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.     

Use of protoplast fusion to introduce methionine overproduction into Saccharomyces cerevisiae

Summary dl-Ethionine-resistant mutants of Saccharomyces uvarum ATCC 26602 were found to overproduce exogenous l-methionine. dl-Ethionine-resistant mutant ER 108, carrying a mutation to chloramphenicol resistance was converted to petite form, and protoplasts obtained from it were fused with protoplasts from antibiotic-sensitive S. cerevisiae X2928 carrying six auxotrophies. The resulting fusants maintained four auxotrophies and were capable of overproducing l-methionine. These fusants were stable after ten passages on complete medium.     

Enantioselective synthesis ofN-acetyl-l-methionine with aminoacylase in organic solvent

We have developed an enzymatic procedure for the enantiospecific synthesis ofN-acetyl-l-methionine with aminoacylase in an organic solvent.N-Acetyl-l-methionine was most effectively synthesized with a yield of about 90% (on the basis of thel-methionine used) when the reaction mixture, composed of 100 mm sodium acetate, 20 MMdl-methionine and aminoacylase (1000 units) immobilized on celite in 1 ml ethyl acetate saturated with 32 l 140mm sodium phosphate buffer (pH 7.0) containing 0.1 mm CoCl₂, was incubated at 30°C for 24 h.N-Acetyl-l-methionine was isolated from the reaction mixture and the enantiomeric excess was 100%.d-Methionine was also isolated from the mixture with a yield of about 95% and 90% enantiomeric excess. The method is applicable to the synthesis of otherN-acetyl-l-amino acids.     

Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes

We derived l-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selected the potent l-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg l-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The l-methionine biosynthetic enzymes, cystathionine γ-synthase and cystathionine β-lyase, of the l-methionine-overproducing mutants were little repressed by l-methionine. To analyse the mechanism of l-methionine overproduction in the mutant strains, the metJ gene coding for the E. colimet repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser → Asn) at position 54 was observed in four independent l-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the l-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the l-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in l-methionine overproduction, probably because of the derepression of l-methionine biosynthetic enzymes. Received: 6 January 1999 / Received last revision: 19 February 1999 / Accepted: 26 February 1999     

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.     

Isolation and Characterization of S-Adenosylmethionine-requiring Mutants and Role of S-Adenosylmethionine in the Regulation of Methionine Biosynthesis in Corynebacterium glutamicum

Using a minimal medium containing a methionine analog together with a small amount of S-adenosylmethionine (SAM), many SAM requiring mutants which responded only to SAM and not to methionine, S-adenosylhomocysteine, or homocysteine were efficiently isolated from Corynebacterium glutamicum TLD-140 after mutagenesis. Among them, SAM-14 and SAM-19 selected from selenomethionine resistant mutants were subjected to further investigation. Both mutants were unable to grow in a minimal medium and had no detectable activity of SAM synthetase. Both mutants acquired higher resistance to methionine hydroxamate and ethionine as well as to selenomethionine than TLD-140 and produced l-methionine in a medium.

Homoserine-O-transacetylase in SAM-19 was subject to full repression by the addition of excess SAM to the growth medium and was not repressed under SAM limitation, whereas addition of excess l-methionine under SAM limitation caused a partial repression of the enzyme. SAM synthetase as well as l-methionine biosynthetic enzymes in a methionine auxotroph of C. glutamicum was repressed by the addition of l-methionine to the growth medium.

These results suggest that SAM is implicated in the repression of l-methionine synthesizing enzymes in C. glutamicum.     

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.     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-dl-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, todl-5-fluorotryptophan anddl-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-dl-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,dl-p-fluorophenylalanine,dl-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-dl-aspartic acid, α-methyl-dl-methionine,N-o-nitrophenylsulfenyl-l-tryptophan, norleucine, andS-2-aminoethyl-l-cysteine did not show any significant effect. Treatment with 7-azatryptophan,dl-β-hydroxynorvaline,l-methionine-dl-sulfoximine,l-methionine sulfone, and β-2-thienyl-dl-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.     

Regulation of biosynthesis of thiolutin and aureothricin inStreptomyces kasugaensis

l-Methionine anddl-ethionine decreased production of thiolutin and aureothricin inStreptomyces kasugaensis. In the presence ofl-methionine the culture also produced 3-methylthioacrylic acid, 3-methylthiopropionic acid and 3,6-bis-(2-methylthioethyl)-2,5-dioxopiperazine. Production of the metabolites depended on the concentration ofl-methionine in the medium.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Altered <Emphasis Type="SmallCaps">d</Emphasis>-methionine kinetics in rats with renal impairment

d-Amino acids are now recognized to be widely present in mammals. In rats, exogenously administered d-methionine is almost converted into the l-enantiomer via 2-oxo-4-methylthiobutylic acid as an intermediate. d-Amino acid oxidase is associated with conversion of d-methionine into the 2-oxo acid. Since d-amino acid oxidase is present at the highest activity in the kidney compared to other organ, kidney injury is suggested to cause accumulation of d-methionine. The purpose of the present study is to assess the role of kidney in the elimination of d-methionine and metabolic conversion into l-methionine in rats using a stable isotope methodology. After a bolus i.v. administration of d-[²H₃]methionine to 5/6-nephrectomized rats, plasma concentrations of d-[²H₃]methionine, l-[²H₃]methionine, and endogenous l-methionine were determined by a stereoselective GC–MS method. Renal mass reduction slowed down the elimination of d-[²H₃]methionine. The clearance values of conversion of d-[²H₃]methionine into the l-enantiomer in 5/6-nephrectomized rats were one-sixth of those in sham-operated rats. The elimination behavior of d-[²H₃]methionine observed in rats demonstrated that kidney was the principal organ responsible for chiral inversion of d-methionine.     

RELATION OF DL-HOMOCYSTEINE,DL-METHIONINE,AND METHYL DONORS TO THE APHANOMYCES ROOT ROT OF PEAS

Davey , C. B., and G. C. Papavizas . (Crops Res. Div., ARS, USDA, Beltsville, Md.) Relation of dl -homocysteine, dl .-methionine, and methyl donors to the Aphanomyces root rot of peas. Amer. Jour. Bot. 50(1): 67–72. 1963.—dl -Homocysteine, a compound lacking a methyl (CH₃) group, completely prevented Aphanomyces root rot of peas, whereas several CH₃ donors (betaine, choline, dimethyl-β-propiothetin chloride, S-methylmethionine) did not. dl -Homocysteine was taken up by pea plants from nutrient solution and converted to methionine. The methylation of dl -homocysteine in pea tissue occurred without an exogenous source of labile CH₃ groups. More d - than l -methionine was taken up by the plants and more methionine of both forms was taken up when methionine served as the sole sulfur source than when it was supplemented with sulfate. dl -Methionine sulfoxide but not dl -methionine sulfone was converted to methionine in pea tissues. Methionine derived from dl -methionine sulfoxide was proportionately more abundant in the aerial portions of the plants.     

Effect of methionine sulfoximine on methylation of guanine residues in astroglial transfer ribonucleic acids

Culture-grown astrocytes derived from 3-day-old rat brain were incubated in the presence of [³H]guanosine and of the convulsant agentl-methionine-dl-sulfoximine (MSO). The resulting [³H]tRNA was purified from control and MSO-exposed cells at several time points during the incubation and was hydrolyzed to [³H]guanine and four [³H]methyl guanines which were separated by high pressure liquid chromatography. Three of the four [³H]methyl guanines were more highly labeled in the [³H]tRNA of the MSO-exposed cells, relative to that of the control cells throughout the entire incubation period. The findings extend to cultured astrocytes, the stimulatory effect of MSO on the methylation of neural tRNA guanines, previouly observed both in vitro using [¹⁴C]S-adenosyl-l-methionine and in vivo using [methyl ³-H]l-methionine.     

Isolation of sulphate transport defective mutants ofCandida utilis: Further evidence for a common transport system for sulphate,sulphite and thiosulphate

Selenate-resistant mutants ofCandida utilis were isolated. They did not take up sulphate while incorporation of an organic sulphur source, such asl-methionine, was similar to the wild-type strain. They grew poorly on sulphate, sulphite and thiosulphate and, as expected, grew well on methionine. Sulphite reductase activities of the mutants were similar to the wild type strain. The properties of these mutants support the view of a common transport system for sulphate, sulphite and thiosulphate.     

Preparation of Cell Wall Fractions by Various Ways and Activity of Bound Saccharase in Sugar Beet Seedlings

Washed cells of facultative methylotrophs which have the serine pathway showed high activities for l-methionine formation from dl-homocysteine, in the presence of methanol as methyl donor. Strain FM 518, isolated from soil and identified as a bacterium belonging to the genus Pseudomonas, showed the highest activity for l-methionine formation and was used as the parental strain for breeding the l-methionine-producing mutants. An ethionine-resistant mutant, FE 244, derived from strain FM 518, accumulated 0.8 mg/ml l-methionine in a methanol-medium under optimum conditions.     

On the turnover of polyamines spermidine and spermine in mouse brain and other organs

The apparent biological half-lives of spermidine and spermine in mouse brain and other organs were determined by measurement of the specific radioactivities of these compounds over long periods of time. The endogenous polyamine pools were labeled by repeated intraperitoneal injections of [1,4-¹⁴C]putrescine·2HCl, [2-¹⁴C]d,l-methionine, [2-³H]l-methionine, andS-adenosyl-[2-³H]l-methionine. Repeated injections were given to ensure labeling of both fast and slow polyamine pools. It was shown that the two parts of the polyamine molecules which derive from ornithine and methionine have significantly different life spans, especially in the brain. Actual turnover rates of polyamines could not be determined because of the active interconversion between spermine and spermidine, and between spermidine and putrescine. The observed reutilization of putrescine originating from spermidine degradation for spermidine biosynthesis, and the analogous reutilization of spermidine in spermine biosynthesis is discussed with respect to its physiological significance and its relationship to cellular organization.     

S-adenosylmethionine and morphogenesis inMucor racemosus

The intracellular concentration of S-adenosylmethionine (SAM) and the specific activity of S-adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6) were examined in wild-typeMucor racemosus, as well as a morphological mutant termedcoy, under conditions designed to prevent the morphogenesis of yeasts to hyphae. When the mutant was grown in a defined medium supplemented with methionine and induced to shift by exposure to air, there was an increase in intracellular SAM analogous to that previously reported with the wild type. However, when thecoy mutant was grown in the absence of methionine, the intracellular concentration decreased dramatically and the mutant failed to undergo the yeast to hypha transition. An inhibitor of SAM synthetase activity, cycloserine, was used to lower the intracellular concentration of SAM in the wild-type organisms. Under these conditions, wild-typeM. racemosus failed to undergo the transition from yeasts to hyphae when exposed to air.