Assay of 6-oxopiperidine-2-carboxylic acid in fermentations of Penicillium chrysogenum PQ-96

Summary 6-Oxopiperidine-2-carboxylic acid (OCA; cyclic -aminoadipic acid) was assayed in fermentations of Penicillium chrysogenum PQ-96 by isotachophoresis and HPLC. From 0.36 mg ml^–1 to 2.41 mg ml^–1 OCA was accumulated in fermentations during penicillin G biosynthesis. Offprint requests to: W. Kurzkowski     

Localization of isopenicillin N synthase in Penicillium chrysogenum PQ-96

Summary For immunoelectron microscopic localization of isopenicillin N synthase (IPNS), glutaraldehyde-fixed mycelium of Penicillium chrysogenum PQ-96 was dehydrated by progressive lowering of the temperature and embedded into Lowicryl K4M at –35° C. This procedure resulted in good structural preservation such that the method of on-section labelling using antibody to IPNS from Cephalosporium acremonium CO 278 with the indirect antibody-gold technique could be successfully applied. By this method IPNS was localized in vesicular compartments belonging probably to the Golgi body and in the cell wall. Offprint requests to: W. Kurzkowski     

alpha-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum

Intracellular amino acid pools in four Penicillium chrysogenum strains, which differed in their ability to produce penicillin, were determined under conditions supporting growth without penicillin production and under conditions supporting penicillin production. A significant correlation between the rate of penicillin production and the intracellular concentration of alpha-aminoadipate was observed, which was not shown with any other amino acid in the pool. In replacement cultivation, penicillin production was stimulated by alpha-aminoadipate, but not by valine or cysteine. Exogenously added alpha-aminoadipate (2 or 3 mM) maximally stimulated penicillin synthesis in two strains of different productivity. Under these conditions intracellular concentrations of alpha-aminoadipate were comparable in the two strains in spite of the higher rate of penicillin production in the more productive strain. Results suggest that the lower penicillin titre of strain Q 176 is due to at least two factors: (i) the intracellular concentration of alpha-aminoadipate is insufficient to allow saturation of any enzyme which is rate limiting in the conversion of alpha-aminoadipate to penicillin and (ii) the level of an enzyme, which is rate limiting in the conversion of alpha-aminoadipate to penicillin, is lower in Q 176 (relative to strain D6/1014/A). Results suggest that the intracellular concentration of alpha-aminoadipate in strain D6/1014/A is sufficiently high to allow saturation of the rate-limiting penicillin biosynthetic enzyme in that strain. The basis of further correlation of intracellular alpha-aminoadipate concentration and penicillin titre among strains D6/1014/A, P2, and 389/3, the three highest penicillin producers studied here, remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

Penicillium chrysogenum mycelial productivity in the 1st phase of penicillin biosynthesis]

The course of the mycelium low productivity during the first phase of the usual two-stage process of penicillin biosynthesis was studied. It was found that the low productivity of the mycelium at the beginning of the fermentation process was probably associated with catabolic regression of the penicillin-producing system. The high specific growth rate registered in the experiments (0.06-0.08 hours-1) had no negative effect on the mycelium productivity. It was not possible to connect the productivity level with the mycelium age, because young and mature mycelium had the same producitivity levels at any developmental stage under the same conditions.     

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.     

Utilization of side-chain precursors for penicillin biosynthesis in a high-producing strain of Penicillium chrysogenum

Utilization of the side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) for penicillin biosynthesis by Penicillium chrysogenum was studied in shake flasks. Precursor uptake and penicillin production were followed by HPLC analysis of precursors and products in the medium and in the cells. P. chrysogenum used both POA and PA as precursors, producing phenoxymethylpenicillin (penicillin V) and benzylpenicillin (penicillin G), respectively. If both precursors were present simultaneously, the formation of penicillin V was blocked and only penicillin G was produced. When PA was added at different times to cells that were induced initially for POA utilization and were producing penicillin V, the POA utilization and penicillin V formation were blocked, whereas the cells started utilizing PA and produced penicillin G. The blocking of the POA turnover lasted for as long as PA was present in the medium. If POA was added to cultures induced initially for PA utilization and producing penicillin G, this continued irrespective of the presence of POA. Utilization of POA increased concomitant with depletion of PA from the medium. Analysis of cellular pools from a growing cell system with POA as precursor to which PA was added after 48 h showed that the cellular concentration of POA was kept high without production of penicillin V and at a concentration comparable to the concentration in the medium. The cellular concentration of POA was higher than the concentration of PA that was utilized for penicillin G production. Correspondence to: S. Havn Eriksen     

Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum.

The inhibitory effect of L-lysine on penicillin biosynthesis by Penicillium chrysogenum has been compared in a low-producing strain (Wis. 54-1255) and a high-producing strain (ASP-78). Lysine inhibited total penicillin synthesis to a similar extent in both strains. However, in the high-producing strain the onset of penicillin synthesis occurred even at a high lysine concentration, whereas in the low-producing strain lysine had to be depleted before penicillin production commenced.     

The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.