Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes

Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.     

Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over that in the untransformed host. Received 5 January 1998/ Accepted in revised form 29 May 1998     

Enrichment of biologically active 18-β glycyrrhetinic acid in Glycyrrhiza glabra root by solid state fermentation

This paper describes the in situ bioconversion of glycyrrhizin of Glycyrrhiza glabra root to 18-β glycyrrhetinic acid by solid state fermentation. Fermentation was carried out with two different fungal strains, Penicillium chrysogenum and Rhizopus oryzae. The solid state fermentation was carried out under stationary state and under rotating state. Penicillium chrysogenum is a better producer of 18-β glycyrrhetinic acid than Rhizopus oryzae. The induced P. chrysogenum seed culture produces higher 18-β glycyrrhetinic acid with 2.955 mg g^?1 and maximum β-glucuronidase activity of 3,583.8 U ml^?1 under stationary solid state fermentation. The mycelium growth and bioconversion rate is highest at pH of 5.5 and 4.5, respectively. G. glabra root supplemented with a solution of dextrose 9 g l^?1, MnSO₄?·?H₂O 3 g l^?1 and (NH₄)₂SO₄ 0.540 g l^?1 produces 48.580 mg of 18-β glycyrrhetinic acid per gram of G. glabra root, i.e. 86.74 % bioconversion by P. chrysogenum in 96 h under stationary state solid state fermentation.     

An Engineered Yeast Efficiently Secreting Penicillin

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS''s.     

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     

Intact cell mass spectrometry as a progress tracking tool for batch and fed-batch fermentation processes

Penicillin production during a fermentation process using industrial strains of Penicillium chrysogenum is a research topic permanently discussed since the accidental discovery of the antibiotic. Intact cell mass spectrometry (ICMS) can be a fast and novel monitoring tool for the fermentation progress during penicillin V production in a nearly real-time fashion. This method is already used for the characterization of microorganisms and the differentiation of fungal strains; therefore, the application of ICMS to samples directly harvested from a fermenter is a promising possibility to get fast information about the progress of fungal growth. After the optimization of the ICMS method to penicillin V fermentation broth samples, the obtained ICMS data were evaluated by hierarchical cluster analysis or an in-house software solution written especially for ICMS data comparison. Growth stages of a batch and fed-batch fermentation of Penicillium chrysogenum are differentiated by one of those statistical approaches. The application of two matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) instruments in the linear positive ion mode from different vendors demonstrated the universal applicability of the developed ICMS method. The base for a fast and easy-to-use method for monitoring the fermentation progress of P. chrysogenum is created with this ICMS method developed especially for fermentation broth samples.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

The effect of dissolved carbon dioxide on penicillin production: Mycelial morphology

The effect of carbon dioxide on the morphology of Penicillium chrysogenum was examined. The scanning electron microscopic (SEM) study indicated that the morphology of P. chrysogenum was subject to change when exposed to various dissolved CO₂ concentrations in the medium. At low influent carbon dioxide partial pressures between 0% and 8%, the predominant morphological form of P. chrysogenum was filamentous. At higher influent carbon dioxide partial pressures of 15% and 20%, the appearance of swollen and stunted hyphae predominated, and a significant quantity of spherical or yeast-like cells were observed. It was evident that for production subject to high dissolved CO₂ concentrations the inhibition of cell growth and penicillin production related strongly to the concomitant morphological changes of P. chrysogenum.     

Morphology of Penicillium chrysogenum strains immobilized in calcium alginate beads and used in penicillin fermentation

Summary The morphology of two strains of Penicillium chrysogenum immobilized in calcium alginate and used in penicillin fermentation was examined. The degree and distribution of mycelial growth inside and on the surface of the beads depended on the strain, the cultivation media and the fermentation time. P. chrysogenum ATCC 12690 developed as a mycelial network inside the beads. The growth tendency of P. chrysogenum S₁ in micropellets was directed to the outer surface of the beads. At the end of the production phase only a trace of mycelia and no micropellets in the center of alginate beads were observed, while the outer surface and the subsurface were completely covered with mycelia.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

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.     

Resolution of four large chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome 1 (10.4 Mb) in Penicillium chrysogenum

Four chromosomes were resolved by pulsed field gel electrophoresis in Penicillium notatum (10.8, 9.6, 6.3 and 5.4 Mb in size) and in five different strains of Penicillium chrysogenum (10.4, 9.6, 7.3 and 6.8 Mb in the wild type). Small differences in size were found between the four chromosomes of the five P. chrysogenum strains. The penicillin gene cluster was localized by hybridization with a pcbAB probe to chromosome II of P. notatum and to chromosome I of all P. chrysogenum strains except the deletion mutant P. chrysogenum npe10, which lacks this DNA region. The pyrG gene was localized to chromosome I in P. notatum and to chromosome II in all P. chrysogenum strains except in the mutant AS-P-78 where the probe hybridized to chromosome 111. A major chromosomal rearrangement seems to have occurred in this high penicillin producing strain. A fast moving DNA band observed in all gels corresponds to mitochondrial DNA. The total genome size has been calculated as 32.1 Mb in P. notatum and 34.1 Mb for the P. chrysogenum strains.     

Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyces vulgaris. IV. Einfluß von Art und Menge des Öles sowie des Zeitpunktes der Ölzugabe

The effect of different quantities and kinds of vegetable and animal oils and the importance of the time of the oil addition on growth and protease synthesis by T. vulgaris was investigated. The used oils stimulate the protease production if they are added in a suitable concentration. However, the stimulation effect of each oil is different. Culture inoculation with spores gives the best results, when oil was added to the medium 1 up to 2 hours after beginning of the fermentation. The enzyme activity is equal to or lower than the control, when the oil addition was carried out before or 3 hours after starting the fermentation.     

A New Enzyme,Agmatine Oxidase,from Fungi

A new enzyme, agmatine oxidase, was found in Penicillium chrysogenum. The oxidation products of agmatine with the enzyme were identified as γ-guanidinobutyraldehyde, NH₃ and H₂O₂. The enzyme rapidly oxidized agmatine, and slightly oxidized histamine, putrescine, 1,3-diaminopropane and cadaverine. Monoamines, polyamines and guanidyl derivatives were not oxidized by the enzyme. Maximal formation of the enzyme of P. chrysogenum was observed in the early stationary phase of growth, and thereafter the enzyme disappeared with consumption of substrate. In addition to agmatine, spermine, spermidine and putrescine were also effective as nitrogen sources. Agmatine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Penicillium, Absidia, Fusarium, Mucor, Gibberella, Cylindrocarpon and Monascus when they were grown in agmatine-containing medium.     

Environmentally Safe Production of 7-ACA by Recombinant Acremonium chrysogenum

7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of β-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris–Cl (pH 9.6), supplement with 7 mM Zn²⁺, slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT–PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO₃ was reduced to 50 mM, ZnSO₄ was increased to 7 mM, CuSO₄ was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 μg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.     

Biotransformation of N-Nitrosodimethylamine by Pseudomonas mendocina KR1

N-Nitrosodimethylamine (NDMA) is a potent carcinogen and an emerging contaminant in groundwater and drinking water. The metabolism of NDMA in mammalian cells has been widely studied, but little information is available concerning the microbial transformation of this compound. The objective of this study was to elucidate the pathway(s) of NDMA biotransformation by Pseudomonas mendocina KR1, a strain that possesses toluene-4-monooxygenase (T4MO). P. mendocina KR1 was observed to initially oxidize NDMA to N-nitrodimethylamine (NTDMA), a novel metabolite. The use of ¹⁸O₂ and H₂¹⁸O revealed that the oxygen added to NDMA to produce NTDMA was derived from atmospheric O₂. Experiments performed with a pseudomonad expressing cloned T4MO confirmed that T4MO catalyzes this initial reaction. The NTDMA produced by P. mendocina KR1 did not accumulate, but rather it was metabolized further to produce N-nitromethylamine (88 to 94% recovery) and a trace amount of formaldehyde (HCHO). Small quantities of methanol (CH₃OH) were also detected when the strain was incubated with NDMA but not during incubation with either NTDMA or HCHO. The formation of methanol is hypothesized to occur via a second, minor pathway mediated by an initial α-hydroxylation of the nitrosamine. Strain KR1 did not grow on NDMA or mineralize significant quantities of the compound to carbon dioxide, suggesting that the degradation process is cometabolic.     

Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>: cross-talk regulation of secondary metabolite pathways

Penicillium chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. chrysogenum. Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.     

Influence of fungal elicitors on biosynthesis of natamycin by Streptomyces natalensis HW-2

To investigate the effect of fungal elicitors on biosynthesis of natamycin in the cultures of Streptomyces natalensis HW-2, the biomass and filtrate of the broth from Aspergillus niger AS 3.6472, Penicillium chrysogenum AS 3.5163, A. oryzae AS 3.2068, and Saccharomyces cerevisiae AS 2.2081 were used to induce natamycin production in S. natalensis HW-2. The results showed that the biomass of P. chrysogenum AS 3.5163 could enhance the yield of natamycin from 0.639 to 0.875 g?l^?1. The elicitor from the fermentation broth of P. chrysogenum AS 3.5163 showed the highest inducing efficiency with the yield of natamycin enhanced from 0.632 to 1.84 g?l^?1. The elicitor that was cultured for 2 days showed the strongest inducing activity during the fermentation of S. natalensis HW-2 for 24 h, and the yield of natamycin was enhanced from 0.637 to 2.12 g?l^?1. The biochemical parameters were examined at the end of fermentation and the results demonstrated that both the growth of cells and the concentration of residual sugar could be influenced. The residual sugar decreased from 5.03 to 4.27 g?l^?1, and the biomass decreased from 10.26 to 6.87 g?l^?1. Finally, the elicitor was identified as a low molecular weight substance with a similar polarity to that of butyl alcohol by primary qualitative analysis.