A novel aspect of NADPH production in ageing Penicillium chrysogenum

NADPH is involved in many basically important anabolic processes. For a long time, pentose phosphate pathway (PPS) was regarded as the most important source of NADPH in fungi. Here we present evidence of a metabolic switch to an alternative NADPH-producing pathway in ageing Penicillium chrysogenum cultures, which involves NADP+ -specific isocitrate dehydrogenase (NADP+ -ID) rather than PPS enzymes. Considering the main biochemical functions of NADPH, we propose that NADP+ -ID could have deep impact on many physiological processes switched on glucose deprivation including proteinase production or penicillin biosynthesis. We also demonstrate that although the alternative pathway was inferior to PPS when the fungus was grown on well-utilisable carbon sources yet it could have an important role in fatty acid biosynthesis as well as in the maintenance of high intracellular NADPH/NADP+ ratios.     

Optimization of a pellicular biocatalyst for penicillin G production by Penicillium chrysogenum

A previously developed immobilization technique involving latex coatings on solid particulate supports was investigated further for penicillin G production by Penicillium chrysogenum. Several modifications were found to decrease the germination lag time, including a higher spore concentration, a thinner latex layer, an increased latex porosity, and a decreased drying time. This approach enabled the development of immobilized mycelial pellets within 2-3 days from the onset of biocatalyst preparation and incubation.A continuous immobilized-cell airlift bioreactor produced penicillin G in a series of runs in which the production phase lasted up to 30 days. The productivity of this system was 3-6 times greater than the productivity of the corresponding free-cell shake flask fermentation.     

Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum

We have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.     

Reduced utilization of side-chain precursor for penicillin biosynthesis by mutants of Penicillium chrysogenum

Summary A high yielding strain of Penicillium chrysogenum was mutated with EMS and investigated for selective effects of semi-continuous fermentations. Preferential growth of a class of mutants with different colony type and having reduced ability to utilize side-chain precursor led to reduced Pen V synthesis by the heterogeneous mycelial population.     

Alternative respiration and antibiotic production of Acremonium chrysogenum

The influence of potassium cyanide (KCN), dissolved O₂ concentration and medium composition on alternative respiration (AR) of Acremonium chrysogenum were investigated. The respiration of the fungus was only partially inhibited by KCN, but completely inhibited by the combination of KCN with salicylhydroxamic acid. It has been proved by in-situ measurements of the NADH-dependent fluorescence that the AR is active at low dissolved O₂ concentrations. The influence of the medium composition and the age of the fungus on the specific oxygen uptake rate is considered. Correpondence to: K. Schügerl     

Energetics of growth and penicillin production in a high-producing strain of Penicillium chrysogenum

The results of a large number of carbon-limited chemostat cultures of Penicillium chrysogenum carried out on glucose, ethanol, and acetate as the growth limiting substrate have been used to obtain an estimation of the adenosine triphosphate (ATP) costs for mycelium growth, penicillin production, and maintenance and the overall stoichiometry of oxidative phosphorylation of the fungus. It was found that penicillin production was accompanied by a significant additional energy drain (73 mol of ATP per mole of penicillin-G) from primary metabolism. This finding has been confirmed in independent experiments and has been shown to result in a significantly lower estimate for the maximum theoretical yield of penicillin-G on the carbon source.     

Impact of the Penicillium chrysogenum genome on industrial production of metabolites

The genome sequence of Penicillium chrysogenum has initiated a range of fundamental studies, deciphering the genetic secrets of the industrial penicillin producer. More than 60 years of classical strain improvement has resulted in major but delicate rebalancing of the intracellular metabolism leading to the impressive penicillin titres of the current production strains. Several leads for further improvement are being followed up, including the use of P. chrysogenum as a cell factory for other products than β-lactam antibiotics.     

Penicillin G production by immobilized whole cells of Penicillium chrysogenum.

Penicillium chrysogenum was immobilized in polyacrylamide gel prepared from 5% acrylamide monomers (85% acrylamide and 15% N,N'-methylene bisacrylamide). Penicillin produced from glucose by the immobilized mycelium was 17% of that produced by washed mycelium. However, the activity of penicillin production of the washed mycelium decreased with repeated use. On the other hand, the activity of the immobilized mycelium increased initially and decreased gradually with repeated use. The rate of oxygen uptake of the immobilized mycelium was about 30% of that of the washed mycelium. The immobilized mycelium required oxygen for the production of penicillin.     

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.     

Continuous penicillin production by Penicillium chrysogenum immobilized in calcium alginate beads

Summary A high penicillin-producing Penicillium chrysogenum strain immobilized in calcium alginate beads was used for continuous penicillin fermentation in a bubble column and in a conical bubble fermentor. The fermentation was limited by the growth rate, dilution rates and the stability of the alginate beads. The immobilized cells lost their ability to produce penicillin in the bubble column after 48 h from beginning of the continuous fermentation. In the conical bubble fermentor the immobilized cells remained active for more than 7 days. This bioreactor ensured a good distribution of nutrients and oxygen as well as a higher mechanical stability of the alginate beads.     

Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine beta-lyase. With this new host-vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein-PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL-SKL produced in H. polymorpha displays normal enzymatic activities.     

Stability of recombinant protein production by Penicillium chrysogenum in prolonged chemostat culture

Abstract A strain (WKW2) of Penicillium chrysogenum transformed with heterologous fungal acetamidase ( amd S) and bacterial β-galactosidase ( lac Z) was grown at a dilution rate of 0.17 h⁻¹ (doubling time of approx. 4.1 h) for 1600 h in a glucose-limited culture. By the end of the experiment the original strain had been almost completely replaced by spontaneous, morphological mutants, but the acetamidase and β-galactosidase activities of the culture were essentially unaltered. Furthermore, when WKW2 and the non-transformed parental strain (NRRL1951) were grown together in glucose- or NH₄⁺-limited chemostat cultures, neither strain had a selective advantage over the other. Thus, heterologous gene expression does not result in NRRL1951 having a selective advantage over WKW2. These results suggest that continuous flow culture systems could be used for efficient (and cost effective) production of recombinant proteins.     

Expression of the transporter encoded by the cefT gene of Acremonium chrysogenum increases cephalosporin production in Penicillium chrysogenum

By introduction of the cefEF genes of Acremonium chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. chrysogenum strains is limited by the ability of the fungus to secrete these compounds.