Exploring and dissecting genome-wide transcriptional responses of Penicillium chrysogenum to phenylacetic acid. Penicillium chrysogenum

In studies on beta-lactam production by Penicillium chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillin-G production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillin-G high-producing strain without a functional penicillin-biosynthesis gene cluster (pcbAB-pcbC-penDE) was constructed. The copy number of this cluster was first reduced to one via spontaneous recombination. The remaining copy was removed by targeted deletion, thereby completely abolishing beta-lactam biosynthesis. 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. A low rate of penicillin-G-independent PAA consumption indicated activity of a PAA-degrading pathway. 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 penicillin-G 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. This study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on transcriptional regulation. Overall design: Mutants of P. chrysogenum impaired in penicillin biosynthesis have been described. Most of these mutants were derived by random mutagenesis from the low-producing strain Wisconsin54-1255, which contains one copy of the penicillin biosynthesis cluster. Although these mutants were very useful for studying gene expression and gene/enzyme relationships, for identification of the factors important for penicillin-G production and PAA consumption a strain obtained from a high-producing strain background by targeted deletion is more beneficial. To this end we constructed a strain in which the tandem-repeated penicillin biosynthesis cluster was specifically deleted, whereas the strain background was retained. In such a way it is possible to identify, by a combinatorial approach, those genes affected by phenylacetic acid and those specifically important for penicillin-G biosynthesis.