Juan F. Martín

Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum

Industrial penicillin production with the filamentous fungus Penicillium chrysogenum is based on an unprecedented effort in microbial strain improvement. To gain more insight into penicillin synthesis, we sequenced the 32.19 Mb genome of P. chrysogenum Wisconsin54-1255 and identified numerous genes responsible for key steps in penicillin production. DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain, grown in the presence and absence of the side-chain precursor phenylacetic acid. Transcription of genes involved in biosynthesis of valine, cysteine and α-aminoadipic acid—precursors for penicillin biosynthesis—as well as of genes encoding microbody proteins, was increased in the high-producing strain. Some gene products were shown to be directly controlling β-lactam output. Many key cellular transport processes involving penicillins and intermediates remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven metabolic engineering.

Phosphate Control of the Biosynthesis of Antibiotics and Other Secondary Metabolites Is Mediated by the PhoR-PhoP System: an Unfinished Story

The biosynthesis of many different types of antibiotics and other secondary metabolites is regulated by phosphate. Production of these valuable compounds occurs only under phosphate-limiting nutritional conditions. In a few cases, there is evidence showing that the negative phosphate control is exerted at the transcriptional level. Recently, it was shown that phosphate control of antibiotic biosynthesis in Streptomyces lividans and Streptomyces coelicolor is mediated by the two-component PhoR-PhoP system that also controls the alkaline phosphatase gene (phoA). The PhoR protein is a standard membrane sensor kinase, whereas PhoP is a member of the DNA-binding response regulators. In Escherichia coli and Bacillus subtilis, the phosphorylated PhoP protein (PhoP∼P) activates, in response to phosphate starvation, expression of the pho regulon genes by binding to consensus phosphate boxes in the promoter regions (PHO boxes). Expression of phoA in S. lividans is induced by PhoP∼P, and mutants lacking phoP (or phoR and phoP) do not form PhoA. These mutants overproduce large amounts of actinorhodin and undecylprodigiosin. No consensus PHO boxes occur in the upstream region of phosphate-regulated secondary metabolism genes. However, pathway-specific activator proteins (ActII-open reading frame 4 [ORF4], RedD, CcaR, and DnrI) are known to bind to these regions. In S. coelicolor, actII-orf4 is positively regulated by the AfsS protein, which, in turn, is induced by the phosphorylated AfsR protein. It is likely that the PhoR-PhoP system exerts its action on actinorhodin and undecylprodigiosin by a cascade mechanism mediated by AfsR and AfsS. Directed phoR-phoP gene disruption will be very useful for the construction of tailored phosphate-deregulated strains overproducing valuable secondary metabolites.

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

BackgroundDuring the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples.ResultsSurprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis.ConclusionsOur study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis

BACKGROUND Polyene macrolides are a class of large macrocyclic polyketides that interact with membrane sterols, having antibiotic activity against fungi but not bacteria. Their rings include a chromophore of 3-7 conjugated double bonds which constitute the distinct polyene structure. Pimaricin is an archetype polyene, important in the food industry as a preservative to prevent mould contamination of foods, produced by Streptomyces natalensis. We set out to clone, sequence and analyse the gene cluster responsible for the biosynthesis of this tetraene. RESULTS A large cluster of 16 open reading frames spanning 84985 bp of the S. natalensis genome has been sequenced and found to encode 13 homologous sets of enzyme activities (modules) of a polyketide synthase (PKS) distributed within five giant multienzyme proteins (PIMS0-PIMS4). The total of 60 constituent active sites, 25 of them on a single enzyme (PIMS2), make this an exceptional multienzyme system. Eleven additional genes appear to govern modification of the polyketide-derived framework and export. Disruption of the genes encoding the PKS abolished pimaricin production. CONCLUSIONS The overall architecture of the PKS gene cluster responsible for the biosynthesis of the 26-membered polyene macrolide pimaricin has been determined. Eleven additional tailoring genes have been cloned and analysed. The availability of the PKS cluster will facilitate the generation of designer pimaricins by combinatorial biosynthesis approaches. This work represents the extensive description of a second polyene macrolide biosynthetic gene cluster after the one for the antifungal nystatin.

Characterization of an Endogenous Plasmid and Development of Cloning Vectors and a Transformation System in Brevibacterium lactofermentum

A cryptic plasmid, pBL1 of 4.3 kb, has been found in lysine-producing Brevibacterium lactofermentum strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for BalI, BclI, HaeII, HindIII and HpaI. It had four sites for AvaI, seven for HaeIII, eight for MboI and a very large number for AluI, but no sites were found for PstI, EcoRI or BamHI. The estimated copy number was 30. Three different pBL1-pBR322 hybrids named pUL1, pUL10 and pUL20 were constructed. Transposon Tn5 was inserted by transposition into either the pBR322 or the pBL1 components of plasmid pUL1, pUL10 and pUL20. A shuttle vector able to replicate in Escherichia coli, Streptomyces lividans and B. lactofermentum was constructed by cloning pBL1 into the plasmid pIJ860, a bifunctional E. coli-S. lividans vector carrying the tsr, bla and kan genes. A polyethylene glycol-assisted transformation system for B. lactofermentum protoplasts was developed. Transformation frequencies of 102 transformants (μg DNA)−1 were obtained. The kan resistance gene from Tn5 was expressed very efficiently in B. lactofermentum (up to 200 μg ml−1). A smaller plasmid, pUL62, was constructed in which the tsr (thiostrepton resistance) gene of pUL61 was deleted.

Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes

The control of phosphate‐regulated genes in Streptomyces coelicolor is mediated by the two‐component system PhoR–PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, the phoRP and the phoU promoters were identified by primer extension. phoRP showed a leaderless transcript. The response‐regulator (DNA‐binding) PhoP protein was overexpressed and purified in Escherichia coli as a GST–PhoP fused protein. The DNA‐binding domain (DBD) of PhoP was also obtained in a similar manner. Both PhoP and its truncated DBD domain were found to bind with high affinity to an upstream region of the pstS and phoRP–phoU promoters close to the −35 sequence of each of these promoters. DNase I protection studies revealed a 29 bp protected stretch in the sense strand of the pstS promoter that includes two 11 bp direct repeat units. Footprinting of the bidirectional phoRP–phoU promoter region showed a 51 bp protected sequence that encompasses four direct repeat units, two of them with high similarity to the protected sequences in the pstS promoter. PHO boxes have been identified by alignment of the six direct repeat units found in those promoter regions. Each direct repeat unit adjusts to the consensus GG/TTCAYYYRG/CG.

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.

Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N.

The tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, an intermediate in the penicillin biosynthetic pathway, is converted to isopenicillin N by isopenicillin N synthetase (cyclase) of Penicillium chrysogenum. The cyclization required dithiothreitol and was stimulated by ferrous ions and ascorbate. Co2+ and Mn2+ completely inhibited enzyme activity. Optimal temperature and pH were 25 degrees C and 7.8, respectively. The reaction required O2 and was stimulated by increasing the dissolved oxygen concentration of the reaction mixture. Purification of the enzyme to a single major band in polyacrylamide gel electrophoresis was achieved by protamine sulfate precipitation, ammonium sulfate fractionation (50 to 80% of saturation), DEAE-Sephacel chromatography, and gel filtration on Sephacryl S-200. The estimated molecular weight was 39,000 +/- 1,000. The apparent Km of isopenicillin N synthetase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.13 mM. The enzyme activity was strongly inhibited by glutathione, which acts as a competitive inhibitor. A good correlation was observed between the isopenicillin N synthetase activity in extracts of four different strains of P. chrysogenum (with widely different penicillin-producing capability) and the amount of penicillin production by these strains. Images

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.

Proteome Analysis of the Penicillin Producer Penicillium chrysogenum CHARACTERIZATION OF PROTEIN CHANGES DURING THE INDUSTRIAL STRAIN IMPROVEMENT

Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by “blue silver” colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin biosynthesis in the high producer strains. These results provide useful information to improve the production of many other bioactive secondary metabolites.