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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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Transcript profiles of the amplified region in penicillinG-producing strains of Penicillium chrysogenum. Transcript level of the penicillin biosynthesis genes embedded in a region that is present in tandem repeats in penicillinG-high-producing strains, the amplified region (Pc21g21280–Pc21g21420, [17,34,40]). 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.
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Figure 4: Transcript profiles of the amplified region in penicillinG-producing strains of Penicillium chrysogenum. Transcript level of the penicillin biosynthesis genes embedded in a region that is present in tandem repeats in penicillinG-high-producing strains, the amplified region (Pc21g21280–Pc21g21420, [17,34,40]). 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.

Mentions: A total number of 409 genes (117 in group 7 and 292 in group 8; Figure 3) showed significantly different transcript levels in cultures of the high-producing and cluster-free strains, irrespective of the presence of the side-chain precursor PAA. As expected, transcripts of the three biosynthesis genes, pcbAB, pcbC and penDE could not be detected in the cluster-free strain. As a result of the strain construction the other genes in the amplified region (Pc21g21280–Pc21g21420) are present as a single copy in the DS50661 strain. The observation that several of these genes showed reduced expression levels in the DS50661 strain would therefore be consistent with a gene-dosage effect (Figure 4). In addition, as confirmed by studies on a different industrial strain of P. chrysogenum [40] and on the laboratory strain Wisconsin54-1255 [34], not all genes in the amplified region are transcriptionally induced under penicillinG producing conditions. In addition to an enrichment of functional categories related to β-lactam biosynthesis (01.20; 01.20.37.05; 11; 11.05, 11.05.05; and 11.05.05.01), group 7 (increased transcript levels in the high-producing strain DS17690) was also enriched for functional categories 01.02 and 01.02.01, which are related to nitrogen and sulfur metabolism (Figure 3). Upregulation of the synthesis of (sulfur-containing) amino acids may be indicative for an increased synthesis of the amino-acid β-lactam precursors. The observation that higher transcript levels of these genes were also observed in the DS17690 strain when it was grown in the absence of the side-chain precursor PAA may indicate that even the low net production of β-lactam intermediates under these conditions has an impact on mRNA level regulation of precursor biosynthesis.


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)

Transcript profiles of the amplified region in penicillinG-producing strains of Penicillium chrysogenum. Transcript level of the penicillin biosynthesis genes embedded in a region that is present in tandem repeats in penicillinG-high-producing strains, the amplified region (Pc21g21280–Pc21g21420, [17,34,40]). 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Transcript profiles of the amplified region in penicillinG-producing strains of Penicillium chrysogenum. Transcript level of the penicillin biosynthesis genes embedded in a region that is present in tandem repeats in penicillinG-high-producing strains, the amplified region (Pc21g21280–Pc21g21420, [17,34,40]). 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.
Mentions: A total number of 409 genes (117 in group 7 and 292 in group 8; Figure 3) showed significantly different transcript levels in cultures of the high-producing and cluster-free strains, irrespective of the presence of the side-chain precursor PAA. As expected, transcripts of the three biosynthesis genes, pcbAB, pcbC and penDE could not be detected in the cluster-free strain. As a result of the strain construction the other genes in the amplified region (Pc21g21280–Pc21g21420) are present as a single copy in the DS50661 strain. The observation that several of these genes showed reduced expression levels in the DS50661 strain would therefore be consistent with a gene-dosage effect (Figure 4). In addition, as confirmed by studies on a different industrial strain of P. chrysogenum [40] and on the laboratory strain Wisconsin54-1255 [34], not all genes in the amplified region are transcriptionally induced under penicillinG producing conditions. In addition to an enrichment of functional categories related to β-lactam biosynthesis (01.20; 01.20.37.05; 11; 11.05, 11.05.05; and 11.05.05.01), group 7 (increased transcript levels in the high-producing strain DS17690) was also enriched for functional categories 01.02 and 01.02.01, which are related to nitrogen and sulfur metabolism (Figure 3). Upregulation of the synthesis of (sulfur-containing) amino acids may be indicative for an increased synthesis of the amino-acid β-lactam precursors. The observation that higher transcript levels of these genes were also observed in the DS17690 strain when it was grown in the absence of the side-chain precursor PAA may indicate that even the low net production of β-lactam intermediates under these conditions has an impact on mRNA level regulation of precursor biosynthesis.

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