Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

<p>Abstract</p> <p>Background</p> <p>β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus <it>Penicillium chrysogenum</it>. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast <it>Hansenula polymorpha</it>. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in <it>H. polymorpha</it>.</p> <p>Results</p> <p>The <it>P. chrysogenum pcbC </it>gene encoding isopenicillin N synthase (IPNS) was successfully expressed in <it>H. polymorpha</it>, but the protein produced was unstable and inactive when the host was grown at its optimal growth temperature (37°C). Heterologously produced IPNS protein levels were enhanced when the cultivation temperature was lowered to either 25°C or 30°C. Furthermore, IPNS produced at these lower cultivation temperatures was functionally active. Localization experiments demonstrated that, like in <it>P. chrysogenum</it>, in <it>H. polymorpha </it>IPNS is located in the cytosol.</p> <p>Conclusion</p> <p>In <it>P. chrysogenum</it>, the enzymes involved in penicillin production are compartmentalized in the cytosol and in microbodies. In this study, we focus on the cytosolic enzyme IPNS. Our data show that high amounts of functionally active IPNS enzyme can be produced in the heterologous host during cultivation at 25°C, the optimal growth temperature for <it>P. chrysogenum</it>. This is a new step forward in the metabolic reprogramming of <it>H. polymorpha </it>to produce penicillin.</p

Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

<p>Abstract</p> <p>Background</p> <p><it>Penicillium chrysogenum </it>converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the <it>penDE </it>gene. <it>In silico </it>analysis of the <it>P. chrysogenum </it>genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding <it>penDE </it>gene. We have termed this gene <it>ial </it>because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the <it>ial </it>gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.</p> <p>Results</p> <p>The IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null <it>ial </it>mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic) and amidohydrolase (6-APA forming) activities <it>in vivo</it>. When the canonical ARL motif (leading to peroxisomal targeting) was added to the C-terminus of the IAL protein (IAL^ARL) by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in <it>Escherichia coli</it>. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive <it>in vitro</it>.</p> <p>Conclusion</p> <p>No activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the <it>P. chrysogenum </it>IAL, the <it>A. nidulans </it>IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the <it>ial </it>gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in <it>A. nidulans</it>, the <it>ial </it>and <it>penDE </it>genes might have been formed from a single ancestral gene that became duplicated during evolution, although a separate evolutive origin for the <it>ial </it>and <it>penDE </it>genes, is also discussed.</p

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

Abstract Background Since the discovery of the antibacterial activity of penicillin by Fleming 80 years ago, improvements of penicillin titer were essentially achieved by classical strain improvement through mutagenesis and screening. The recent sequencing of Penicillium chrysogenum strain Wisconsin1255-54 and the availability of genomics tools such as DNA-microarray offer new perspective. Results In studies on β-lactam production by P. chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillinG production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillinG high-producing strain without a functional penicillin-biosynthesis gene cluster was constructed. In glucose-limited chemostat cultures of the high-producing and cluster-free strains, PAA addition caused a small reduction of the biomass yield, consistent with PAA acting as a weak-organic-acid uncoupler. Microarray-based analysis on chemostat cultures of the high-producing and cluster-free strains, grown in the presence and absence of PAA, showed that: (i) Absence of a penicillin gene cluster resulted in transcriptional upregulation of a gene cluster putatively involved in production of the secondary metabolite aristolochene and its derivatives, (ii) The homogentisate pathway for PAA catabolism is strongly transcriptionally upregulated in PAA-supplemented cultures (iii) Several genes involved in nitrogen and sulfur metabolism were transcriptionally upregulated under penicillinG producing conditions only, suggesting a drain of amino-acid precursor pools. Furthermore, the number of candidate genes for penicillin transporters was strongly reduced, thus enabling a focusing of functional analysis studies. Conclusion This study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on gene expression regulation. This study provides for the first time clear-cut target genes for metabolic engineering, beyond the three genes of the β-lactam pathway.</p