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Resolving phenylalanine metabolism sheds light on natural synthesis of benzylpenicillin in Penicillium chrysogenum

ABSTRACT: Industrial production of penicillin G by Penicillium chrysogenum requires medium supplementation with the side chain precursor phenylacetate. However, P.chrysogenum grown in presence of phenylalanine as sole nitrogen source formed detectable extracellular amounts of phenylacetate and penicillin G. To get more insights in the metabolism implicated, chemostat-cultivation in presence of 13C9-phenylalanine were carried out. Quantification and modeling of the labeled metabolite pools indicated that phenylalanine was i) incorporated in nascent protein, ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation and iii) oxidized into tyrosine and subsequently assimilated in the homogentisate pathway. Comparative transcriptome analysis of phenylalanine and (NH4)2SO4 grown P.chrysogenum cultures enabled to identify two putative 2-oxo acid decarboxylases Pc13g9300 and Pc18g01490. Both cDNAs were cloned and expressed in the decarboxylase-free Saccharomyces cerevisiae CEN.PK711-7C (pdc1delta, 5delta, 6delta, aro10delta, thi3delta) strain that has lost the ability to grow on glucose as sole carbon source or on phenylalanine as sole nitrogen source. Only Pc13g09300 was able to restore growth on glucose and on phenylalanine, demonstrating that this gene encodes a dual substrate pyruvate, phenylpyruvate decarboxylase. This newly identified thiamine-dependent 2-oxo acid decarboxylase provides clues to explain the formation of phenylacetate via an Ehrlich-like pathway in P.chrysogenum.

ORGANISM(S): Penicillium chrysogenum

PROVIDER: GSE32097 | GEO | 2011/11/05

SECONDARY ACCESSION(S): PRJNA147549

REPOSITORIES: GEO

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Project description:The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbonÃ¢â¬âlimited chemostat with phenylalanine as nitrogen source indicated that:; i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate ÃÂ±-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and; iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any ÃÂ±-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Experiment Overall Design: Comparison of glucose limited chemostat cultivations with phenylalanine as sole nitrogen sources of the following strains: Experiment Overall Design: - CENPK113-7D MATa MAL2-8c SUC2 Experiment Overall Design: - CENPK555-4A MATa MAL2-8c SUC2 ydr380w delta Experiment Overall Design: - CENPK5632-3B MATalpha MAL2-8c SUC2 ydr380w delta ydl080c delta Experiment Overall Design: - CENPK609-11A MATa MAL2-8c SUC2 pdc1, 5, 6 delta ydr380w delta

Project description:Its characteristic rose-like aroma makes phenylethanol a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbor an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source such as glucose provides an economically attractive alternative for phenylalanine bioconversion. In this study, a Synthetic Genetic Array screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to cause a transcriptional upregulation of ARO10 during growth with ammonium sulfate as the sole nitrogen source. Physiological characterization revealed that the aro8M-oM-^AM-^D mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8 mutation also stimulated phenylethanol production when combined with other, previously documented mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced over 3 mM of phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. The goal of the present study was to identify genes that influence the transcriptional (de)repression of the Ehrlich pathway during growth with ammonium as the nitrogen source. With the aid of Synthetic Genetic Array technology, we constructed a strain collection in which deletions in the non-essential genes in the S. cerevisiae genome were combined with a reporter plasmid comprising the ARO10 promoter fused to a reporter gene (egfp) encoding a fluorescent reporter protein. After screening by flow cytometry, deletion of ARO8 led to a deregulated expression from the ARO10 promoter. The impact of this deletion was further studied by transcriptome and intracellular metabolite analyses. Furthermore, phenylethanol production was measured in strains that combined the aro8 mutation with mutations that were previously shown to deregulate aromatic amino acid biosynthesis.

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Resolving phenylalanine metabolism sheds light on natural synthesis of benzylpenicillin in Penicillium chrysogenum

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