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.     

Scale-down of penicillin production in Penicillium chrysogenum

In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.     

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.     

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.     

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.     

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     

Optimization of culture conditions for glucose oxidase production by a Penicillium chrysogenum SRT 19 strain

The enzyme glucose oxidase (GOD) has been used for a variety of biotechnological applications in food and pharmaceutical industries. In this study, the optimization of extracellular GOD production was carried out in a Penicillium chrysogenum SRT 19 strain isolated from contaminated and decaying cheese samples. Maximum GOD production was attained at pH 6 and 20°C in fermentation broth after 72 h of incubation. The effects of metal ions and sugars were screened for the induction of higher GOD production. The results revealed that glucose and lactose give the highest production of enzyme (0.670 and 0.552 U/mL, respectively) as compared with other sugars (sucrose, cellulose, mannitol and fructose). Out of the seven metal ions studied, CaCO₃ (1.123 U/mL) and FeSO₄ (0.822 U/mL) act as modulators, while MgSO₄ (0.535 U/mL), CuSO₄ (0.498 U/mL), HgCl₂ (0.476 U/mL), ZnSO₄ (0.457 U/mL) and BaSO₄ (0.422 U/mL) yield lower production. The study therefore suggests that a strain of P. chrysogenum SRT 19 can be used as a new strain for GOD production.     

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.     

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q_p) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q_p(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed‐batch experiments, leads to errors in the prediction, because q_p is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate‐limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin‐N synthase (IPNS) was the main rate‐limiting enzyme for penicillin‐G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady‐state as well as the dynamics during chemostat start‐up and fed‐batch cultivation. Biotechnol. Bioeng. 2010;106: 608–618. © 2010 Wiley Periodicals, Inc.     

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.