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 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.

Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

“…The decarboxylase activity with phenylpyruvate, ketoisovalerate, ketomethylvalerate, ketoisocaproate, or methylthio-2-oxo-butanoate as the substrate was measured by monitoring NAD ϩ reduction in the presence of excess aldehyde dehydrogenase from yeast, using a Tecan GENios Pro (Tecan) (57). The reaction mixture contained, in a total volume of 300 l, 100 mM KH 2 PO 4 -K 2 HPO 4 buffer (pH 7.0), 2 mM NAD ϩ , 5 mM MgCl 2 , 15 mM pyrazole, 0.2 mM thiamine diphosphate, and 1.75 U of aldehyde dehydrogenase from yeast (Sigma-Aldrich) (dissolved in 1 mM dithiothreitol).…”

The old 3-oxoadipate pathway revisited: New insights in the catabolism of aromatics in the saprophytic fungus Aspergillus nidulans

“…The homogentisate degradation pathway (involved e.g. in the phenylalanine metabolism) has been recently resolved in Penicillium chrysogenum (Veiga et al, 2012). On the contrary, the 3-oxoadipate pathway that combines the protocatechuate and the catechol branches, remains largely overlooked, notwithstanding its significance in the end degradation of numerous exogenous aromatic hydrocarbons (Harwood and Parales, 1996).…”

Functional characterization of a Penicillium chrysogenum mutanase gene induced upon co-cultivation with Bacillus subtilis

“…P. chrysogenum is able to synthesize phenylacetic acid by itself, using L-phenylalanine as a precursor [10]. However, co-cultivation did not affect expression of genes involved in phenylalanine production or catabolism.…”

“…Most of these genes are not significantly transcribed under standard laboratory conditions [5-10] and, consequently, their functional and physiological roles remains to be determined.…”