A new class of IMP dehydrogenase with a role in self-resistance of mycophenolic acid producing fungi

<p>Abstract</p> <p>Background</p> <p>Many secondary metabolites produced by filamentous fungi have potent biological activities, to which the producer organism must be resistant. An example of pharmaceutical interest is mycophenolic acid (MPA), an immunosuppressant molecule produced by several <it>Penicillium </it>species. The target of MPA is inosine-5'-monophosphate dehydrogenase (IMPDH), which catalyses the rate limiting step in the synthesis of guanine nucleotides. The recent discovery of the MPA biosynthetic gene cluster from <it>Penicillium brevicompactum </it>revealed an extra copy of the IMPDH-encoding gene (<it>mpaF</it>) embedded within the cluster. This finding suggests that the key component of MPA self resistance is likely based on the IMPDH encoded by <it>mpaF</it>.</p> <p>Results</p> <p>In accordance with our hypothesis, heterologous expression of <it>mpaF </it>dramatically increased MPA resistance in a model fungus, <it>Aspergillus nidulans</it>, which does not produce MPA. The growth of an <it>A. nidulans </it>strain expressing <it>mpaF </it>was only marginally affected by MPA at concentrations as high as 200 μg/ml. To further substantiate the role of <it>mpaF </it>in MPA resistance, we searched for <it>mpaF </it>orthologs in six MPA producer/non-producer strains from <it>Penicillium </it>subgenus <it>Penicillium</it>. All six strains were found to hold two copies of IMPDH. A cladistic analysis based on the corresponding cDNA sequences revealed a novel group constituting <it>mpaF </it>homologs. Interestingly, a conserved tyrosine residue in the original class of IMPDHs is replaced by a phenylalanine residue in the new IMPDH class.</p> <p>Conclusions</p> <p>We identified a novel variant of the IMPDH-encoding gene in six different strains from <it>Penicillium </it>subgenus <it>Penicillium</it>. The novel IMPDH variant from MPA producer <it>P. brevicompactum </it>was shown to confer a high degree of MPA resistance when expressed in a non-producer fungus. Our study provides a basis for understanding the molecular mechanism of MPA resistance and has relevance for biotechnological and pharmaceutical applications.</p

Systems Analysis Unfolds the Relationship between the Phosphoketolase Pathway and Growth in Aspergillus nidulans

Background: Aspergillus nidulans is an important model organism for studies on fundamental eukaryotic cell biology and on industrial processes due to its close relation to A. niger and A. oryzae. Here we identified the gene coding for a novel metabolic pathway in A. nidulans, namely the phosphoketolase pathway, and investigated the role of an increased phosphoketolase activity. Methodology/Principal Findings: Over-expression of the phosphoketolase gene (phk) improved the specific growth rate on xylose, glycerol and ethanol. Transcriptome analysis showed that a total of 1,222 genes were significantly affected by overexpression of the phk, while more than half of the affected genes were carbon source specific. During growth on glucose medium, the transcriptome analysis showed that the response to phk over-expression is targeted to neutralize the effect of the over-expression by regulating the acetate metabolism and initiate a growth dampening response. Conclusions/Significance: Metabolic flux analysis using 13C-labelled glucose, showed that over-expression of phosphoketolase added flexibility to the central metabolism. Our findings further suggests that A. nidulans is not optimized for growth on xylose, glycerol or ethanol as the sole carbon sources. © 2008 Panagiotou et al.published_or_final_versio

Studies of the Production of Fungal Polyketides in Aspergillus nidulans by Using Systems Biology Tools▿ †

Many filamentous fungi produce polyketide molecules with great significance as human pharmaceuticals; these molecules include the cholesterol-lowering compound lovastatin, which was originally isolated from Aspergillus terreus. The chemical diversity and potential uses of these compounds are virtually unlimited, and it is thus of great interest to develop a well-described microbial production platform for polyketides. Using genetic engineering tools available for the model organism Aspergillus nidulans, we constructed two recombinant strains, one expressing the Penicillium griseofulvum 6-methylsalicylic acid (6-MSA) synthase gene and one expressing the 6-MSA synthase gene and overexpressing the native xylulose-5-phosphate phosphoketolase gene (xpkA) for increasing the pool of polyketide precursor levels. The physiology of the recombinant strains and that of a reference wild-type strain were characterized on glucose, xylose, glycerol, and ethanol media in controlled bioreactors. Glucose was found to be the preferred carbon source for 6-MSA production, and 6-MSA concentrations up to 455 mg/liter were obtained for the recombinant strain harboring the 6-MSA gene. Our findings indicate that overexpression of xpkA does not directly improve 6-MSA production on glucose, but it is possible, if the metabolic flux through the lower part of glycolysis is reduced, to obtain quite high yields for conversion of sugar to 6-MSA. Systems biology tools were employed for in-depth analysis of the metabolic processes. Transcriptome analysis of 6-MSA-producing strains grown on glucose and xylose in the presence and absence of xpkA overexpression, combined with flux and physiology data, enabled us to propose an xpkA-msaS interaction model describing the competition between biomass formation and 6-MSA production for the available acetyl coenzyme A

Versatile Enzyme Expression and Characterization System for Aspergillus nidulans, with the Penicillium brevicompactum Polyketide Synthase Gene from the Mycophenolic Acid Gene Cluster as a Test Case▿†

Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid