Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies

Snoek, I.S.I.; Krogt, Zita A. van der; Touw, Hesselien; Kerkman, Richard; Pronk, Jack T.; Bovenberg, Roel A. L.; Berg, M. A.; Daran, Jean‐Marc

doi:10.1016/j.fgb.2009.02.008

Cited by 73 publications

(78 citation statements)

References 35 publications

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“…The average coefficient of variation for biological replicates (triplicates for the carbon-limited cultures with ammonium sul- a Three different network scenario hypotheses were employed to obtain an optimal fit for flux distribution: scenario i, phenylalanine was incorporated into protein, incorporated into penicillin G, metabolized via the homogentisate pathway, or metabolized via tyrosine; scenario ii, an unidentified pathway for phenylalanine metabolism, which did not involve any of the measured metabolites, was introduced into the model together with exchanges of phenylalanine and tyrosine with the protein pool (synthesis and degradation); and scenario iii, hypothetical hydroxylase activities that convert phenylpyruvate to 4-hydroxyphenylpyruvate and phenylacetaldehyde to 4-hydroxyphenylacetaldehyde were included. (14,26,27,36,68,71).…”

Section: Resultsmentioning

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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Section: Resultsmentioning

confidence: 99%

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

et al. 2012

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“…Although the targeting efficiency obtained is less than in a DPcku70 strain (Hoff et al, 2009) the observed frequencies of homologous recombination are clearly above values that were observed with the wild-type strain (e.g. Casqueiro et al, 1999;Hoff et al, 2009;Naranjo et al, 2004;Snoek et al, 2009). The use of the non-integrative RNAi vector leads to the construction of single and double mutants that will be a valuable tool for further functional and physiological studies.…”

Section: Discussionmentioning

confidence: 81%

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

2009

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RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrial β-lactam antibiotic producer Penicillium chrysogenum using the DsRed reporter gene under the control of the constitutive trpC promoter or the inducible xylP promoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific 32P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenous PcbrlA morphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi in P. chrysogenum is dependent on a Dicer peptide was provided with a strain lacking Pcdcl2. In the ΔPcdcl2 background, silencing of the PcbrlA gene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system in P. chrysogenum was finally demonstrated by silencing the Pcku70 gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.

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“…To enable site-specific gene deletion in P. chrysogenum, strain DS54465, an ⌬hdfA derivative of DS17690, was used (23). Since this strain cannot be provided with fluorescent markers by random integration, we constructed an niaD-[P gpdA -GFP-SKL-T penDE ]-niaD cassette that enables the specific integration of the fluorescent marker in the P. chrysogenum niaD locus.…”

Section: Methodsmentioning

confidence: 99%

Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum

Meijer

Gidijala

Fekken

et al. 2010

Appl Environ Microbiol

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In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1.

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Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies

Cited by 73 publications

References 35 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

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum

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