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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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Related in: MedlinePlus

Cross-sections, profiles and overrepresented functional categories of the pair-wise comparisons. Transcript data from independent chemostat cultures of P. chrysogenum strains DS17690 and DS50661 grown at D = 0.03 h-1 in the presence and absence of phenylacetic acid (PAA) were compared in four pairwise comparisons (DS17690 + PAA versus DS17690 - PAA; DS50661 + PAA versus DS50661 - PAA; DS17690 + PAA versus DS50661 + PAA and DS17690 - PAA versus DS50661 - PAA. Genes whose transcript level was significantly different in at least one of the four pairwise comparisons were overlapped as shown in A, resulting in 12 different groups of genes: 1–6: compare the response to PAA in DS17690 and DS50661; 7–12 compare the effect of the cluster removal. B shows the gene-transcript profiles of the groups of specific interest with the results of the hypergeometric distribution analysis for enrichment of functional categories. The thick line represents the average of the mean normalized transcript data of the genes comprising the cluster. The y-axis represents 10log transcript values. Functional categories are mentioned together with their P-value and the number of genes in the respective functional category in the group of genes with a higher transcript level compared to the prevalence of this functional category in the whole genome. Due to a large redundancy in the functional categories some categories might appear without having a significant biological relevance.
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Figure 3: Cross-sections, profiles and overrepresented functional categories of the pair-wise comparisons. Transcript data from independent chemostat cultures of P. chrysogenum strains DS17690 and DS50661 grown at D = 0.03 h-1 in the presence and absence of phenylacetic acid (PAA) were compared in four pairwise comparisons (DS17690 + PAA versus DS17690 - PAA; DS50661 + PAA versus DS50661 - PAA; DS17690 + PAA versus DS50661 + PAA and DS17690 - PAA versus DS50661 - PAA. Genes whose transcript level was significantly different in at least one of the four pairwise comparisons were overlapped as shown in A, resulting in 12 different groups of genes: 1–6: compare the response to PAA in DS17690 and DS50661; 7–12 compare the effect of the cluster removal. B shows the gene-transcript profiles of the groups of specific interest with the results of the hypergeometric distribution analysis for enrichment of functional categories. The thick line represents the average of the mean normalized transcript data of the genes comprising the cluster. The y-axis represents 10log transcript values. Functional categories are mentioned together with their P-value and the number of genes in the respective functional category in the group of genes with a higher transcript level compared to the prevalence of this functional category in the whole genome. Due to a large redundancy in the functional categories some categories might appear without having a significant biological relevance.

Mentions: The set of differentially expressed genes was distributed over 12 groups following a two-way comparison (Figure 3) [see also Additional file 1]. Groups 1 and 2 (Figure 3) contained genes whose transcript levels were consistently higher or lower in the presence of PAA, irrespective of the strain background. Similarly, groups 7 and 8 harboured genes whose transcript levels were consistently higher or lower in the DS17690 strain, irrespective of the presence of PAA (Figure 3). Groups 5 and 6 represent genes that show a higher and a lower transcript level in the presence of PAA, but only in the DS17690 strain. These latter profiles are consistent with genes whose transcription is responsive to the production of penicillinG.


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)

Cross-sections, profiles and overrepresented functional categories of the pair-wise comparisons. Transcript data from independent chemostat cultures of P. chrysogenum strains DS17690 and DS50661 grown at D = 0.03 h-1 in the presence and absence of phenylacetic acid (PAA) were compared in four pairwise comparisons (DS17690 + PAA versus DS17690 - PAA; DS50661 + PAA versus DS50661 - PAA; DS17690 + PAA versus DS50661 + PAA and DS17690 - PAA versus DS50661 - PAA. Genes whose transcript level was significantly different in at least one of the four pairwise comparisons were overlapped as shown in A, resulting in 12 different groups of genes: 1–6: compare the response to PAA in DS17690 and DS50661; 7–12 compare the effect of the cluster removal. B shows the gene-transcript profiles of the groups of specific interest with the results of the hypergeometric distribution analysis for enrichment of functional categories. The thick line represents the average of the mean normalized transcript data of the genes comprising the cluster. The y-axis represents 10log transcript values. Functional categories are mentioned together with their P-value and the number of genes in the respective functional category in the group of genes with a higher transcript level compared to the prevalence of this functional category in the whole genome. Due to a large redundancy in the functional categories some categories might appear without having a significant biological relevance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Cross-sections, profiles and overrepresented functional categories of the pair-wise comparisons. Transcript data from independent chemostat cultures of P. chrysogenum strains DS17690 and DS50661 grown at D = 0.03 h-1 in the presence and absence of phenylacetic acid (PAA) were compared in four pairwise comparisons (DS17690 + PAA versus DS17690 - PAA; DS50661 + PAA versus DS50661 - PAA; DS17690 + PAA versus DS50661 + PAA and DS17690 - PAA versus DS50661 - PAA. Genes whose transcript level was significantly different in at least one of the four pairwise comparisons were overlapped as shown in A, resulting in 12 different groups of genes: 1–6: compare the response to PAA in DS17690 and DS50661; 7–12 compare the effect of the cluster removal. B shows the gene-transcript profiles of the groups of specific interest with the results of the hypergeometric distribution analysis for enrichment of functional categories. The thick line represents the average of the mean normalized transcript data of the genes comprising the cluster. The y-axis represents 10log transcript values. Functional categories are mentioned together with their P-value and the number of genes in the respective functional category in the group of genes with a higher transcript level compared to the prevalence of this functional category in the whole genome. Due to a large redundancy in the functional categories some categories might appear without having a significant biological relevance.
Mentions: The set of differentially expressed genes was distributed over 12 groups following a two-way comparison (Figure 3) [see also Additional file 1]. Groups 1 and 2 (Figure 3) contained genes whose transcript levels were consistently higher or lower in the presence of PAA, irrespective of the strain background. Similarly, groups 7 and 8 harboured genes whose transcript levels were consistently higher or lower in the DS17690 strain, irrespective of the presence of PAA (Figure 3). Groups 5 and 6 represent genes that show a higher and a lower transcript level in the presence of PAA, but only in the DS17690 strain. These latter profiles are consistent with genes whose transcription is responsive to the production of penicillinG.

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