Formation Of Secondary Metabolites Research Articles

Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system

A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression. The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis. A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription. The StrR product, in turn, activates the other Sm-biosynthesis genes. A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene. Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes. It is most likely that AfsR and AfsK compose a two-component regulatory system. Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1. In addition to the afsR system, another pair of closely located genes that encode proteins with great similarities to the typical proteins of the prokaryotic two-component regulatory system was cloned by utilizing their abilities to confer pigment production on Streptomyces lividans.

Enhancement of phytoalexin accumulation in cultured plant cells by oxalate

Oxalate (0.2–2.0 mM), a compound which induces systemic resistance, can enhance secondary product synthesis 10-fold in cotton cell suspension cultures. To accomplish this, cells require induction with a low level (10 μ of protein per 15 ml incubation) of elicitor from Verticillium dahliae. In the presence of oxalate, these minimal concentrations of elicitor required to induce phytoalexin formation did not have any necrotic effect on the growth of the cotton cultures. Even at higher concentrations of elicitor (80 μg of protein per 15 ml incubation), oxalate was able to reduce the detrimental effect of elicitor on cell suspension growth rates indicating oxalate may have a direct effect on growth. Therefore, in cotton cell suspension cultures the addition of an enhancer of elicitor-induced phytoalexin formation allows optimum stimulation of secondary metabolite formation without affecting cell mass accumulation.

Gene probes for the detection of 6-deoxyhexose metabolism in secondary metabolite-producing streptomycetes

DNA probes were designed from the streptomycin production genes strDELM of Streptomyces griseus involved in the biosynthesis of the 6-deoxyhexose (6DOH) dihydrostreptose which could detect the genomic fragments coding for 6DOH formation in other actinomycetes strains. In about 70% of the 43 strains tested at least one signal could be detected with strD-, strE- or strLM-specific probes. Evidence is presented that the hybridizing genes are mostly clustered and probably engaged in the formation of secondary metabolites. Because of the wide-spread use of 6DOH constituents in natural products these probes should allow to detect a vast array of different secondary metabolic gene clusters in actinomycetes.

Regulation by A-factor and afsR of Secondary Metabolism and Morphogenesis in Streptomyces.

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a microbial hormone that functions as a key switch for secondary metabolite formation and cell differentiation in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases its repression. The positive A-factor signal is transferred, probably via some additional unknown regulatory proteins, to the strR gene, a putative regulator for Sm biosynthesis. The StrR product, in turn, activates the other Sm production genes.A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase present in the same organism. Characterization of AfsR by means of site-directed mutagenesis has revealed that phosphorylated AfsR stimulates globally transcription of antibiotic production genes. It is most likely that the AfsR protein and the AfsR-phosphokinase compose a two-component regulatory system.

Enhanced anthocyanin production in grape cell cultures

Cultured grape cells (Bailey Alicant A Vitis hybrid) that produce and accumulate anthocyanins provide a convenient model system to study secondary metabolite formation and compartmentation. A reduction in anthocyanin production in suspension cultures occurred after a series of subcultures in liquid Gamborg's B5 (GB5) maintenance medium. In contrast, newly derived suspensions from selected red calluses retained their ability to produce anthocyanin at a sustained high level. A production medium with reduced nitrate and elevated sucrose induced very high levels of anthocyanin (up to 2.9 g/l) in suspension cultures derived from selected callus. This medium also induced increased pigment production in faded suspension cultures and in callus cultures of Vignoles [Ravat 51, Vitis hybrid] which normally do not produce anthocyanin in culture. Reduction of nitrate to a critical level resulted in a ‘switchlike’ (rather than gradual) enhancement of anthocyanin production which may represent the removal of an inhibitory effect. Improvements obtained with sucrose were probably due to increased medium osmolarity. The combined effects of low nitrate and high sucrose synergistically improved anthocyanin volumetric productibility (up to 0.15 g/l · day) and surpassed previously published values for cultured grape cells. Our results indicate a possible involvement of a nitrate-sensitive tonoplast ATPase in the accumulation of anthocyanin in grape vacuoles.

Primary structure of AfsR, a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2)

The afsR gene of Streptomyces coelicolor A3(2) complements afsB mutations affecting production of pigmented antibiotics. It also directs pigment production in Streptomyces lividans when carried on a plasmid vector. Nucleotide sequencing of the afsR gene revealed that it codes for a 993-amino acid protein ( M r 105 600) with A- and B-type ATP-binding consensus sequences at its N-terminal portion and two DNA-binding consensus sequences with a helix-turn-helix motif at its C-terminal portion. Each of the N- and C-terminal halves was capable of conferring pigment production, to some extent, in S. lividans, when carried separately on a multicopy plasmid. In addition, expression in trans of the two regions on the same plasmid conferred pigment production to almost the same extent as did the intact afsR gene. Mutations at the two ATP-binding consensus sequences, that were generated by in vitro site-directed mutagenesis, revealed their functional importance. Disruption of the S. coelicolor A3(2) chromosomal afsR gene in either the N- or C-terminal half using phage φC31 KC515 resulted in significant, but not complete, loss of pigment production. These data suggest that the AfsR protein comprises two domains, viz., an ATP-binding and a DNA-binding domain, each of which could function as a positive regulator for pigment production. These afsR mutants sporulate normally. In addition to an internal promoter, which we previously detected in the middle of the AfsR coding region, S1 nuclease mapping revealed two tandem transcriptional start points, separated by 64 bp, upstream from a putative ATG start codon of the AfsR product.

The Pharmacokinetics of Omeprazole in Humans—A Study of Single Intravenous and Oral Doses

The pharmacokinetics of omeprazole, hydroxyomeprazole, omeprazolesulfone, and "remaining metabolites" have been studied in eight young healthy subjects following an acute i.v. and oral dose of 10 and 20 mg of 14C-labeled drug, respectively. The oral dose was given as a buffered solution. Two subjects exhibited essentially higher and more sustained plasma levels of omeprazole than the others. This was due to a higher bioavailability, lower clearance, and longer t1/2 of omeprazole in these two subjects. Maximum concentration (0.7-4.6 mumol/L) was reached between 10 and 25 min after oral dosing. The median bioavailability was 39% (25-117%) and the median systemic plasma clearance was 624 ml/min (range of 59-828 ml/min). The corresponding t1/2 for the i.v. dose was 35 min (16-150 min) and 39 min (14-186 min) after oral administration. The drug was rapidly distributed to extravascular sites (mean t1/2 lambda 1 = 3.0 +/- 0.8 min). Mean Vss was 0.23 +/- 0.04 L/kg. Low systemic clearance of omeprazole was associated with a decreased formation rate of hydroxyomepraxole and "remaining metabolites" while omeprazolesulfone formation seemed to be less affected. However, there was a clear-cut correlation between the t1/2 of omeprazole and of its omeprazolesulfone metabolite, indicating that the elimination of these two compounds is mediated by the same isoenzyme. The mean urinary recovery of the radioactive dose during 96 h was 78.3 +/- 2.3 and 75.7 +/- 2.6% for the i.v. and oral dose, respectively. Insignificant amounts were due to unchanged drug and omeprazolesulfone. The excretion of hydroxyomeprazole during the first 12 h varied between 4.6 to 15.5% of a given dose. The mean recovery of radioactivity in the feces was 19.3 +/- 3.1% of a given i.v. dose and 18.2 +/- 2.3% when given orally. It is concluded that omeprazole is mainly eliminated metabolically and that there is a substantial interindividual variation in the rate of formation of primary and secondary metabolites. This variation in omeprazole disposition is probably of limited clinical importance. The half-life, with a maximum of approximately 3 h, is too short to cause accumulation when the drug is administered in a once-daily regimen.

NEW DIRECTIONS IN THE AETIOLOGY OF ULCERATIVE COLITIS

The causative factors of ulcerative colitis remain unknown. Cellular biochemistry has revealed altered oxidative metabolism, membrane function and synthesis of mucus in colonic epithelial cells in the early stages of ulcerative colitis. Immune studies have highlighted more precise changes in neutrophil and macrophage function in cells of the lamina propria while microbiological evidence has shown a dysbiosis of colonic bacteria related to entero-adhesion, release of bacterial peptides and formation of secondary bacterial metabolites (mercapto fatty acids). A precise sequence of pathological events still needs to be established to account for an acute attack of ulcerative colitis.

Kinetics of secondary metabolite formation by wild type and mutant strains of Streptomyces griseus

In a particle containing medium, the kinetics of formation of secondary metabolites of a Streptomyces wild type and a mutant strain were determined by spectrophotometric measurements of the anthracyclinone pigments and by the analysis of antibiotic activity of the substances produced. The results were related to the physiological state of the culture derived from the oxygen supply. The total cultivation time was about 400 hours. The production of secondary metabolites started at the end of the logarithmic growth phase (after about 30 hours). During the following cultivation remarkable differences between the two strains regarding the formation of pigments and antibiotics occurred. In the wild type strain producing daunomycinone glycosides and related compounds with antibiotic activity, these antibiotics are active as inhibitors (negative feedback). Therefore, at a certain concentration of antibiotics, the de-novo formation of anthracycline molecules was impaired. In the mutant strain, as the result of a genetic change, the secondary metabolite production was shifted to nonglycosylated compounds. The production of these substances is not influenced by feedback inhibition as in the case of the wild type strain. From that reason the amount of pigment increased slightly throughout the course of the experiment. Under our experimental conditions the measurement of oxygen supply was a suitable indicator of the physiological state of culture.

Cell and tissue cultures ofCatharanthus roseus (L.) G. Don: a literature survey

The literature concerning the formation of secondary metabolites in cell and tissue cultures ofCatharanthus roseus has been reviewed. Several aspects involved in the formation of secondary metabolites are discussed; e.g. regulation of secondary metabolism, environmental factors influencing secondary metabolism, biosynthesis and enzymology of the products, analysis of product formation, immobilization of cultured cells and stability of cell lines. Some economical aspects of production processes are discussed.

Antibiotics: opportunities for genetic manipulation

New antibiotics can still be discovered by the development of novel screening procedures. Notable successes over the last few years include the monobactams, beta-lactamase inhibitors (clavulanic acid) and new glycopeptides in the antibacterial field; antiparasitic agents such as avermectins; and herbicidal antibiotics like bialaphos. In the future we can expect the engineering of genes from 'difficult' pathogens, including mycobacteria and fungi, and cancer cells, to provide increasingly useful in vitro targets for the screening of antibiotics that can kill pathogens and tumours. There will also be a greater awareness of the need to reveal the full potential for antibiotic production on the part of microorganisms by the physiological and/or genetic awakening of 'silent' genes. Nevertheless, the supply of natural antibiotics for direct use or chemical modification is not infinite and there will be increasing scope for widening the range of available antibiotics by genetic engineering. 'Hybrid' antibiotics have been shown to be generated by the transfer of genes on suitable vectors between strains producing chemically related compounds. More exciting is the possibility of generating novelty by the genetic engineering of the synthases that determine the basic structure of antibiotics belonging to such classes as the beta-lactams and polyketides. Research in this area will certainly yield knowledge of considerable scientific interest and probably also of potential applicability. In the improvement of antibiotic titre in actinomycetes, protoplast fusion between divergent selection lines has taken a place alongside random mutation and screening. In some cases the cloning of genes controlling metabolic 'bottlenecks' in fungi and actinomycetes will give an immediate benefit in the conversion of accumulated biosynthetic intermediates to the desired end product. However, the main impact of genetic engineering in titre improvement will probably come only after a further use of this technology to understand and manipulate the regulation of antibiotic biosynthesis as a facet of the general challenge of understanding differential gene expression. Streptomyces offers a particularly fertile field for such research, following the isolation of DNA segments that carry groups of closely linked operons for the biosynthesis of and resistance to particular antibiotics, and of genes with pleiotropic effects on morphological differentiation and secondary metabolite formation.

Primary, secondary, and tertiary metabolite kinetics.

Because of the propensity of nascently formed metabolites towards sequential metabolism within formation organs, theoretical and experimental treatments that achieve mass conservation must recognize the various sources contributing to primary, secondary, and tertiary metabolite formation. A simple one-compartment open model, with first-order conditions and the liver as the only organ of drug disappearance and metabolite formation, was used to illustrate the metabolism of a drug to its primary, secondary, and tertiary metabolites, encompassing the cascading effects of sequential metabolism. The concentration-time profiles of the drug and metabolites were examined for two routes of drug administration, oral and intravenous. Formation of the primary metabolite from drug in the gut lumen, with or without further absorption, and metabolite formation arising from first-pass metabolism of the drug and the primary metabolite during oral absorption were considered. Mass balance equations, incorporating modifications of the various absorption and conversion rate constants, were integrated to provide the explicit solutions. Simulations, with and without consideration of the sources of metabolite formation other than from its immediate precursor, were used to illustrate the expected differences in circulating metabolite concentrations. However, a simple relationship between the area under the curve of any metabolite, M, or [AUC (m)], its clearance [CL(m)], and route of drug administration was found. The drug dose, route, fraction absorbed into the portal circulation, Fabs, fraction available of drug from the liver, F, availabilities of the metabolites F(m) from formation organs, and CL(m) are determinants of the AUC(m)'s. After iv drug dosing, the area of any intermediary metabolites is determined by the iv drug dose divided by the (CL(m)/F(m] of that metabolite. When a terminal metabolite is not metabolized, its area under the curve becomes the iv dose of drug divided by the clearance of the terminal metabolite since the available fraction for this metabolite is unity. Similarly, after oral drug administration, when loss of drug in the gut lumen does not contribute to the appearance of metabolites systematically, the general solution for AUC(m) is the product of Fabs and oral drug dose divided by [CL(m)/F(m)]. A comparison of the area ratios of any metabolite after po and iv drug dosing, therefore, furnishes Fabs. When this fraction is divided into the overall systemic availability or Fsys, the drug availability from the first-pass organs, F, may be found.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g.

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.

Book Review: Regulation of Secondary Metabolite Formation. Proceedings of the Sixteenth Workshop Conference, Hoechst, Gracht Castle, 12–16 May 1985. Edited by H. Kleinkauf, H. von Döhren, H. Dornauer, and G. Nesemann

Book Review: Regulation of Secondary Metabolite Formation. Proceedings of the Sixteenth Workshop Conference, Hoechst, Gracht Castle, 12–16 May 1985. Edited by H. Kleinkauf, H. von Döhren, H. Dornauer, and G. Nesemann

Regulation of Secondary Metabolite Formation. Workshop Conferences Hoechst, Vol. 16. Edit. by H. Kleinkauf, H. v. Döhren, H. Dornauer and G. Nesemann. VCH Verlagsges. Weinheim 1986; 402 S., geb. DM 108,–

Pharmazie in unserer ZeitVolume 16, Issue 3 p. 94-94 Bücher Regulation of Secondary Metabolite Formation. Workshop Conferences Hoechst, Vol. 16. Edit. by H. Kleinkauf, H. v. Döhren, H. Dornauer and G. Nesemann. VCH Verlagsges. Weinheim 1986; 402 S., geb. DM 108,– I. Graf, I. GrafSearch for more papers by this author I. Graf, I. GrafSearch for more papers by this author First published: 1987 https://doi.org/10.1002/pauz.19870160306AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume16, Issue31987Pages 94-94 RelatedInformation

Biosynthesis of 3-acetyldeoxynivalenol and other metabolites by Fusarium culmorum HLX 1503 in a stirred jar fermentor

The formation of 3-acetyldeoxynivalenol (ADON) and other secondary metabolites of Fusarium culmorum in a stirred jar fermentor is described in relation to changes in concentrations of sugars, N, P, and O2, and in pH in the medium as well as changes in cellular parameters. The addition of sodium [1-13C]acetate to the fermentation demonstrated that the conditions used resulted in the biosynthesis of ADON instead of other primary and secondary metabolites such as culmorin, dihydroxycalonectrin, and sambucinol for all but the early stages of the fermentation. Enrichment studies also revealed that dihydroxycalonectrin was not an important intermediate of ADON. Biosynthesis of ADON was apparently induced by nitrogen limitation. At the end of the fermentation, ca. 710 mg L−1 ADON was obtained.

Effect of 5-Azacytidine on the Formation of Secondary Metabolites in Catharanthus roseus Cell Suspension Cultures

A cell suspension culture of Catharanthus roseus was treated during the growth phase with the nucleoside analogue 5-azacytidine, a powerful inducer of cell differentiation, as evidenced with animal and human cell lines. After induction the cell culture was further incubated in a production medium in the absence of the inducer for 10 days. The extraction of the freeze dried cells and analysis by high performance liquid chromatography showed an additional intense peak in the elution profile, not present in extracts of untreated cells. The structure of the newly synthesized metabolite, elucidated by mass spectrometry and 1H and 13C NMR, was a lignan type compound, lirioresinol B mono-β-D-glucoside. This compound has hitherto not been found in Catharanthus roseus plants or cell cultures.

Multistage continuous culture to examine secondary metabolite formation in plant cells: Phenolics from Nicotiana tabacum

The feasibility of operating a multistage continuous culture of plant cells was demonstrated for Nicotiana tabacum. Cells in the second stage of a two-stage chemostat were morphologically distinct from cells in the first stage or cells in a single-stage unit with a holding time equal to the combined holding times in the two-stage system. Cells in the second stage produced much higher levels of phenolics per unit weight of cells than cells in either the first-stage or single-stage unit. The steady-state was reproduced. When a glucose side stream was fed to the second stage, an increase in apparent cell division was observed with a simultaneous decrease in phenolics productivity. When the toxic precursor phenylalanine was pulsed into the reactor, the quantity of biomass decreased temporarily while phenolic productivity increased. These experiments demonstrate that multistage continuous culture may be useful in increasing secondary metabolite formation in cells and in exploring mechanisms controlling secondary metabolite formation.

Continuous production of patulin by immobilized cells ofPenicillium urticae in a stirred tank reactor

Production of the mycotoxin, patulin, by immobilized cells ofPenicillium urticae was chosen as a model system to study the formation of secondary metabolites on a long term basis. Spores of the fungus were immobilized in carrageenan beads and allowed to germinate in situ by incubation in a growth medium. Production of the antibiotic by the populated biocatalyst was then followed in a continuous stirred tank reactor (CSTR) under three different regimes of nutrient feed. Using a nitrogen free feed, antibiotic production gradually declined but the cells could be re-activated by addition of a diluted growth medium. Prolonged production of patulin (up to 440 h) was observed when a source of nitrogen (ie. yeast extract) was supplied to the feed medium.

The application of materials balancing to the characterization of sequential secondary metabolite formation in Streptomyces cattleya NRRL 8057.

The high substrate yield factor (0.73 g biomass g glucose-1) and low R.Q. (respiratory quotient, i.e. mol CO2 evolved per mol O2 consumed) value (0.8) measured during growth-phase batch cultures of Streptomyces cattleya could be rationalized in terms of the fermentation mass balance when the oxidized elemental composition of biomass was considered. R.Q. was also indicative of the sequence of secondary metabolite formation, the value rising in steps as each new product was formed. The period of maximum respiratory activity and phosphate uptake preceded maximum growth and glucose uptake. At the end of the lytic phase, a cyclopentenedione cobalt chelator was produced. The termination of lysis coincided with melanin production. Sequential cephamycin C and thienamycin production then took place. Specific hyphal protein content (per unit RNA) peaked before the production of each new metabolite. Melanin, cephamycin C and thienamycin production were initiated when glucose, ammonia and phosphate, respectively, became growth-limiting.