Respiration activity of suspension cell culture of <Emphasis Type="Italic">Polyscias filicifolia</Emphasis> bailey, <Emphasis Type="Italic">Stephania glabra</Emphasis> (Roxb.) miers,and <Emphasis Type="Italic">Dioscorea deltoidea</Emphasis> wall

Peculiarities of respiration of cells cultures producing biologically active compounds (isoprenoids and alkaloids) were investigated in order to optimize productivity of culture growth and biosynthesis. It had been revealed that studied cells cultures of Dioscorea deltoidea Wall (producer of furistanol glycosides), Stephania glabra (Roxb.) Miers (producer of stepharin alkaloid) and Polyscias filicifolia Bailey (complex of biologically active agents) differ both in joint respiration activity and in ratio between cytochrome and cyanide-resistant respiration, while changes of rate of total oxygen consumption and activity of alternative oxidase during growth were found to be individual for every investigated culture. Maximum rate of oxygen consumption for cells of D. deltoidea and S. glabra was marked in the period preceding active synthesis of secondary metabolites (lag phase for D. deltoidea and exponential phase for S. glabra). The revealed trends can be used for further monitoring and regulation of growth and biosynthesis of secondary metabolites in producing cell cultures during deep cultivation.     

Fungi of the genus Penicillium as producers of physiologically active compounds (Review)

Fungi of the genus Penicillium isolated from little studied habitats are able to synthesize both previously known and new physiologically active compounds with diverse structures. They include secondary metabolites of alkaloid nature, i.e., ergot alkaloids, diketopiperazines, quinolines, quinazolines, benzodiazepines, and polyketides. We discuss the use of profiles of secondary metabolites for taxonomy purposes. Studying the physicochemical characteristics of producers of biologically active compounds showed that the biosynthesis of alkaloids is initiated on the first days of cultivation and proceeds simultaneously with growth. The cyclic character of alkaloid accumulation was recorded related to the processes of alkaloid biosynthesis, excretion from cells, degradation in culture fluid, and consumption by cells. Synchronic variations in the concentrations of intracellular tryptophan and alkaloids are necessary for the regulation of the optimal quantity of tryptophan necessary for the culture.     

Penicillium expansum, a resident fungal strain of the orbital complex Mir, producing xanthocillin X and questiomycin A

It was demonstrated that the fungus Penicillium expansum 2-7, a resident strain of the orbital complex Mir, which became dominating at the end of a long-term space flight, formed biologically active secondary metabolites (antibiotics). Using physicochemical methods, these metabolites were identified as xanthocyllin X and questiomycin A. Time courses of their biosyntheses during growth and development of the producer culture were studied. Addition of zinc to the culture medium affected both the growth of the culture and the biosyntheses of the antibiotics. The concentrations of zinc in the medium, optimum for xanthocyllin X and questiomycin A production, amount to 0.3 and 3.0 mg/l, respectively.     

Growth kinetics of Dioscorea deltoidea and Catharanthus roseus in batch culture

Sugar-limited batch growth of Dioscorea deltoidea and Catharanthus roseus plant cell cultures was studied in a 14-L stirred tank fermentor. With dissolved oxygen concentration monitored and maintained at nonlimiting levels, growth rates and ratios of dry weight to fresh weight were found to be strongly influenced by sugar concentration. Linear relationships between respiration rate and growth rate were observed, and respiration rate was found to drop to a maintenance level after sugar had been fully depleted from the medium. Diosgenin biosynthesis by D. deltoidea was shown to be independent of growth rate. Ajmalicine biosynthesis in C. roseus was negligible during sugar-limited growth but was induced by inoculation into an 80-g/L glucose solution.     

Penicillium expansum, a Resident Fungal Strain of the Orbital Complex Mir, Producing Xanthocyllin X and Questiomycin A

It was demonstrated that the fungus Penicillium expansum 2-7, a resident strain of the orbital complex Mir, which became dominant at the end of a long-term space flight, formed biologically active secondary metabolites (antibiotics). Using physicochemical methods these metabolites were identified as xanthocyllin X and questiomycin A. The time courses of their biosyntheses during the growth and development of the producer culture were studied. The addition of zinc to the culture medium affected both the growth of the culture and the biosyntheses of the antibiotics. The concentrations of zinc in the medium, optimum for xanthocyllin X and questiomycin A production, were 0.3 and 3.0 mg/l, respectively.     

Fungus Penicillium citrinum, isolated from permafrost sediments, as a producer of ergot alkaloids and new quinoline alkaloids quinocitrinines

Quinocitrinines and ergot alkaloids are synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grows. The major part of these secondary metabolites are secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

The Fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>, Isolated from Permafrost Sediments,as a Producer of Ergot Alkaloids and New Quinoline Alkaloids Quinocitrinines

Quinocitrinines and ergot alkaloids were synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grews. The major part of these secondary metabolites was secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

New producers of biologically active compounds--fungal strains of the genus Penicillium isolated from permafrost

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazines, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxiagroclavine-I and quinocitrinins. The main physiological and biochemical characteristics of this producer were investigated.     

Biochemical parameters of Saccharopolyspora Erythraea during feeding ammonium sulphate in erythromycin biosynthesis phase

The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.     

Secondary metabolism of hairy root cultures in bioreactors

Summary In vitro cultures are being considered as an alternative to agricultural processes for producing valuable secondary metabolites. Most efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Bioreactors used to culture hairy roots can be roughly divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both. The growth and productivity of hairy root cultures are reviewed with an emphasis on successful bioreactors and important culture considerations. The latter include strain selection, production of product in relation to growth phase, media composition, the gas regime, use of elicitors, the role of light, and apparent product loss. Together with genetic engineering and process optimization, proper reactor design plays a key role in the development of successful large scale production of secondary metabolites from plant cultures.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Identification and activation of novel biosynthetic gene clusters by genome mining in the kirromycin producer <Emphasis Type="Italic">Streptomyces collinus</Emphasis> Tü 365

Streptomycetes are prolific sources of novel biologically active secondary metabolites with pharmaceutical potential. S. collinus Tü 365 is a Streptomyces strain, isolated 1972 from Kouroussa (Guinea). It is best known as producer of the antibiotic kirromycin, an inhibitor of the protein biosynthesis interacting with elongation factor EF-Tu. Genome Mining revealed 32 gene clusters encoding the biosynthesis of diverse secondary metabolites in the genome of Streptomyces collinus Tü 365, indicating an enormous biosynthetic potential of this strain. The structural diversity of secondary metabolisms predicted for S. collinus Tü 365 includes PKS, NRPS, PKS-NRPS hybrids, a lanthipeptide, terpenes and siderophores. While some of these gene clusters were found to contain genes related to known secondary metabolites, which also could be detected in HPLC–MS analyses, most of the uncharacterized gene clusters are not expressed under standard laboratory conditions. With this study we aimed to characterize the genome information of S. collinus Tü 365 to make use of gene clusters, which previously have not been described for this strain. We were able to connect the gene clusters of a lanthipeptide, a carotenoid, five terpenoid compounds, an ectoine, a siderophore and a spore pigment-associated gene cluster to their respective biosynthesis products.     

Influence of nutrient excess on intracellular proteins composition in relation to spiramycin production,in Streptomyces ambofaciens

The protein profile on media favouring or reducing spiramycin biosynthesis in S. ambofaciens, was examined using SDS-polyacrylamide gradient gel electrophoresis over a time course of 144 h. Changes in the pattern of protein synthesis were observed which varied according to the nutrient medium conditions and appeared to suggest the presence of protein subsets specific to the production phase, which were probably involved in the switch to secondary metabolism and the onset of antibiotic synthesis. Excess of ammonium, glycerol or phosphate provoked the persistence of a protein band during the whole culture period, which was detected only during growth phase in the control producer culture. In addition, the nutrient excess caused the suppression of spiramycin production and the absence of two protein bands which appeared only in production phase in the control producer culture.     

Isolation and characterization of an intermediate steroid metabolite in diosgenin biosynthesis in suspension cultures of Dioscorea deltoidea cells.

The aglycon form of the steroidal sapogenin furost -5-ene-3 beta, 22,26-triol, 3 beta- chacotrioside 26 beta-D-glucopyranoside was isolated from cell suspension cultures of Dioscorea deltoidea and its molecular structure was determined by mass spectrometry and 1H and 13C n.m.r. spectroscopy. From kinetic studies and incorporation experiments with [1-14C]acetate it was concluded that the steroidal compound (in the glycoside form) is an intermediate in vivo in diosgenin biosynthesis. It accumulated in growing cells of D. deltoidea and was metabolized to diosgenin (in the glycoside form, i.e. dioscin ) in non-dividing cells.     

New producers of biologically active compounds—fungal strains of the genus <Emphasis Type="Italic">Penicillium</Emphasis> isolated from permafrost

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazinees, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxyagroclavine-I and quinocitrinines. The main physiological and biochemical characteristics of this producer were investigated.     

Eliciting secondary metabolism in plant cell cultures

Plant cell cultures are potentially rich sources of valuable pharmaceuticals and other biologically active phytochemicals, but relatively few cultures synthesize secondary compounds over extended periods in amounts comparable to those found in the whole plant. Frequently, no secondary metabolites characteristic of the intact plant are produced. So far, the manipulation of culture media, culture conditions and phytohormone levels have, in general, failed to permit commercial production of those phytochemicals useful in medicine and industry. This almost certainly reflects the lack of understanding of basic secondary metabolic regulation in cultured plant cells.

Microbial insult can induce antibiotic phytochemical synthesis in cultured plant cells: the microbial molecules which stimulate synthesis have been called ‘elicitors’. Increased synthesis of secondary products in response to elicitation of various types appear to be the general response of cultured cells. This paper illustrates the immense biotechnological potential of plant cell culture—‘elicitor’ (inducer) interactions to the large scale production of secondary metabolites, and suggests several lines of enquiry that remain to be authoritatively treated.     

Cystine/cysteine metabolism in cultured Sf9 cells: influence of cell physiology on biosynthesis, amino acid uptake and growth

Spodoptera frugiperda (Sf9) insect cells proliferate in a cystine-free medium, with the same growth rate, reaching the same final cell density, as in a cystine-containing medium, provided that the inoculum is taken from a pre-culture sufficiently early, at 47–53 h. With an inoculum from a 103 h culture an extended lag phase accompanied by cell death was observed during the first 50 h of cystine-free culture, even though the culture had been adapted to cystine-free conditions for 10 passages. Cystine-free cultures seeded with a 103 h inoculum had lower growth rates and reached lower final cell densities than corresponding cystine-supplied cultures. Cysteine biosynthesis occurs from methionine via the β-cystathionine pathway. More methionine was consumed by the cells in cystine-free media, and cystathionine was secreted when methionine and cystine were supplied in excess. The data suggest that cysteine biosynthesis is up-regulated in proliferating cells but down-regulated when the cells enter the stationary phase. In cultures supplied with cystine (10–100 mg 1^-1), the specific uptake rate and total consumption of cystine, as well as the uptake of glutamate, glutamine and glucose increased with increasing cystine concentrations. These results are interpreted in view of system x _c ^– , a concentration dependent amino acid transporter. Similarly, the consumption of amino acids transported by system L (ile, leu, val, tyr) was enhanced in cystine-containing cultures, as compared to cystine-free cultures. Uptake of cystine, methionine and system L amino acids ceases abruptly in all cultures, even before growth ceased. The specific growth rate starts to decline early during the growth phase, but this growth behaviour could not be correlated to the depletion of nutrients. We therefore propose that the observed growth pattern is a result of (auto)regulatory events that control both proliferation and metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.     

Pulsed electric field stimulates plant secondary metabolism in suspension cultures of Taxus chinensis

The effects of pulsed electric field (PEF) on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. Cultured cells in different growth phases were exposed to a PEF (50 Hz, 10 V/m) for various periods of time. A significant increase in intracellular accumulation of taxuyunnanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF. The Tc content (i.e., the specific production based on dry cell weight) was increased by 30% after exposure to PEF, without loss of biomass, compared with the control. The combination of PEF treatment and sucrose feeding proved useful for improving secondary metabolite formation. Production levels of reactive oxygen species, extracellular Tc, and phenolics were all increased, whereas cell capacitance was decreased with PEF treatment. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane's dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures.