Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the β-lactam producer Penicillium chrysogenum

Gombert, Andreas; Veiga, Tânia; Puig-Martinez, M.; Lamboo, F.; Nijland, Jeroen G.; Driessen, Arnold J. M.; Pronk, Jack T.; Daran, Jean‐Marc

doi:10.1016/j.fgb.2011.04.007

Cited by 22 publications

(11 citation statements)

References 44 publications

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“…For each strain, transcriptome analysis was performed from at least two independent replicate chemostat cultures. The average coefficient of variation for transcriptome data from replicated cultures was below 20%, in accordance with previous chemostat-based transcriptome analyses in P. chrysogenum (Douma et al, 2011;Gombert et al, 2011;Harris et al, 2009aHarris et al, , 2009bKoetsier et al, 2010;Snoek et al, 2009;van den Berg et al, 2008).…”

Section: Chemostat-based Transcriptome Analysis: Data Quality and Ovesupporting

confidence: 90%

Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

Veiga

Nijland

Driessen

et al. 2012

OMICS: A Journal of Integrative Biology

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The multicomponent global regulator Velvet complex has been identified as a key regulator of secondary metabolite production in Aspergillus and Penicillium species. Previous work indicated a massive impact of PcvelA and PclaeA deletions on penicillin production in prolonged batch cultures of P. chrysogenum, as well as substantial changes in transcriptome. The present study investigated the impact of these mutations on product formation and genome-wide transcript profiles under glucose-limited aerobic conditions, relevant for industrial production of b-lactams. Predicted amino acid sequences of PcVelA and PcLaeA in this strain were identical to those in its ancestor Wisconsin54-1255. Controls were performed to rule out transformation-associated loss of penicillin-biosynthesis clusters. The correct PcvelA and PclaeA deletion strains revealed a small reduction of penicillin G productivity relative to the reference strain, which is a much smaller reduction than previously reported for prolonged batch cultures of similar P. chrysogenum mutants. Chemostat-based transcriptome analysis yielded only 23 genes with a consistent differential response in the PcvelAD and PclaeAD mutants when grown in the absence of the penicillin G side-chain precursor phenylacetic acid. Eleven of these genes belonged to two small gene clusters, one of which contained a gene with high homology to the aristolochene synthase. These results provide a clear caveat that the impact of the Velvet complex on secondary metabolism in filamentous fungi is strongly context dependent.

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Section: Chemostat-based Transcriptome Analysis: Data Quality and Ovesupporting

confidence: 90%

Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

Veiga

Nijland

Driessen

et al. 2012

OMICS: A Journal of Integrative Biology

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“…Biomass dry weight was measured in duplicate samples via filtration and drying as described previously for P. chrysogenum (23) and S. cerevisiae (6). For sampling from labeling experiments, filters with mycelia were dried for 24 h at 70°C.…”

Section: Methodsmentioning

confidence: 99%

Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

et al. 2012

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The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-13 C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH 4 ) 2 SO 4 as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6⌬ aro10⌬ thi3⌬). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate.

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“…Deletion strains Genomic DNA fragments used in the construction of genedeletion cassettes for Pc13g14410, Pc22g25150, Pc20g15640, Pc20g07920 and Pc20g01800 deletion strains were amplified from genomic DNA of P. chrysogenum Wisconsin54-1255 using Phusion Hot-Start Polymerase (Finnzymes, Landsmeer, The Netherlands) and the oligonucleotides listed in Table S1. Plasmid construction was performed with the Multisite Gateway s Three-Fragment Vector Construction Kit (Invitrogen, Breda, The Netherlands) as previously described (Gombert et al, 2011). The destination vectors pDEST43-KO Pc13g14410, pDEST43-KO Pc22g25150 and pDEST43-KO Pc20g15640 contained HindIII, Mph1103I and Cfr42I restriction sites, respectively, that were used to cut out the deletion cassettes.…”

Section: Strain Constructionmentioning

confidence: 99%

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Veiga¹,

Gombert²,

Landes³

et al. 2012

Metabolic Engineering

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Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via β-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of β-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of β-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis.

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Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the β-lactam producer Penicillium chrysogenum

Cited by 22 publications

References 44 publications

Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

Impact of Velvet Complex on Transcriptome and Penicillin G Production in Glucose-Limited Chemostat Cultures of a β-Lactam High-ProducingPenicillium chrysogenumStrain

Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

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