The transport of phenylacetic acid across the peroxisomal membrane is mediated by the PaaT protein in Penicillium chrysogenum

Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl–CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT–DsRed and EGFP–SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.     

Expression of a cephalosporin C esterase gene in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.     

A vacuolar membrane protein affects drastically the biosynthesis of the ACV tripeptide and the beta-lactam pathway of Penicillium chrysogenum

The knowledge about enzymes’ compartmentalization and transport processes involved in the penicillin biosynthesis in Penicillium chrysogenum is very limited. The genome of this fungus contains multiple genes encoding transporter proteins, but very little is known about them. A bioinformatic search was made to find major facilitator supefamily (MFS) membrane proteins related to CefP transporter protein involved in the entry of isopenicillin N to the peroxisome in Acremonium chrysogenum. No strict homologue of CefP was observed in P. chrysogenum, but the penV gene was found to encode a membrane protein that contained 10 clear transmembrane spanners and two other motifs COG5594 and DUF221, typical of membrane proteins. RNAi-mediated silencing of penV gene provoked a drastic reduction of the production of the δ-(l-α-aminoadipyl-l-cysteinyl-d-valine) (ACV) and isopenicillin N intermediates and the final product of the pathway. RT-PCR and northern blot analyses confirmed a reduction in the expression levels of the pcbC and penDE biosynthetic genes, whereas that of the pcbAB gene increased. Localization studies by fluorescent laser scanning microscopy using Dsred and GFP fluorescent fusion proteins and the FM 4-64 fluorescent dye showed clearly that the protein was located in the vacuolar membrane. These results indicate that PenV participates in the first stage of the beta-lactam biosynthesis (i.e., the formation of the ACV tripeptide), probably taking part in the supply of amino acids from the vacuolar lumen to the vacuole-anchored ACV synthetase. This is in agreement with several reports on the localization of the ACV synthetase and provides increased evidence for a compartmentalized storage of precursor amino acids for non-ribosomal peptides. PenV is the first MFS transporter of P. chrysogenum linked to the beta-lactam biosynthesis that has been located in the vacuolar membrane.     

Heterologous Protein Production in Acremonium chrysogenum: Expression of Bacterial Cephalosporin C Acylase and Human Thrombomodulin Genes

We have developed an efficient expression system for foreign genes in Acremonium chrysogenum. After inserting the foreign gene between the phosphoglycerate kinase (PGK) promoter and a terminator derived from A. chrysogenum, multiple copies of this expression unit are tandemly ligated into cosmids and the resultant cosmids are introduced into A. chrysogenum.

We expressed Pseudomonas cephalosporin C acylase and a human thrombomodulin mutant protein containing the fourth, fifth, and sixth epidermal growth factor (EGF)-like structures (E456). The acylase activity in the transformants obtained using our system was several times higher than that in the transformants without the use of the system. The acylase proteins expressed had enzymatic and immunochemical properties identical to those of authentic acylase. The transformants with the expression plasmid for E456 secreted biologically active E456 protein into the culture medium. The amino terminal sequence of the purified E456 was identical to that of recombinant E456 obtained using mammalian cells.     

An Efficient Fungal RNA-Silencing System Using the DsRed Reporter Gene

In filamentous fungi, RNA silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level of expression of the DsRed gene were constructed, resulting in red fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by Northern hybridization, real-time PCR quantification, and spectrofluorometric measurement of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of the DsRed silencing system was demonstrated by investigating cosilencing of DsRed together with pcbC, encoding the isopenicillin N synthase, an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can be detected easily by a bioassay measuring the antibiotic activity of individual strains. In addition, the presence of the isopenicillin N synthase was investigated by Western blot hybridization. All transformants having a colorless phenotype showed simultaneous downregulation of the pcbC gene, albeit at different levels. The RNA-silencing system presented here should be a powerful genetic tool for strain improvement and genome-wide analysis of this biotechnologically important filamentous fungus.     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

Genetic transformation of the mycelium fungi <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

The system of transformation of heterologous genes under the method of agrobacterial transfer into Acremonium chrysogenum ATCC 11550 wild-type strains, natural producents of beta-lactam antibiotic cephalosporin C, and strains highly producing cephalosporin C no. 26/8 revealed by the multistage selection on its basis were developed. Vectors for agrobacterial transformation of A. chrysogenum containing expression cassettes of genes encoding resistance to geneticin (G418) and bleomicin (Zeocin^TM) antibiotics under control of Ashbya gossypii and Saccharomyces cerevisiae TEF1 promoters were constructed. A comparable assessment of agrotransformation methods while co-cultivating fungi and agrobacterial cells on filters and in deep culture was conducted. Transformants, selected by resistance to geneticin and bleomicin, were characterized by PCR and Southern blot analyses.     

Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C

Using pulse electrophoresis in controlled homogenous electric field we performed molecular karyotyping of cephalosporin C-producing industrial and laboratory strains of Acremonium chrysogenum. Differences in size of several chromosomes of high-producing strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the high-producing strain was not associated with alteration of localization and copy number of cephalosporin C (CPC) biosynthesis and transport genes. A cluster of ??early?? CPC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the ??late genes??, on chromosome II (2.3 Mb). Both clusters are presented as a single copy per A. chrysogenum genome in the wild-type and in CB26/8 high-producing strains. Based on comparative analysis of laboratory and industrial CPC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.     

Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.     

头孢菌素C生物合成调控研究进展北大核心CSCD

头孢菌素C由丝状真菌顶头孢霉产生,属于β-内酰胺类抗生素。其经改造后的7-氨基头孢烷酸是头孢类抗生素的重要中间体。头孢类抗生素在国内外抗生素市场中占有巨大的份额,是临床上的主要抗感染药物。随着分子生物学的发展,头孢菌素C的生物合成途径已基本阐明。为提高头孢菌素C的产量和降低生产成本,越来越多的研究者开始关注其较为精细、复杂的调控机制。本文重点对头孢菌素C生物合成及其调控机制的最新进展进行了简述,希望为今后头孢菌素C生产菌株的菌种改造和传统产业的升级换代提供一定的借鉴。     

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells

In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of cephalosporin C using crude glycerol in fed-batch culture of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> M35

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.     

Production of cephalosporin C,and its intermediates,by raised-titre strains of Acremonium chrysogenum

Three different strains of Acremonium chrysogenum have been grown under identical fermentation conditions and their profiles with respect to cephalosporin C and its intermediates were compared. Clear differences were found between the strains; one notably accumulated a large pool of penicillin N, showing a reduced ability to convert this antibiotic to the later intermediates in the pathway, deacetoxycephalosporin C, deacetylcephalosporin C and cephalosporin C.     

Process Development in Biotechnology – A Re‐Evaluation

This review considers some process development problems in biotechnology and presents examples of solutions, which were developed in cooperation with industrial partners. These processes include the production of restriction endonuclease EcoRI by recombinant Escherichia coli, which is toxic to the cell, penicillin V by Penicillium chrysogenum, xylanase by Aspergillus awamori, cephalosporin C by Acremonium chrysogenum, erythritol by Moniliella tomentosa var pollinis, and alkaline serine protease by Bacillus licheniformis. Special attention is given to the practical aspects of product development.