Fatty acids reduce the tensile strength of fungal hyphae during cephalosporin C production in Acremonium chrysogenum

Fragmentation rate constants, which can be used to estimate the tensile strength of fungal hyphae, were used to elucidate relationships between morphological changes and addition of fatty acids during cephalosporin C production in Acremonium chrysogenum M35. The number of arthrospores increased gradually during fermentation, and, in particular, was higher in the presence of rice oil, oleic acid or linoleic acid than in their absence. Because supplementation of rice oil or fatty acids increased cephalosporin C, we concluded that differentiation to arthrospores is related to cephalosporin C production. To estimate the relative tensile strengths of fungal hyphae, fragmentation rate constants (k _frag) were measured. When rice oil, oleic acid, or linoleic acid were added into medium, fragmentation rate constants were higher than for the control, and hyphal tensile strengths reduced. The relative tensile strength of fungal hyphae, however was not constant presumably due to differences in physiological state.     

Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95

Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri. Pune 411 018, India     

Morphology and kinetics studies on cephalosporin C production by Cephalosporium acremonium M25 in a 30-l bioreactor using a mixture of inocula

AIMS: In this study, the relationship between morphology and cephalosporin C (CPC) production in a 30-l bioreactor culture of Cephalosporium acremonium M25 using a 3:7 seed mixture was investigated. In addition, the kinetic model was established and applied. METHODS AND RESULTS: CPC production was performed in a 30-l bioreactor using a 3:7 seed mixture. It was recognized that a 3:7 seed mixture was able to reduce lag phase and enhance CPC production. The maximum CPC production and cell mass were 1.96 and 81.5 g l-1 respectively. Through a morphology study by observation using image analysis, it was concluded that changes of morphological features predicted the progressive production of CPC and that a morphology study could be useful in monitoring the CPC fermentation by C. acremonium M25. In the kinetics study, a kinetic model of CPC fermentation was developed and applied. The proposed model could adequately describe the fermentation of C. acremonium M25 in a 30-l bioreactor. CONCLUSIONS: CPC productivity was improved by using a 3:7 seed mixture in a 30-1 bioreactor. The changes in morphological features showed a very similar tendency with CPC production. A kinetic model of CPC fermentation was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study suggest that the use of a 3:7 seed mixture inocula has considerable possibilities for improving CPC productivity if applied to industrial scale fermentations. Through morphology and kinetics study, the kinetic model to describe the morphological differentiation and CPC production by C. acremonium M25 was established.     

头孢菌素C生物合成调控研究进展北大核心CSCD

头孢菌素C由丝状真菌顶头孢霉产生,属于β-内酰胺类抗生素。其经改造后的7-氨基头孢烷酸是头孢类抗生素的重要中间体。头孢类抗生素在国内外抗生素市场中占有巨大的份额,是临床上的主要抗感染药物。随着分子生物学的发展,头孢菌素C的生物合成途径已基本阐明。为提高头孢菌素C的产量和降低生产成本,越来越多的研究者开始关注其较为精细、复杂的调控机制。本文重点对头孢菌素C生物合成及其调控机制的最新进展进行了简述,希望为今后头孢菌素C生产菌株的菌种改造和传统产业的升级换代提供一定的借鉴。     

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.     

Optimization of feed conditions in a 2.5-l fed-batch culture using rice oil to improve cephalosporin C production by <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> M25

Summary Rice oil significantly affected cephalosporin C production in a 2.5-l bioreactor culture of Cephalosporium acremonium M25. To improve cephalosporin C production, the feed conditions of rice oil were optimized. Reducing the feed rate of rice oil improved cephalosporin C production to 1.01 g/l when the consumption rate of rice oil decreased. Overall, under optimal feed conditions in the 2.5-l fed-batch culture, cephalosporin C production increased about four times compared to before optimization.     

Production of cephalosporin C using crude glycerol in fed-batch culture of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> M35

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.     

Comparative lipidomic analysis of <Emphasis Type="Italic">Cephalosporium acremonium</Emphasis> insights into industrial and pilot fermentations

Cephalosporium acremonium has been widely applied in industrial cephalosporin C fermentation. However, little is known about the molecular basis of fermentation behavior of this strain. In this study, comparative lipidomic analysis using LC/ESI/MSⁿ technology was employed to investigate responses of Cephalosporium acremonium to multiple environment variations in realistic industrial cephalosporin C fermentation process and provide molecular basis for the discrepancies between industrial and pilot fermentations. Totally 77 phospholipids species were detected and 65 species were further quantified. Score plot revealed that phospholipids metabolism differed in industrial and pilot process. Loading pilot indicated that the main variables responsible for the discrimination of industrial and pilot process were phosphatidylinositols (PIs), phosphatidylserines (PSs) and phosphatic acids (PAs). Higher PIs content in industrial process indicated that cells were more vigorous in industrial process than those in pilot process. Larger increases of PSs, PAs and ratio of oleic acid to linoleic acid coincided well with the earlier and more thorough cellular morphological differentiation in industrial process. The synergetic reaction between cellular behavior and cells living environment led to titer discrepancies between industrial and pilot process. These findings provided lipidomic insights into industrial cephalosporin C production.     

Kinetic model for biotransformation of digitoxin in plant cell suspension culture ofDigitalis lanata

Batch suspension cultures ofDigitalis lanata plant cell were performed to investigate the biotransformation of digitoxin.Digitalis lanata K3OHD plant cells were used to biotransform digitoxin into deacetyllanatoside C. A kinetic model was proposed to describe cell growth, substrate consumption, depletion of digitoxin, formation and depletion of digoxin and purpureaglycoside A, and formation of deacetyllanatoside C. The digoxin and purpureaglycoside A are intermediates of deacetyllanatoside C formation from digitoxin. Interactions between extracellular and intracellular compounds were considered. The proposed model could accurately predict cell growth, substrate consumption and product synthesis. And it can provide a useful framework for quantitative analysis of biotransformation in a plant cell culture system.     

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

A role for alanine in the ammonium regulation of cephalosporin biosynthesis inStreptomyces clavuligerus

Summary It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We foundl-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.     

A Population-Based Morphologically Structured Model for Hyphal Growth and Product Formation in Streptomycin Fermentation

Summary A population model discriminating the hyphae according to the hyphal length and a morphologically structured model considering the specific function of different morphological forms of a hypha are combined together to describe mycelial growth, substrate consumption and secondary metabolite formation in streptomycin fermentation. In the population model, the growth modes of hyphae with different age or length are considered, while in the morphologically structured model, the morphological forms of hyphae and their functions in growth and metabolism are described. The population model and the morphologically structured model are interrelated by a branching function and a differentiation function. In the model, the growth rate of immature apical compartment is distinguished from those of matured ones, branching is proposed to occur only in the subapical region, and the hyphal compartment is assumed to synthesize secondary metabolites. The model is successfully applied to simulate the batch fermentation process of streptomycin production. The growth characteristics of filamentous microorganisms are also discussed using the model predictions.     

Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor

The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity.     

Production of cephalosporin C in a fluidized-bed bioreactor

Production of cephalosporin C was investigated in a fluidized-bed bioreactor using bioparticles of Cephalosporium acremonium. Bioparticles were developed by forming a biofilm of growing hyphae around celite particles which contained spores of the microorganism. Production of the antibiotic was significantly improved by using bioparticles over the free mycelial culture, possibly due to the enhanced mass transfer capacity of the bioreactor system and successive generation of highly productive morphological forms of the microorganism. The maximum attainable titer of cephalosporin C from the bioreactor system was almost double that from a jar fermentor operation with a free mycelial culture of the same strain. The biofilm of the bioparticles became unstable as the fermentation proceeded. Morphological differentiation of the microorganism caused a gradual loss of biofilm and an increase of free cells in the culture broth. Additional feeding of a limited amount of methionine to the fermentation broth was not as effective as expected for improving the bioparticle stability. However, repeated use of the bioparticles revealed a strong possibility to improved the overall reactor performance since it allowed an enhanced production of the antibiotic with fewer free cells.     

Morphological characterization of <Emphasis Type="Italic">Cupriavidus necator</Emphasis> DSM 545 flocs through image analysis

Flocculating agents are used as auxiliary to recover bacterial cells in downstream processes for polyhydroxyalkanoate production. However little is known about the Curpiavidus necator flocs. In this work a new procedure for floc characterization through digital image analysis is presented and validated using the batch settling test. Average diameter, particle size distribution and morphological characteristics of the microbial aggregates were obtained from the flocculation/sedimentation process of the Cupriavidus necator DSM 545 cells by the use of tannin as flocculating agent. The experimental results demonstrated that the proposed method is adequate to determine the average floc diameter with values around 150 μm in accordance with the value obtained from the batch settling test. Nevertheless a morphological characterization of Cupriavidus necator DSM 545 bioaggregates in terms of size distribution and regularity could only be performed by an image analysis procedure. The procedure allowed us to describe the regularity of bacterial flocs through the quantification of morphological parameters of Euclidean [convexity (Conv) and form factor (FF)] and fractal geometry [surface fractal dimension (D _BS)], which are important factors to be considered in the settling efficiency of aggregates.     

Increase in activity of acetylcholinesterase by 20-OH-ecdysone in a Chironomus tentans cell line

Summary An epithelial cell line from Chironomus tentans exhibits acetylcholinesterase activity (specific activity 0.05–0.2 nkat/mg protein), which rises 30– to 40-fold after addition of 10^–6 M 20-OH-ecdysone. The first visible increase occurs after 4 days of incubation with hormone. The enzyme has an apparent K _m of 2.3±0.2×10^–4 M for acetylthiocholine iodide as substrate and is inhibited by eserine and BW284 C51 (50% inhibition at 5×10^–7 M for both inhibitiors) as well as by high concentrations of substrate, but not by tetraisopropylpyrophosphamide. The sensitivity against inhibitors is the same in extracts from hormone-treated cells and from controls. The cholinesterase activity correlates with morphological changes (shape and cell arrangement) and is indepenent of neuronal differentiation. We therefore propose a function for this activity during morphogenesis.     

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.     

Endogenous Inhibitors for Pollen Germination and Pollen Tube Elongation in Pinus densiflora SIEB,et Zucc.

Sulfur metabolism in Cephalosporium acremonium was investigated using a mutant, 8650⁺/ OAH^?/SeMe^R, which could not convert cysteine or inorganic sulfur to methionine. The production of cephalosporin by the mutant depended on the amount of S-sulfocysteine in a chemically defined medium supplemented with a low level of methionine sufficient to support optimal growth. S-Sulfocysteine was detected in an extract of cells grown in the presence of sodium thiosulfate and l-serine. Furthermore, an NADPH-linked reduction of S-sulfocysteine to cysteine was demonstrated in a cell-free extract. These facts suggest that S-sulfocysteine is a direct precursor in cysteine biosynthesis in C. acremonium and an alternative pathway involving the compound is one of the most important ones in cephalosporin C production by this fungus.     

Relationships among species inCarex sect.Phyllostachys (Cyperaceae) based on allozyme divergence

Allozyme variation was examined inCarex sect.Phyllostachys (Cyperaceae) to provide insight into phylogenetic relationships hypothesized in an earlier study and to determine the degree of genetic differentiation within and between taxa. Genetic identity values are concordant with the morphological differences found between species. The lowest values are found between species with the greatest morphological dissimilarity. Conversely, the highest values are associated with species pairs distinguished by relatively few morphological differences. Conspecific populations possess high genetic identities, although interpopulation differentiation has characterized the evolutionary history of some species. Geographic patterning is also evident within species, with geographically proximate populations often having the highest identity values. Phylogenetic trees produced using different cladistic methods were poorly supported and varied in their depiction of relationships among species. One cladogram produced using presence/absence allelic data is more or less congruent with a topology recovered from an earlier analysis utilizing molecular and morphological data. The wide- and narrow-scaled clades are maintained as are the sister species pairsC. backii/C. saximontana, C. basiantha/C. superata, andC. jamesii/C. juniperorum. Contrary to the finding of our previous study, however,C. willdenowii is aligned withC. jamesii/C. juniperorum.