Development of the cephalosporin C fermentation taking into account the instability of cephalosporin C

Summary Cephalosporin C undergoes chemical hydrolysis during fermentation at 25°C at a rate of 0.48% per hour to give increasing amounts of 2-(D-4-amino-4-carboxybutyl)thiazole-4-carboxylic acid (compound X). Although the cephalosporin C titer in a fermentation levels off at 160–170h, the total of C+X rises almost linearly up to 200h. Cells, therefore, biosynthesize cephalosporin C at an almost constant rate up to 200h. This instability greatly influences attempts to improve the cephalosporin strain by genetic manipulation, or the process by alterations in technology.     

Constant dissolved oxygen concentrations in cephalosporin C fermentation: Applicability of different controllers and effect on fermentation parameters

Summary The behaviour and applicability of several controllers for maintaining a constant dissolved oxygen concentration (DO) during the cephalosporin C production with Cephalosporium acremonium in a laboratory fermentor is described. The process controllers were realized on a MC 68000 based process computer using the real-time language PEARL. The discrete signum integral controller showed the best control action. In addition some derived fermentation data were calculated on-line by the process computer.The results obtained by comparison of fermentations carried out at DO between 10% and 40% saturation during ideophase indicate that high DO leads to a high specific production rate for cephalosporin C and a low specific production rate for penicillin N and vice versa. In the range of DO investigated the production of deacetyl and deacetoxy cephalosporin C is not affected by DO. A direct correlation between DO and the yield coefficients Y _P/S and Y _P/X could be established. The yield coefficient Y _P/O for cephalosporin C is constant in the DO range from 10%–40%.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Cybernetic modeling of the cephalosporin C fermentation process by Cephalosporium acremonium

A cybernetic mathematical model has been developed to describe the production of cephalosporin C. In developing the model, diauxic behavior of substrate consumption, morphological differentiation of cells, and catabolite repression of cephalosporin C production by the preferred substrate, glucose, were considered. The proposed model was tested on the experimental data from the literature and could adequately describe the morphological differentiation of cells, the sequential utilization of carbon sources and the production of cephalosporin C. It could be a useful tool to optimize the production of cephalosporin C by Cephalosporium acremonium in batch, fed-batch or continuous operations.     

Comparison of plant secondary metabolites and digestibility of three different boreal coniferous trees

Moose (Alces alces L.) prefer to browse Pinus sylvestris over other coniferous trees. One reason for this preference might be due to differences in the chemical composition of various coniferous plants, and/or differences in digestibility. We evaluated the in-sacco digestibility of P. sylvestris, Pinus contorta and Picea abies using a fistulated cow (Bos taurus). Additionally, we determined the chemical profile of the different plant species (with emphasis on specific phenolics) and we modified the concentration of the individual phenolic (+)catechin in plant material of P. sylvestris to test the influence of this particular phenolic on digestibility in vitro (IVDMD). The in-sacco experiment revealed the highest digestibility of dry matter, and neutral detergent fibre for P. sylvestris. In addition, the concentration of nitrogen was significantly higher, and the concentrations of total phenolics and condensed tannins were significantly lower in P. sylvestris than in the other species. Individual phenolics showed high variation but were low in P. sylvestris, whereas P. contorta and P. abies had at least one species-specific phenolic compound of high concentration each. We found no changes in the digestibility of samples in which we experimentally elevated (+)catechin content.     

Modeling, optimization, and computer control of the cephalosporin C fermentation process

In this study, a cephalosporin C producing strain, Cephalosporium acremonium (ATCC 36225), was chosen to determine the optimal conditions that maximize antibiotic production in a mixed substrate of glucose and sucrose. A model for cell growth and cephalosporin C production at different pH and temperature was developed and the associated parameters were evaluated experimentally. Pontryagin's maximum principle, in conjunction with the model, was used to predict the optimal temperature and pH control profiles to maximize the production of antibiotic.     

Novel benzene ring biosynthesis from C(3) and C(4) primary metabolites by two enzymes

The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, l-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn(2+) ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C(3) and C(4) primary metabolites.     

Evolution of secondary metabolites in legumes (Fabaceae)

Legumes produce a high diversity of secondary metabolites which serve as defence compounds against herbivores and microbes, but also as signal compounds to attract pollinating and fruit-dispersing animals. As nitrogen-fixing organisms, legumes produce more nitrogen containing secondary metabolites than other plant families. Compounds with nitrogen include alkaloids and amines (quinolizidine, pyrrolizidine, indolizidine, piperidine, pyridine, pyrrolidine, simple indole, Erythrina, simple isoquinoline, and imidazole alkaloids; polyamines, phenylethylamine, tyramine, and tryptamine derivatives), non-protein amino acids (NPAA), cyanogenic glucosides, and peptides (lectins, trypsin inhibitors, antimicrobial peptides, cyclotides). Secondary metabolites without nitrogen are phenolics (phenylpropanoids, flavonoids, isoflavones, catechins, anthocyanins, tannins, lignans, coumarins and furanocoumarins), polyketides (anthraquinones), and terpenoids (especially triterpenoid, steroidal saponins, tetraterpenes). While some secondary metabolites have a wide distribution (flavonoids, triterpenes, pinitol), however, others occur in a limited number of taxa. The distributions of secondary metabolites with an irregular occurrence are mapped on a molecular phylogeny of the Fabaceae, reconstructed from a combined data set of nucleotide sequences from rbcL, matK and ITS genes. In most cases, the distribution patterns of secondary metabolites do not agree with the phylogeny of the plants producing them. In contrary, the distribution of many secondary metabolites is patchy and irregular. Thus, the use of phytochemical data to reconstruct a phylogeny of plants is often not informative and can be misleading. The patchy distribution may be due to convergent evolution, a contribution of endophytic fungi or more likely, to an early acquisition of the key genes of secondary metabolism in the evolution of land plants among others by horizontal gene transfer from bacteria. Thus it would be a matter of gene regulation whether these genes are active in some but not all taxa.     

Correlations between primary and secondary metabolites in Ceramiales (Rhodophyta)

A correlation between the presence of terpenes and floridosides and between the presence of bromophenols and digeneaside was observed in species of red algae belonging to the order Ceramiales. This observation suggests a division in Ceramiales that can be easily detected through the presence of the secondary metabolites, terpenes and bromophenols.     

Seasonal variation of secondary metabolites in nine different bryophytes

Bryophytes occur in almost all land ecosystems and contribute to global biogeochemical cycles, ecosystem functioning, and influence vegetation dynamics. As growth and biochemistry of bryophytes are strongly dependent on the season, we analyzed metabolic variation across seasons with regard to ecological characteristics and phylogeny. Using bioinformatics methods, we present an integrative and reproducible approach to connect ecology with biochemistry. Nine different bryophyte species were collected in three composite samples in four seasons. Untargeted liquid chromatography coupled with mass spectrometry (LC/MS) was performed to obtain metabolite profiles. Redundancy analysis, Pearson's correlation, Shannon diversity, and hierarchical clustering were used to determine relationships among species, seasons, ecological characteristics, and hierarchical clustering. Metabolite profiles of Marchantia polymorpha and Fissidens taxifolius which are species with ruderal life strategy (R‐selected) showed low seasonal variability, while the profiles of the pleurocarpous mosses and Grimmia pulvinata which have characteristics of a competitive strategy (C‐selected) were more variable. Polytrichum strictum and Plagiomnium undulatum had intermediary life strategies. Our study revealed strong species‐specific differences in metabolite profiles between the seasons. Life strategies, growth forms, and indicator values for light and soil were among the most important ecological predictors. We demonstrate that untargeted Eco‐Metabolomics provide useful biochemical insight that improves our understanding of fundamental ecological strategies.     

On the evolution of functional secondary metabolites (natural products)

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.     

Modelling the effect of directional spatial ecological processes at different scales

Understanding the effects of disturbance and secondary succession on spatio-temporal patterns in the abundance of species is stymied by a lack of long-term demographic data, especially in response to infrequent and high intensity disturbances, such as hurricanes. Moreover, resistance and resilience to hurricane-induced disturbance may be mediated by legacies of previous land use, although such interactive effects are poorly understood, especially in tropical environments. We address these central issues in disturbance ecology by analyzing an extensive dataset, spanning the impacts of Hurricanes Hugo and Georges, on the abundance of a Neotropical walking stick, Lamponius portoricensis, in tabonuco rainforest of Puerto Rico during the wet and dry seasons from 1991 to 2007. By synthesizing data from two proximate sites in tabonuco forest, we show that resistance to Hurricane Hugo (97% reduction in abundance) was much less than resistance to Hurricane Georges (21% reduction in abundance). Based on a powerful statistical approach (generalized linear mixed-effects models with Poisson error terms), we documented that the temporal trajectories of abundance during secondary succession (i.e., patterns of resilience) differed between hurricanes and among historical land use categories, but that the effects of hurricanes and land use histories were independent of each other. These complex results likely arise because of differences in the intensities of the two hurricanes with respect to microclimatic effects (temperature and moisture) in the forest understory, as well as to time-lags in the response of L. portoricensis to changes in the abundance and distribution of preferred food plants (Piper) in post-hurricane environments.     

Imprinted polymers as tools for the recovery of secondary metabolites produced by fermentation

Imprinted polymers were synthesized using a mixture of pigments, N‐glutamyl‐rubropuctamine, and N‐glutamyl‐monascorubramine (I) as the template, and 2‐methacrylamido‐6‐picoline or 4‐aminostyrene as functional monomers, to obtain recognition materials capable of forming hydrogen bonds and charge interactions, respectively, with carboxyl groups of target I in the binding sites. The polymers were prepared thermally at a template loading of 5 mol% using ethylene‐glycol dimethacrylate or trimethylolpropane trimethacrylate as crosslinkers and acetonitrile or tetrahydrofuran as porogens. The selective binding of I to both types of polymer was demonstrated, although aminostyrene‐based materials showed higher overall adsorption and were studied in more detail. It was shown that the kinetics of binding of I from ethyl‐acetate extracts of fermented Monascus sp. was very rapid and virtually all the pigment adsorbed can be released by washing the polymer with ethanol–water mixtures. The feasibility of reusing imprinted polymer in consecutive adsorption/desorption cycles was also demonstrated. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 232–239, 1999.     

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10

Aims: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid‐state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. Methods and Results: Solid‐state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7·8, but also under 6·4. Using initial pH lower than 6·8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 μg gdm^?1 (g^?1 of dry matter). Conclusion: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. Significance and Impact of the Study: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.     

Growth and bioactive secondary metabolites of arctic Protoceratium reticulatum (Dinophyceae)

Harmful algal blooms are mainly caused by marine dinoflagellates and are known to produce potent toxins that may affect the ecosystem, human activities and health. Such events have increased in frequency and intensity worldwide in the past decades. Numerous processes involved in Global Change are amplified in the Arctic, but little is known about species specific responses of arctic dinoflagellates. The aim of this work was to perform an exhaustive morphological, phylogenetical and toxinological characterization of Greenland Protoceratium reticulatum and, in addition, to test the effect of temperature on growth and production of bioactive secondary metabolites. Seven clonal isolates, the first isolates of P. reticulatum available from arctic waters, were phylogenetically characterized by analysis of the LSU rDNA. Six isolates were further characterized morphologically and were shown to produce both yessotoxins (YTX) and lytic compounds, representing the first report of allelochemical activity in P. reticulatum. As shown for one of the isolates, growth was strongly affected by temperature with a maximum growth rate at 15 °C, a significant but slow growth at 1 °C, and cell death at 25 °C, suggesting an adaptation of P. reticulatum to temperate waters. Temperature had no major effect on total YTX cell quota or lytic activity but both were affected by the growth phase with a significant increase at stationary phase. A comparison of six isolates at a fixed temperature of 10 °C showed high intraspecific variability for all three physiological parameters tested. Growth rate varied from 0.06 to 0.19 d⁻¹, and total YTX concentration ranged from 0.3 to 15.0 pg  YTX cell⁻¹ and from 0.5 to 31.0 pg YTX cell⁻¹ at exponential and stationary phase, respectively. All six isolates performed lytic activity; however, for two isolates lytic activity was only detectable at higher cell densities in stationary phase.     

Primary and secondary growth phenology of Schizolobium parahyba (Vell.) Blake at different time scales

Schizolobium parahyba is a tropical species that has expanded its distribution. However, little is known about its ecological performance beyond the tropics. Therefore, the objective of this research was to describe for this species, south of its original distribution, intra-annual variations in primary growth phenology (leaf flushing, leaf shedding, flowering and fruiting) and secondary growth (radial increase of the trunk), as well as secondary growth on an interannual scale (radial xylem increase) and to analyze the influences of climatic conditions on the evidenced phenological patterns. For this purpose, on an intra-annual scale, ten individuals had their phenophases observed fortnightly for 23 months and were described and correlated to climatic variables of photoperiod, temperature and precipitation. Inter-annual activity samples were obtained from the growth rings of 16 individuals, which were evaluated according to dendrochronological protocols and correlated to climatic variables of temperature and precipitation. South of its original distribution, S. parahyba trees presented seasonal growth rates in all observed scales and associations with variations in phenological rhythms and climatic conditions. Differences were found in the climatic response of the species in comparison to studies already performed in the tropics. In this study, on an intra-annual scale, this association is manifested by relationships with photoperiod, being positive for leaf flushing and fruiting and negative for leaf shedding. In the secondary growth, the intra-annual scale positive relationship was found with temperature, but on an inter-annual scale a positive influence of winter rainfall was found. The consistence of those patterns in broader spatial scales deserves further investigation.     

Identification of two major ammonia-releasing reactions involved in secondary natto fermentation

Natto is a traditional Japanese food made from soybeans fermented by strains of Bacillus subtilis natto. It gives off a strong ammonia smell during secondary fermentation, and the biochemical basis for this ammonia production was investigated in this study. When natto was fermented by strain r22, ammonia production was shown to involve degradation of soybean proteins releasing amino acids, and only the glutamate contained in the natto obviously decreased, while the other amino acids increased during secondary fermentation. Strain r22 has two active glutamate dehydrogenase genes, rocG and gudB, and inactivating both genes reduced ammonia production by half, indicating that deamination of glutamate was one of the major ammonia-releasing reactions. In addition, urease encoded by ureABC was found to degrade urea during secondary fermentation. A triple mutant lacking rocG, gudB, and ureC exhibited minimal ammonia production, suggesting that the degradation of urea might be a further ammonia-releasing reaction.     

Comparison of the effect of two types of whole mushroom (Agaricus bisporus) powders on intestinal fermentation in rats

The effects of two types of mushroom (Agaricus bisporus; white, WM; brown, BM) powders on intestinal fermentation in rats were investigated in terms of the physical characteristics of animals and by bacterial and HPLC analyses of cecal contents. Short-chain fatty acid levels were found to be significantly higher in the WM group than in the BM and the control (CN) groups; coliform bacteria levels in the BM group were significantly lower than those in the CN group, with the WM group inducing an apparent but insignificant decrease in coliforms. Anaerobe levels in the WM group were significantly higher than those in the CN group and, compared with the CN group, the BM and WM groups exhibited significantly increased feces weight and cecum weight, respectively. These results indicate that the mushroom powders, and in particular the WM powder, have beneficial effects on the intestinal environment in rats.     

Measurement of penicillin acylase and cephalosporin acylase in fermentation broths

Summary The p-dimethylamino-benzaldehyde method of Bomstein and Evans, the ninhydrin method of Marrelli and the new p-dimethyl-amino-benzaldehyde method of Kornfeld were evaluated for analysis of penicillin-V acylase and cephalosporin-V acylase in fermentation broths. The ninhydrin method was the method of choice for cephalosporin-V acylase, whereas the Kornfeld method had certain advantages with respect to penicillin-V acylase.