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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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Global gene expression response of DS17690 and DS50661 strains to the presence and absence of PAA. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-limited chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. A: Pie chart of overall transcript differences of the DS50661 and DS17690 strains grown in the absence and presence of PAA. B: Results of the pairwise comparisons of the two strains and the two conditions. Red arrows indicate genes with a higher transcript level in the respective pairwise comparison, green arrows indicate genes with a lower transcript level.
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Figure 2: Global gene expression response of DS17690 and DS50661 strains to the presence and absence of PAA. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-limited chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. A: Pie chart of overall transcript differences of the DS50661 and DS17690 strains grown in the absence and presence of PAA. B: Results of the pairwise comparisons of the two strains and the two conditions. Red arrows indicate genes with a higher transcript level in the respective pairwise comparison, green arrows indicate genes with a lower transcript level.

Mentions: Genome-wide transcriptome analysis was carried out on mycelium of both strains grown in glucose-limited chemostat cultures in the presence and absence of PAA at a dilution rate of 0.03 h-1. The average coefficient of variation of the transcriptome data derived from independent triplicate cultures did not exceed 0.21 (Table 2), which is similar to the reproducibility obtained with chemostat cultures of the non-filamentous yeast Saccharomyces cerevisiae [37]. The level of the actA [38] and gdh2 transcripts, which are commonly used loading standards for .Northern analysis, varied by less than 14% over the four situations tested. Chemostat experiments were designed to dissect gene expression responses to PAA and penicillinG production. Addition of PAA to growing DS17690 strain may induce two types of responses. Firstly, the presence of PAA itself may affect cellular processes outside β-lactam biosynthesis and thus affect the transcriptome of the fungus. Secondly, availability of a side-chain precursor enables the penicillinG formation, thus resulting in the induction and/or repression of the transcription of genes that are (in)directly related to β-lactam production. To dissect these two types of responses, we used the cluster-free strain DS50661 as a filter for the PAA response, as this strain neither produces penicillinG nor intermediates. Comparison of the transcript data of the two strains grown under the same conditions (DS17690+PAA versus DS50661+PAA; DS17690-PAA versus DS50661-PAA) will provide information on the effect of the removal of the penicillin cluster (and of possible unintended genetic changes resulting from the gene-cluster removal procedure) (Figure 2).


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)

Global gene expression response of DS17690 and DS50661 strains to the presence and absence of PAA. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-limited chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. A: Pie chart of overall transcript differences of the DS50661 and DS17690 strains grown in the absence and presence of PAA. B: Results of the pairwise comparisons of the two strains and the two conditions. Red arrows indicate genes with a higher transcript level in the respective pairwise comparison, green arrows indicate genes with a lower transcript level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Global gene expression response of DS17690 and DS50661 strains to the presence and absence of PAA. Total RNA was obtained from P. chrysogenum strains DS17690 and DS50661, grown in the presence and absence of phenylacetic acid (PAA) in independent glucose-limited chemostat cultures at D = 0.03 h-1 and hybridized to Affymetrix GeneChip® microarrays. A: Pie chart of overall transcript differences of the DS50661 and DS17690 strains grown in the absence and presence of PAA. B: Results of the pairwise comparisons of the two strains and the two conditions. Red arrows indicate genes with a higher transcript level in the respective pairwise comparison, green arrows indicate genes with a lower transcript level.
Mentions: Genome-wide transcriptome analysis was carried out on mycelium of both strains grown in glucose-limited chemostat cultures in the presence and absence of PAA at a dilution rate of 0.03 h-1. The average coefficient of variation of the transcriptome data derived from independent triplicate cultures did not exceed 0.21 (Table 2), which is similar to the reproducibility obtained with chemostat cultures of the non-filamentous yeast Saccharomyces cerevisiae [37]. The level of the actA [38] and gdh2 transcripts, which are commonly used loading standards for .Northern analysis, varied by less than 14% over the four situations tested. Chemostat experiments were designed to dissect gene expression responses to PAA and penicillinG production. Addition of PAA to growing DS17690 strain may induce two types of responses. Firstly, the presence of PAA itself may affect cellular processes outside β-lactam biosynthesis and thus affect the transcriptome of the fungus. Secondly, availability of a side-chain precursor enables the penicillinG formation, thus resulting in the induction and/or repression of the transcription of genes that are (in)directly related to β-lactam production. To dissect these two types of responses, we used the cluster-free strain DS50661 as a filter for the PAA response, as this strain neither produces penicillinG nor intermediates. Comparison of the transcript data of the two strains grown under the same conditions (DS17690+PAA versus DS50661+PAA; DS17690-PAA versus DS50661-PAA) will provide information on the effect of the removal of the penicillin cluster (and of possible unintended genetic changes resulting from the gene-cluster removal procedure) (Figure 2).

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