Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production

Harris, Diana M.; Krogt, Zita A. van der; Klaassen, Paul; Raamsdonk, Léonie M.; Hage, Susanne; Berg, M. A.; Bovenberg, Roel A. L.; Pronk, Jack T.; Daran, Jean‐Marc

doi:10.1186/1471-2164-10-75

Cited by 75 publications

(103 citation statements)

References 82 publications

(98 reference statements)

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“…Although P. chrysogenum cannot grow on phenylacetate as the sole carbon source, it can efficiently oxidize it (61). This ability has been severely reduced in modern production strains, which preferentially incorporate phenylacetate to penicillin G instead of catabolizing it (26,72). In contrast to the P. chrysogenum NRRL 1951 strain, which is the ancestor of modern penicillinproducing strain lineages, the model strain Wisconsin 54-1255, which represents an early stage in strain improvement, already exhibits the reduced consumption of phenylacetate and stable penicillin G production.…”

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

et al. 2012

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“…To produce other pharmaceuticals in this species, an isolate completely devoid of β-lactam antibiotics was needed. Deleting all penicillin biosynthetic genes in a high producing strain led to such a host (15). To test whether the deletions had any adverse effect on secondary metabolite production potential, we reintroduced the biosynthetic genes, resulting in the recovery of penicillin production (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…P. chrysogenum is well known for β-lactam production, but this is an undesirable feature when producing other pharmaceuticals (29). Therefore, we used a variant in which the β-lactam biosynthetic genes are removed (15), essentially putting into practice the suggestion made by Jami et al (30) to use P. chrysogenum for other biotechnological purposes. Reintroduction of the penicillin biosynthetic genes proved that all capabilities were retained in this mutant, and introducing the compactin cluster demonstrated that beneficial mutations are not restricted to a single compound of interest but have general advantages for production of secondary metabolites.…”

Section: Discussionmentioning

confidence: 99%

“…E. coli DH10B and TOP10 (Invitrogen) were used for gene cloning. P. chrysogenum strains used were laboratory strain Wisconsin 54-1255 (44), the high penicillin producer DS17690 (16), the single penicillin gene cluster derivative DS47274 (15), and the β-lactam free platform strain DS50662 (15). For compactin reference fermentations and genomic DNA isolation, P. citrinum NRRL8082 was used (NRRL).…”

Section: Methodsmentioning

confidence: 99%

“…All PCR amplified fragments were verified via sequencing. Linearized plasmids were transformed to P. chrysogenum as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

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Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

McLean

Hans

Meijrink

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

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The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the β-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6-epi-pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450 Prava bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450 Prava fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.pravastatin | P450 engineering | fermentation | statin | cholesterol

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Fungal biomolecules as modulators of growth and pathogenesis

Patkar

Naqvi

2015

Fungal Biomolecules

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Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production

Cited by 75 publications

References 82 publications

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

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

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum

Fungal biomolecules as modulators of growth and pathogenesis

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