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

Harris DM, van der Krogt ZA, Klaassen P, Raamsdonk LM, Hage S, van den Berg MA, Bovenberg RA, Pronk JT, Daran JM - BMC Genomics (2009)

Bottom Line: 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.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 gene expression regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. DHarris@cntnl.jnj.com

ABSTRACT

Background: 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. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective.

Results: In studies on beta-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.

Conclusion: This 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 beta-lactam pathway.

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Detoxification of phenylacetic acid via incorporation into penicillinG or the homogentisate pathway. Those genes (putatively) related to penicillin, biosynthesis and phenylacetate catabolism are depicted. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. The color bar indicates the range of the mean normalized transcript level value per gene. αAAA: α-aminoadipate, Cys: cysteine and Val: valine.
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Figure 6: Detoxification of phenylacetic acid via incorporation into penicillinG or the homogentisate pathway. Those genes (putatively) related to penicillin, biosynthesis and phenylacetate catabolism are depicted. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. The color bar indicates the range of the mean normalized transcript level value per gene. αAAA: α-aminoadipate, Cys: cysteine and Val: valine.

Mentions: 329 genes (groups 1 and 2, Figure 3) showed a consistently different transcript level in the presence and absence of PAA. Remarkably, genes belonging to the penicillin biosynthesis gene cluster were not differentially expressed in the presence and absence of PAA, with the pcb genes still being highly expressed in the absence of PAA. Only the gene encoding the PAA CoA ligase (phl, Pc22g14900) [46] showed a significantly higher transcript level in the presence of PAA (increase of 70 and 100% relative to cultures lacking PAA for the DS17690 and DS50661 strains, respectively). Recent functional characterization of phl revealed that upon its deletion, the PAA-CoA ligase activity decreased by only 40% [46], indicating the existence of one or more additional PAA-CoA ligases. Five genes with similarity to aryl- or fatty-acid CoA ligases were expressed to a higher level in cultures of both strains grown in the presence of PAA. The two closest phl homologues (Pc22g24780 and Pc22g20270) showed over 65% identity with Aspergillus proteins of unknown function, as well as a strong similarity with Arabidopsis thaliana 4-coumarate-CoA ligase (30%) (Figure 6). Further inspection of the predicted amino acid sequences of both these genes identified the presence of PTS1 peroxisomal targeting sequences. Based on these observations, Pc22g24780 and Pc22g20270 represent interesting candidates for further functional analysis aimed at identifying alternative PAA CoA-ligases in phlΔ strains.


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

Harris DM, van der Krogt ZA, Klaassen P, Raamsdonk LM, Hage S, van den Berg MA, Bovenberg RA, Pronk JT, Daran JM - BMC Genomics (2009)

Detoxification of phenylacetic acid via incorporation into penicillinG or the homogentisate pathway. Those genes (putatively) related to penicillin, biosynthesis and phenylacetate catabolism are depicted. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. The color bar indicates the range of the mean normalized transcript level value per gene. αAAA: α-aminoadipate, Cys: cysteine and Val: valine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657799&req=5

Figure 6: Detoxification of phenylacetic acid via incorporation into penicillinG or the homogentisate pathway. Those genes (putatively) related to penicillin, biosynthesis and phenylacetate catabolism are depicted. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. The color bar indicates the range of the mean normalized transcript level value per gene. αAAA: α-aminoadipate, Cys: cysteine and Val: valine.
Mentions: 329 genes (groups 1 and 2, Figure 3) showed a consistently different transcript level in the presence and absence of PAA. Remarkably, genes belonging to the penicillin biosynthesis gene cluster were not differentially expressed in the presence and absence of PAA, with the pcb genes still being highly expressed in the absence of PAA. Only the gene encoding the PAA CoA ligase (phl, Pc22g14900) [46] showed a significantly higher transcript level in the presence of PAA (increase of 70 and 100% relative to cultures lacking PAA for the DS17690 and DS50661 strains, respectively). Recent functional characterization of phl revealed that upon its deletion, the PAA-CoA ligase activity decreased by only 40% [46], indicating the existence of one or more additional PAA-CoA ligases. Five genes with similarity to aryl- or fatty-acid CoA ligases were expressed to a higher level in cultures of both strains grown in the presence of PAA. The two closest phl homologues (Pc22g24780 and Pc22g20270) showed over 65% identity with Aspergillus proteins of unknown function, as well as a strong similarity with Arabidopsis thaliana 4-coumarate-CoA ligase (30%) (Figure 6). Further inspection of the predicted amino acid sequences of both these genes identified the presence of PTS1 peroxisomal targeting sequences. Based on these observations, Pc22g24780 and Pc22g20270 represent interesting candidates for further functional analysis aimed at identifying alternative PAA CoA-ligases in phlΔ strains.

Bottom Line: 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.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 gene expression regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. DHarris@cntnl.jnj.com

ABSTRACT

Background: 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. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective.

Results: In studies on beta-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.

Conclusion: This 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 beta-lactam pathway.

Show MeSH
Related in: MedlinePlus