Marco van den Berg

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

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.

The Sequence of a 1.8-Mb Bacterial Linear Plasmid Reveals a Rich Evolutionary Reservoir of Secondary Metabolic Pathways

Plasmids are mobile genetic elements that play a key role in the evolution of bacteria by mediating genome plasticity and lateral transfer of useful genetic information. Although originally considered to be exclusively circular, linear plasmids have also been identified in certain bacterial phyla, notably the actinomycetes. In some cases, linear plasmids engage with chromosomes in an intricate evolutionary interplay, facilitating the emergence of new genome configurations by transfer and recombination or plasmid integration. Genome sequencing of Streptomyces clavuligerus ATCC 27064, a Gram-positive soil bacterium known for its production of a diverse array of biotechnologically important secondary metabolites, revealed a giant linear plasmid of 1.8 Mb in length. This megaplasmid (pSCL4) is one of the largest plasmids ever identified and the largest linear plasmid to be sequenced. It contains more than 20% of the putative protein-coding genes of the species, but none of these is predicted to be essential for primary metabolism. Instead, the plasmid is densely packed with an exceptionally large number of gene clusters for the potential production of secondary metabolites, including a large number of putative antibiotics, such as staurosporine, moenomycin, β-lactams, and enediynes. Interestingly, cross-regulation occurs between chromosomal and plasmid-encoded genes. Several factors suggest that the megaplasmid came into existence through recombination of a smaller plasmid with the arms of the main chromosome. Phylogenetic analysis indicates that heavy traffic of genetic information between Streptomyces plasmids and chromosomes may facilitate the rapid evolution of secondary metabolite repertoires in these bacteria.

Matching the proteome to the genome : the microbody of penicillin-producing Penicillium chrysogenum cells

In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.

Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum

ABSTRACT In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1.

Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production

BackgroundSince the discovery of the antibacterial activity of penicillin by Fleming 80 years ago, improvements of penicillin titer were essentially achieved by classical strain improvement through mutagenesis and screening. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective.ResultsIn studies on β-lactam production by P. chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillinG production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillinG high-producing strain without a functional penicillin-biosynthesis gene cluster was constructed. In glucose-limited chemostat cultures of the high-producing and cluster-free strains, PAA addition caused a small reduction of the biomass yield, consistent with PAA acting as a weak-organic-acid uncoupler. Microarray-based analysis on chemostat cultures of the high-producing and cluster-free strains, grown in the presence and absence of PAA, showed that: (i) Absence of a penicillin gene cluster resulted in transcriptional upregulation of a gene cluster putatively involved in production of the secondary metabolite aristolochene and its derivatives, (ii) The homogentisate pathway for PAA catabolism is strongly transcriptionally upregulated in PAA-supplemented cultures (iii) Several genes involved in nitrogen and sulfur metabolism were transcriptionally upregulated under penicillinG producing conditions only, suggesting a drain of amino-acid precursor pools. Furthermore, the number of candidate genes for penicillin transporters was strongly reduced, thus enabling a focusing of functional analysis studies.ConclusionThis study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on gene expression regulation. This study provides for the first time clear-cut target genes for metabolic engineering, beyond the three genes of the β-lactam pathway.

Repeat induced point mutation in two asexual fungi, Aspergillus niger and Penicillium chrysogenum

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.

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.

Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor

Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P.chrysogenum. Growth of the engineered strain in the presence of adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA-producing strain and a strain lacking key genes in beta-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. Transcriptome analysis revealed that adipate catabolism in P.chrysogenum occurs via beta-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses.

δ-(l-α-Aminoadipyl)-l-cysteinyl-d-valine synthetase, that mediates the first committed step in penicillin biosynthesis, is a cytosolic enzyme

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.