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

Diana M Harris,Roel A L Bovenberg,Susanne Hage,Paul Klaassen,Zita A van der Krogt,Marco A van den Berg,Jack T Pronk,Leonie M Raamsdonk,Jean-Marc Daran

doi:10.1186/1471-2164-10-75

Diana M Harris, Roel A L Bovenberg + Show 7 more

Open Access

https://doi.org/10.1186/1471-2164-10-75

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Abstract

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.

Highlights

Since 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
Isolates that underwent spontaneous recombinations were obtained via protoplast formation and subsequent sporulation of regenerating colonies. 27 random isolates were selected for Southern analysis to estimate the relative gene copy numbers of the penicillin biosynthesis gene pcbC and the single-copy niaA gene (Figure 1)
It was assumed that these three isolates are derivatives of P. chrysogenum DS17690 that carry a single copy of the penicillin biosynthesis gene cluster (Figure 1C)

Summary

Introduction

Since 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. Biosynthesis starts with the condensation of the three amino acids cysteine, valine and α-aminoadipic acid to form the tripeptide ACV. Penicillins can be produced from isopenicillinN by the exchange of the α-aminoadipic acid moiety for a CoA activated side-chain, such as phenylacetic acid or phenoxyacetic acid, by acyl-CoA: isopenicillinN acyltransferase (penDE), which results in the production of penicillinG or penicillinV [6,7]. These three biosynthesis genes were shown to be physically linked in a penicillin biosynthesis gene cluster (pcbAB-pcbC-penDE) [7,1013]. The enzymes involved in the first steps of biosynthesis, ACVS and IPNS, are localised in the cytosol, whereas the final steps, acyltransferase and the activation of the side-chain precursor by phenylacetyl-CoA ligase, take place in peroxisomes [16]

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