A chemically defined medium for cephalosporin C production by Paecilomyces persicinus

A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.     

Effect of methionine on the growth and morphogenesis ofSaccharomycopsis fibuligera in defined medium

Summary The growth ofSaccharomycopsis fibuligera on defined media has been investigated. Growth on a minimal medium was stimulated by the addition of methionine but not by the addition of norleucine or other amino acids including cysteine. Growth in the presence of methionine produced pseudomycelium rather than the true mycelium which is produced during growth on complex media A requirement for methionine for growth and pseudomycelium formation inS. fibuligera has been demonstrated.     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.     

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.     

Defining an optimal carbon source/methionine feed strategy for growth and cephalosporin C formation by Cephalosporium acremonium.

The effect of the method of methionine addition, growth-limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose-limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to mumax (mu approximately 0.036 h-1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (mu approximately 0.01 h-1) with sucrose for the duration of the fermentation.     

Synthesis of cephalosporin C by a methionine analogue resistant mutant of Cephalosporium acremonium

Summary DL-seleno-methionine resistant mutants of Cephalosporium acremonium were isolated which have an enhanced capacity to utilized sulfate for the synthesis of cephalosporin C. Of these mutants, one designated as SMR-I3 produced three-fold more cephalosporin C from sulfate than its parent CW19. Mutant SMR-I3 required less dl-methionine for maximal synthesis of cephalosporin C, but an excess of dl-methionine inhibited the synthesis of the antibiotic. Furthermore, the mutant accumulated excessive methionine in the amino acid pool and possessed superior activity for sulfate uptake. These observations indicate that in the mutant SMR-I3, the biosynthesis of methionine from sulfate is very active and excess methionine becomes available for the synthesis of cephalosporin C.     

Biosynthesis of Cephalosporin C

The superiority of d-methionine over l-methionine for stimulation of cephalosporin C synthesis in a crude medium was confirmed. The optimal level of dl-methionine was 0.5%. Methionine stimulates growth slightly but this is not thought to be the cause of the marked stimulation of antibiotic synthesis. Of a large number of sulfur compounds tested, only dl-methionine-dl-sulfoxide and S-methyl-l-cysteine showed considerable methionine-replacing activity. Lysine and α-aminoadipic acid were inactive.     

Nitrogen nutrition and regulation of cephalosporin production in Streptomyces clavuligerus.

When used as sole nitrogen source, certain amino acids (e.g., proline, asparagine) supported both growth and sporulation by Streptomyces clavuligerus streaked onto solid defined medium. Ammonium supported growth but suppressed sporulation. Amino nitrogen was best for cephalosporin production in liquid defined medium, although urea was almost as useful. A comparison of amino acids showed asparagine and glutamine to be the best nitrogen sources and arginine to be almost as good. Ammonium salts supported a somewhat lower growth rate than asparagine, but antibiotic production was very poor on these inorganic nitrogen sources. Addition of ammonium to asparagine did not affect growth rate but increased mycelial mass; cephalosporin production was reduced by about 75%. Antibiotic production was more closely associated with growth in the absence of ammonium than in its presence, indicating a strong inhibitory and (or) repressive effect of NH4+ on antibiotic production. Ammonium exerted its negative effect when added at 24h or earlier, i.e. before antibiotic formation began.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Influence of inorganic phosphate and organic buffers on cephalosporin production by Streptomyces clavuligerus

A high concentration of potassium phosphate (75–100 mM) stabilized pH and supported extensive growth of Streptomyces clavuligerus in a chemically defined medium; such a concentration also inhibited cephalosporin production. Although Tris buffer was found to have detrimental effects on growth and antibiotic production, 3-(N-morpholino)-propane sulfonate (MOPS) or 2-(N-morpholino)-ethane sulfonate (MES) buffer provided a nontoxic buffering system. In the presence of MOPS buffer, cephalosporin production was optimal at 25 mM phosphate, whereas higher concentrations of phosphate progressively inhibited antibiotic production up to 85% without modifying the pH pattern. MOPS buffer can be used to conduct fermentations at a relatively constant pH value in shake flasks.List of Non-Common Abbreviations MOPS 3-(N-morpholino)propane sulfonic acid - MES 2-(N-morpholino)ethane sulfonic acid     

Microbiological studies on the formation of gramicidin S synthetases

Rapid and extensive growth of Bacillus brevis ATCC 9999 was obtained in a complex medium containing yeast extract and peptone. Gramicidin S (GS) production in this medium reached 2.5 g/liter and 0.25 g/g dry cell weight. GS synthetase I production was also high in this complex medium. Chemically defined media were also developed for this strain. In a glycerol-ammonium sulfate-Tris-salts medium, the culture grew about 40% as well (rate and extent) as in complex medium. Although GS production was low (0.23 g GS/liter), peak specific activity of GS synthetase I was as high as on complex medium. Nutritional experiments showed that growth was stimulated by glutamine, methionine, proline, arginine, and histidine. Addition of these amino acids almost doubled the rate and extent of growth and GS production on a volumetric basis. However the increase in GS was due merely to the increased cell density; GS synthetase I specific activity was in fact decreased by the supplement. Complex medium is better than defined medium for GS and GS synthetase production due to increased cell density and a slower rate of synthetase disappearance.     

Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase

Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.     

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:204–211, 2015     

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.     

Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosynthesis.

Hydrogen cyanide (HCN) production by Pseudomonas aeruginosa in a synthetic medium is stimulated by the presence of glycine. Methionine enhances this stimulation but will not substitute for glycine as a stimulator of cyanogenesis. Threonine and phenylalanine are effective substitutes for glycine in the stimulation of HCN production. Glycine, threonine, and serine are good radioisotope precursors of HCN, but methionine and phenylalanine are not. Cell extracts of P. aeruginosa convert [14C]threonine to [14C]glycine. H14CN is produced with low dilution of label from either [1-14C]glycine or [2-14C]glycine, indicating a randomization of label either in the primary or secondary metabolism of glycine. When whole cells were fed [1,2-14C]glycine, cyanide and bicarbonate were the only radioactive extracellular products observed.