title: Resolving phenylalanine metabolism sheds light on natural synthesis of penicillin G in Penicillium chrysogenum.
abstract: 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 pdc6Δ aro10Δ thi3Δ). 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.
authors.affiliation: Industrial Microbiology Section, Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft, The Netherlands.
authors.forename: Tânia
authors.initials: T
authors.lastname: Veiga
authors.forename: Daniel
authors.initials: D
authors.lastname: Solis-Escalante
authors.forename: Gabriele
authors.initials: G
authors.lastname: Romagnoli
authors.forename: Angela
authors.initials: A
authors.lastname: ten Pierick
authors.forename: Mark
authors.initials: M
authors.lastname: Hanemaaijer
authors.forename: Amit T
authors.initials: AT
authors.lastname: Deshmukh
authors.forename: Amit
authors.initials: A
authors.lastname: Deshmuhk
authors.forename: Aljoscha
authors.initials: A
authors.lastname: Wahl
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
mesh_terms: D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003653:Decarboxylation; D060847:Metabolic Engineering; D010400:Penicillin G / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000201:enzymology / Q000378:metabolism*; D010648:Phenylacetates / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D010667:Phenylpyruvic Acids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D059467:Transcriptome
chemical_list: D010648:Phenylacetates; D010667:Phenylpyruvic Acids; D010649:Phenylalanine; D002262:Carboxy-Lyases; D010400:Penicillin G; C031606:phenylpyruvic acid