Penicillium chrysogenum Takes up the Penicillin G Precursor Phenylacetic Acid by Passive Diffusion

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.     

Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase

Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.     

Reduced Function of a Phenylacetate-Oxidizing Cytochrome P450 Caused Strong Genetic Improvement in Early Phylogeny of Penicillin-Producing Strains

The single-copy pahA gene from Penicillium chrysogenum encodes a phenylacetate 2-hydroxylase that catalyzes the first step of phenylacetate catabolism, an oxidative route that decreases the precursor availability for penicillin G biosynthesis. PahA protein is homologous to cytochrome P450 monooxygenases involved in the detoxification of xenobiotic compounds, with 84% identity to the Aspergillus nidulans homologue PhacA. Expression level of pahA displays an inverse correlation with the penicillin productivity of the strain and is subject to induction by phenylacetic acid. Gene expression studies have revealed a reduced oxidative activity of the protein encoded by pahA genes from penicillin-overproducing strains of P. chrysogenum compared to the activity conferred by phacA of A. nidulans. Sequencing and expression of wild-type pahA from P. chrysogenum NRRL 1951 revealed that an L181F mutation was responsible for the reduced function in present industrial strains. The mutation has been tracked down to Wisconsin 49-133, a mutant obtained at the Department of Botany of the University of Wisconsin in 1949, at the beginning of the development of the Wisconsin family of strains.     

Quantitative analysis of Penicillium chrysogenum Wis54-1255 transformants overexpressing the penicillin biosynthetic genes

The low penicillin-producing, single gene copy strain Wis54-1255 was used to study the effect of overexpressing the penicillin biosynthetic genes in Penicillium chrysogenum. Transformants of Wis54-1255 were obtained with the amdS expression-cassette using the four combinations: pcbAB, pcbC, pcbC-penDE, and pcbAB-pcbC-penDE of the three penicillin biosynthetic genes. Transformants showing an increased penicillin production were investigated during steady-state continuous cultivations with glucose as the growth-limiting substrate. The transformants were characterized with respect to specific penicillin productivity, the activity of the two pathway enzymes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthetase (IPNS) and the intracellular concentration of the metabolites: delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV), isopenicillin N (IPN), glutathione (GSH), and glutathione disulphide (GSSG). Transformants with the whole gene cluster amplified showed the largest increase in specific penicillin productivity (r(p))-124% and 176%, respectively, whereas transformation with the pcbC-penDE gene fragment resulted in a decrease in r(p) of 9% relative to Wis54-1255. A marked increase in r(p) is clearly correlated with a balanced amplification of both the ACVS and IPNS activity or a large amplification of either enzyme activity. The increased capacity of a single enzyme occurs surprisingly only in the transformants where all the three biosynthetic genes are overexpressed but is not found within the group of pcbAB or pcbC transformants. The indication of the pcbAB and pcbC genes being closely regulated in fungi might explain why high-yielding strains of P. chrysogenum have been found to contain amplifications of a large region including the whole penicillin gene cluster and not single gene amplifications. Measurements of the total ACV concentration showed a large span of variability, which reflected the individual status of enzyme overexpression and activity found in each strain. The ratio ACV:bisACV remained constant, also at high ACV concentrations, indicating no limitation in the capacity of the thioredoxin-thioredoxin reductase (TR) system, which is assumed to keep the pathway intermediate LLD-ACV in its reduced state. The total GSH pool was at a constant level of approx. 5.7 mM in all cultivations.     

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.     

Effects of lysine analogs on Penicillium chrysogenum.

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.     

Effects of lysine analogs on Penicillium chrysogenum.

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.     

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.     

产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定

从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.     

Behaviour of tritium-labelled isopenicillin N and 6-aminopenicillanic acid as potential penicillin precursors in an extract of Penicillum chrysogenum.

1. 3H was incorporated into solvent-soluble penicillin from isopenicillin N and 6-aminopenicillanic acid 3H-labelled in the 2beta-methyl group when the labelled compounds were incubated with a crude extract of Penicillum chrysogenum. 2. With a soluble protein fraction of the extract incorporation from isopenicillin N occurred on addition of phenyl-acetyl-CoA. 3. Labelled benzylpenicillin was isolated after incubation of the crude extract with phenylacetyl-CoA and isopenicillin and the addition of unlabelled benzylpenicillin as a carrier. 4. No incorporation of 3H into solvent-soluble penicillin was detected on incubation of these extracts with penicillin N.     

Strain improvement of Penicillium chrysogenum by recombinant DNA techniques.

The penDE gene from Penicillium chrysogenum has been isolated; the gene is located in close vicinity of the pcbC gene. Amplification of the pcbC-penDE gene cluster in Penicillium chrysogenum Wis54-1255 leads to a significant increase in penicillin production. In selected transformants an increase of up to 40% is observed.     

Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Enzymic acylation of 6-aminopenicillanic acid

The enzymic synthesis of benzylpenicillin from 6-aminopenicillanic acid in the presence of poly (ethylene glycol) has been studied. With equimolar initial concentrations (20 mM) of 6-aminopenicillanic acid and phenylacetic acid a 60% conversion to benzylpenicillin can be achieved at 10°C and pH 5.2 in the presence of 45% (w/v) poly(ethylene glycol). Under these conditions the lactam ring of the benzylpenicillin and 6-aminopenicillanic acid and the enzyme, penicillin acylase (penicillin amidase, penicillin amidohydrolase, EC 3.5.1.11), were more stable than in the absence of the polyol.     

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.     

Formation of bulges associated with penicillin production in high-producing strains ofPenicillium chrysogenum

Bulges were formed in the hyphae of a high penicillin-producing strain ofPenicillium chrysogenum AS-P-78 when penicillin was accumulated in the broth. The mycelium of cultures grown in the presence of 50 mM lysine, which specifically inhibits penicillin formation, showed a greatly reduced number of bulges. The size and number of bulges increased during the fermentation in parallel with penicillin accumulation. A smaller number of bulges was formed in the mycelium of the low-producing strain Wis 54-1255 than in the high-producing mutant. Three mutants blocked in penicillin biosynthesis did not form these globose structures. Bulges were not osmotically sensitive, although some of them burst out. They may be formed by weakening of the cell wall of hyphae following accumulation of high concentrations of penicillin.     

Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum.

The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum. Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis. The culture response was assessed chemically and morphologically, using computerised image analysis. High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis. However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss. Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process. Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P. chrysogenum. By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P. chrysogenum.