstrate the posttranslational activation and the requirement of specific amino acid residues in the activation domain of the AAR of C. albicans.