An efficient fungal RNA-silencing system using the DsRed reporter gene

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.     

Establishment of an efficient RNA silencing system in Trichoderma koningii using DsRed as a reporter

We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.     

Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions

Two cephalosporin genes from Acremonium chrysogenum, pcbAB and pcbC encode the ACV (-aminoadipyl-cysteinyl-valine) synthetase and isopenicillin N-synthetase, respectively. The two adjacent genes are orientated in opposite directions on the chromosomal DNA, separated by a 1.2-kb non-translated sequence, carrying the putative promoter sequences. Complete sequencing of this intergenic region revealed differences from homologous sequences from other strains. To assess the putative promoter strength, we constructed an expression vector carrying the -glucuronidase (gusA) and -galactosidase (lacZ) genes in opposite orientation. Fusion of the pcbAB-pcbC promoter region resulted in recombinant vector molecules, which were used for in-vivo expression studies. Using the co-transformation procedure, the reporter gene fusions were transferred into A. chrysogenum recipient strains together with vector pMW1. Individual transformants were used for protein preparations to measure specific activities of the enzymes coded by the reporter genes. The data provide in-vivo evidence that the pcbC promoter is at least five times stronger than the pcbAB promoter. Our approach should prove useful in evaluating regulatory sequences that govern gene expression in A. chrysogenum.     

Regulation and compartmentalization of β‐lactam biosynthesis

Penicillins and cephalosporins are β-lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l -α-aminoadipic acid, l -cysteine and l -valine to form the tripeptide δ-l -α-aminoadipyl-l -cysteinyl-d -valine catalysed by the non-ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two-component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.     

Expression of<Emphasis Type="Italic"> cefD2</Emphasis> and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A. chrysogenum C10, and produced 80 to 100% more cephalosporin C and deacetylcephalosporin C than the parental strain. A second class of transformants, including TMCD2, TMCD32 and TMCD39, in contrast, showed a drastic reduction in cephalosporin biosynthesis relative to the untransformed control. These transformants had no detectable IPN epimerase activity and did not produce cephalosporin C or deacetylcephalosporin C. They also expressed both endogenous and exogenous cefD2 genes only after long periods (72–96 h) of incubation, as shown by Northern analysis, and were impaired in mycelial branching in liquid cultures. The negative effect of amplification of the cefD1 - cefD2 gene cluster in this second class of transformants is not correlated with high gene dosage, but appears to be due to exogenous DNA integration into a specific locus, which results in a pleiotropic effect on growth and cefD2 expression. Communicated by P. J. Punt     

Molecular analysis of the gene cluster involved in cephalosporin biosynthesis from Lysobacter lactamgenus YK90

Determination of the nucleotide sequence downstream from the Lysobacter lactamgenus pcbC gene encoding isopenicillin N synthase revealed that five open-reading frames (ORF) including the pcbC gene were tightly linked in the same orientation. Each ORF has the remarkable feature of the protein-coding frame in the DNA sequence with a high G + C content. Expression in Escherichia coli and a comparison of the deduced amino acid sequences with published sequences showed that the gene cluster contained a deacetoxycephalosporin C synthetase (DAOCS) gene (cefE), an ORF having homology with the Cephalosporium acremonium DAOCS/deacetylcephalosporin C synthetase gene (cefEF), an isopenicillin N epimerase gene(cefD), and a -lactamase gene. The gene order was pcbC-cefE-ORF3-cefD--lactamase.     

BioBrickTM compatible vector system for protein expression in Rhodobacter sphaeroides

We report here the creation of a modular, plasmid-based protein expression system utilizing elements of the native Rhodobacter puf promoter in a BioBrick^TM-based vector system with DsRed encoding a red fluorescent reporter protein. A suite of truncations of the puf promoter were made to assess the influence of different portions of this promoter on expression of heterologous proteins. The 3′ end of puf was found to be particularly important for increasing expression, with transformants accumulating significant quantities of DsRed under both aerobic and anaerobic growth conditions. Expression levels of this reporter protein in Rhodobacter sphaeroides were comparable to those achieved in Escherichia coli using the strong, constitutive P _lac promoter, thus demonstrating the robustness of the engineered system. Furthermore, we demonstrate the ability to tune the designed expression system by modulating cellular DsRed levels based upon the promoter segment utilized and oxygenation conditions. Last, we show that the new expression system is able to drive expression of a membrane protein, proteorhodopsin, and that membrane purifications from R. sphaeroides yielded significant quantities of proteorhodopsin. This toolset lays the groundwork for the engineering of multi-step pathways, including recalcitrant membrane proteins, in R. sphaeroides.     

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

Isolation of large linear plasmids from β-lactam producing actinomycete strains

Summary Sixteen -lactam producing actinomycete strains were screened for the presence of large linear plasmids. Among these, four strains contained linear plasmids of 12–450 kb in size. Southern blot analysis using a synthetic oligonucleotide for the isopenicillin N synthase gene and plasmid pBROC137 containing thecefD andcefE genes in the cephalosporin biosynthesis, respectively, showed no hybridization. This result suggests that these linear plasmids are not involved in the biosynthesis of -lactam antibiotics.     

Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)_n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.     

A vacuolar membrane protein affects drastically the biosynthesis of the ACV tripeptide and the beta-lactam pathway of Penicillium chrysogenum

The knowledge about enzymes’ compartmentalization and transport processes involved in the penicillin biosynthesis in Penicillium chrysogenum is very limited. The genome of this fungus contains multiple genes encoding transporter proteins, but very little is known about them. A bioinformatic search was made to find major facilitator supefamily (MFS) membrane proteins related to CefP transporter protein involved in the entry of isopenicillin N to the peroxisome in Acremonium chrysogenum. No strict homologue of CefP was observed in P. chrysogenum, but the penV gene was found to encode a membrane protein that contained 10 clear transmembrane spanners and two other motifs COG5594 and DUF221, typical of membrane proteins. RNAi-mediated silencing of penV gene provoked a drastic reduction of the production of the δ-(l-α-aminoadipyl-l-cysteinyl-d-valine) (ACV) and isopenicillin N intermediates and the final product of the pathway. RT-PCR and northern blot analyses confirmed a reduction in the expression levels of the pcbC and penDE biosynthetic genes, whereas that of the pcbAB gene increased. Localization studies by fluorescent laser scanning microscopy using Dsred and GFP fluorescent fusion proteins and the FM 4-64 fluorescent dye showed clearly that the protein was located in the vacuolar membrane. These results indicate that PenV participates in the first stage of the beta-lactam biosynthesis (i.e., the formation of the ACV tripeptide), probably taking part in the supply of amino acids from the vacuolar lumen to the vacuole-anchored ACV synthetase. This is in agreement with several reports on the localization of the ACV synthetase and provides increased evidence for a compartmentalized storage of precursor amino acids for non-ribosomal peptides. PenV is the first MFS transporter of P. chrysogenum linked to the beta-lactam biosynthesis that has been located in the vacuolar membrane.     

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.     

Microbial isopenicillin N synthase genes: structure, function, diversity and evolution.

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.