Amino Acid Metabolism in Microorganisms

Certain strains of Streptomyces were found to convert l-methionine into 3-methylthio-propylamine (MTPA), but not d-methionine. Now, optical resolution of DL-methionine was attempted using this phenomenon. Streptomyces sp. K37 was cultured in a medium containing DL-methionine (10 mg/ml). The culture filtrate was applied to a column of Diaion SA-21A (OH form). MTPA was recovered from the effluent by ether exraction. The Diaion SA-21A was eluted with 1N HCl and the eluate was applied to a column of Diaion SK-1 (H form). d-Methionine was eluted from the column with 1N NH₄OH and recovered after concentration, decolorization with active carbon, and precipitation with ethanol. The yields of MTPA and d-methionine from the broth were 69.5% and 89.5%, respectively.     

Phyllostine,a New Phytotoxic Compound Produced by Phyllosticta sp.

Ethionine-resistant mutants derived from Corynebacterium glutamicum KY 9276 (Thr^?) were found to accumulate l-methionine in culture media. One of the mutants, ER-107-4, which produced 250 μg/ml of l-methionine was subjected to further mutagenesis to obtain better l-methionine producers. l-Methionine production increased stepwise by successive endowing such markers as selenomethionine, 1,2,4-triazole, trifluoromethionine and methionine hydroxamate resistance. Thus, a mutant multi-resistant to ethionine, selenomethionine and methionine hydroxamate, ESLMR-724, produced 2 mg/ml of l-methionine in a medium containing 10% glucose.

Increase of l-methionine production was accompanied by increased levels and reduced repressibility of methionine-forming enzymes. The levels of methionine enzymes in ESLMR-724 increased to 2.5~4.2 fold of those in KY9276, In addition, homoserine-O-trans-acetylase and cystathionine γ-synthase which were strongly repressed by l-methionine in KY 9276 were stimulated by exogenous l-methionine in ESLMR-724. Implications of these results were discussed in relation to the productivity of l-methionine and the regulation of l-methionine biosynthesis.     

Amino Acid Metabolism in Microorganisms

3-Methylthiopropylamine (MTPA) formation from l-methionine in Streptomyces sp. K37 was studied in detail. The reaction was confirmed to be catalyzed by the decarboxylase of l-methionine. The properties of the enzyme were studied in detail using acetone dried cells or cell-free extract. The enzyme was specific for l-methionine. Pyridoxal phosphate stimulated the reaction and protected the enzyme against heat inactivation. The optimum pH for the reaction was 6.0~8.0 and the optimum temperature was about 40°C. Carbonyl reagents (10^?2~10^?3 m) inhibited the reaction completely, and silver nitrate and mercuric chloride (10^?3~10^?4 m) markedly inhibited the reaction. Km value for the reaction was 1.21 × 10^?5 m. l-Methionine assay using the decarboxylase was attempted and was found to be applicable to practical use.     

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.     

Inhibition of Yeast Growth by Methionine

The accumulation of S-adenosylmethionine in adenine-requiring yeast cells grown in a culture medium containing dl-, l-, or d-methionine was much larger than that in cells grown in a methionine-free medium. The accumulation of S-adenosyl-d-methionine in the cells was significantly lower than that of S-adenosyl-l-methionine. When yeast cells containing a large amount of S-adenosyl-l-methionine were incubated in an adenine-free medium, adenosylmethionine was degraded, but poor and insignificant growth was observed indicating the meager nature of this compound as an adenine source. No degradation of accumulated S-adenosyl-d-methionine was detected. Isotopic experiment revealed that S-adenosyl-l-methionine in the yeast cells turned over at a considerable rate when the medium contained both adenine and l-methionine. Most of the l-methionine assimilated appears to be metabolized via S-adenosyl-l-methionine.     

Microbial Production of l-Threonine

l-Threonine production by strain BB-69, which was derived from Brevibacterium flavum No. 2247 as a α-amino-β-hydroxyvaleric acid resistant mutant and produced about 12 g/liter of l-threonine, was reduced by the addition of l-lysine or l-methionine in the culture medium. Many of lysine auxotrophs but not methionine auxotrophs derived from strain B–2, which produced about 7 g/liter of l-threonine, produced more l-threonine than the parental strain. Except only one methionine auxotroph (BBM–21), none of lysine and methionine auxotrophs derived from BB–69 produced more l-threonine than the parental strain. Homoserine dehydrogenase of crude extract from strain B–2 was inhibited by l-threonine more strongly than that from BB–69. Strain BBM–21, a methionine auxotroph derived from BB–69, produced about 18 g/liter of l-threonine, 50% more than BB–69, while accumulation of homoserine decreased remarkably as compared with BB–69. l-Threonine production by BBM–21 was increased by the addition of l-homoserine, a precursor of l-threonine, while that by BB–69 was not. No difference was found among BBM–21, BB–69 and No. 2247 in the degree of inhibition of homoserine kinase by l-threonine. l-Threonine production by revertants of BBM–21, that is, mutants which could grow without methionine, were all lower than that of BBM–21. Correlation between l-threonine production and methionine or lysine auxotrophy was discussed.     

On the Mechanism of l-Prolyl Diketopiperazine Formation by Streptomyces

Since l-prolyl diketopiperazines, l-prolyl-l-valine anhydride and l-leucyl-l-proline anhydride, had been isolated from the culture filtrate of Streptomyces sp. S-580, the mechanism of l-prolyl diketopiperazine formation by Streptomyces has been studied. These two l-prolyl diketopiperazines were not formed from their constituent amino acids incubated with intact cell or cell free homogenate of this strain in buffered salt solution containing energy source. However, from milk casein, poly peptone or gelatin, the former two were components of the culture medium of this strain, hydrolyzed with the pure streptomyces-protease, these l-prolyl diketopiperazines were obtained (only from gelatin, glycyl-l-proline anhydride were obtained in addition to these two). Furthermore, in hydrolysis of some synthetic l-prolyl peptides with this enzyme, l-prolyl diketopiperazine formation were also studied, and as the result, glycyl-l-proline anhydride was obtained from glycyl-l-prolyl-l-leucine but no l-prolyl diketopiperazine was formed from l-prolyl-l-leucyl-glycine. From these evidences, the possible route of l-prolyl diketopiperazine formation by Streptomyces has been discussed.     

Inhibition by Hadacidin,Duazomycin A,and Other Amino Acid Derivatives of de novo Starch Synthesis

Two amino acid derivative antibiotics, hadacidin and duazomycin A, were isolated as inhibitors of de novo starch synthesis in excised leaf segments from culture filtrates of Penicillium No. 467 and Streptomyces No. 317, respectively. In addition, azaserine, 6-diazo-5-oxo-l-norleucine (DON), and trifluoro-dl-methionine were found to be potent inhibitors among about 70 kinds of commercial amino acid derivatives. These amino acid derivatives inhibited de novo starch synthesis at concentrations ranging from 1 to 10ppm but did not inhibit photosynthetic oxygen evolution at a concentration of 100ppm. The inhibition caused by these diazo compounds was reversed by a supplement of l-glutamine. With hadacidin and trifluoro-dl-methionine, however, the reversal was not observed upon the addition of l-aspartic acid or l-methionine, respectively. Among these active compounds, hadacidin was herbicidal against lettuce and barnyard millet by foliar treatment at a concentration of more than 1000 ppm.     

Microbial Production of l-Threonine

l-Threonine producing α-amino-β-hydroxyvaleric acid resistant mutants were derived from E. coli K-12 with 3 x 10^-5 frequency. One of mutants, strain β-101, accummulated maximum amount of l-threonine (1. 9 g/liter) in medium. Among isoleucine, methionine and lysine auxotrophs derived from E. coli K-12, only methionine auxotrophs produced l-threonine. In contrast, among isoleucine, methionine and lysine auxotrophs derived from β-101, l-threonine accumulation was generally enhanced in isoleucine auxotrophs. One of isoleucine auxotrophs, strain βI-67, produced maximum amount of l-threonine (4. 7 g/liter). Methionine auxotroph, βM-7, derived from β-101 produced 3.8 g/liter, and βIM-4, methionine auxotroph derived from β1-67, produced 6.1 g/liter, when it was cultured in 3% glucose medium supplemented with 100 μg/ml of l-isoleucine and l-methionine, respectively. These l-threonine productivities of E. coli mutants were discussed with respect to the regulatory mechanisms of threonine biosynthesis. A favourable fermentation medium for l-threonine production by E. coli mutants was established by using strain βM-4.     

Accumulation of S-Adenosylmethionine by Molds

Aspergillus tamani accumulated about 20 μmoles of S-adenosylmethionine (SAM) in 1 g of dry cells when cultured secondarily in a medium containing more than 10 mm of l- methionine. The accumulation was not so high when l-methionine was replaced by d- methionine. Addition of nucleic acid-related substances was not effective for the accumulation. Addition of d, l-ethionine in place of methionine caused accumulation of S-adenosylethionine (SAE) in place of SAM. Among 100 strains of molds tested, a number of strains belonging to the genera Penicillium, Aspergillus, Rhizopus and Mucor could accumulate SAM in their mycelia. Especially Mucor jansseni had the highest ability; it accumulated 45 μmoles of SAM in 1 g of dry cells.     

2-Keto-l-gulonic Acid Fermentation

l-Sorbose metabolism in Pseudomonas aeruginosa IFO 3898 was studied. When the strain was cultivated in l-sorbose medium, l-idonic and 2-keto-l-gulonic acids were detected in the culture broth.

From the results on the metabolism of various sugars and sugar acids with the cell suspension and the metabolites accumulated, the following pathway was proposed for the l-sorbose metabolism in Ps. aeruginosa IFO 3898.

l-Sorbose → l-idose → l-idonic acid → 2-keto-l-gulonic acid.     

SILAR-10 C as a Liquid Phase for Gas-liquid Chromatography of Alditol Acetates

It is known that amino acid esters added in a protein hydrolysate are covalently incorporated during the plastein reaction. As its modification, a novel simplified process was developed which permitted incorporation of l-methionine directly into soy protein during treatment with papain. This process is characterized by its requirement for a very high substrate (protein) concentration and an alkaline pH condition. Racemic dl-methionine ethyl ester could be used to incorporate the l-isomer only. The amount of l-methionine incorporated varied depending on the amount of formulation of l-methionine ethyl ester; it was easily feasible to obtain final products with almost expected methionine contents. From an economic point of view, defatted soy flour was used as starting material, with a satisfactory result that the methionine level was enhanced in a large measure.     

Production of O-Acetyl-l-homoserme by Methionine Analogresistant Mutants and. Regulation of Homosenne-O-transacetylase in Gorynebacterium glutamicum

A large amount of O-acetyl-l-homoserine (OAH) was found to be produced by trifluo-romethionine-resistant mutants derived from Corynebacterium glutamicum ESLR–146 (Thr?,ethionine^R, selenomethionine^R) and ET_zR–606(Thr?,ethionine^R, 1,2,4-triazole^R) by mutational treatment with ethyl methanesulfonate. Some cultural conditions for OAH production were examined with one of the mutants, ESLFR-736, which was derived from ESLR–146. Addition of l-methionine or l-serine decreased OAH production. Optimal level of l-threo- nine, a growth factor in ESLFR–736, for OAH production was about 200 μg/ml, and further addition of excess l-threonine repressed OAH production. Corn steep liquor (CSL) and yeast extract added simultaneously enhanced OAH production to a great extent. Thus, the amount of OAH production reached to a level of 10.5 mg/ml with a medium containing 10% glucose and 0.01 % of both CSL and yeast extract after 2 days incubation.

Cell-free extract of C. glutamicum catalyzed the formation of OAH from acetyl CoA and l-homoserine, while a corresponding reaction with succinyl CoA was hardly detected. These observations indicate that OAH but not O-succinyl-l-homoserine is an intermediate of l-methionine biosynthesis in C. glutamicum.

The regulation of homoserine-O-transacetylase was examined in a methionine requiring mutant of C. glutamicum. The enzyme activity was not inhibited by l-methionine, S-adenosyl-methionine and S-adenosylhomocysteine, separately or in combination. The synthesis of homoserine-O-transacetylase was strongly repressed by l-methionine. The enzyme level in an OAH producer, ESLFR–736, increased to about 2-fold of that in ESLR–146, the parental strain.     

Studies on the Formation of the Cheese-like Flavor

Solution containing l-leucine and l-methionine cultured by Aspergillus flavus were found to develop cheese-like flavor.

α-Keto-isocaproic acid was isolated and identified from the culture of l-leucine and α-keto-β-methylmercaptobutyric acid from that of l-methionine. The flavor was also developed from the mixture of the synthetic sample of α-ketoisocaproic acid and α-keto-β-methylmercaptobutyric acid.     

Essential Role of Aspartokinase in L-Threonine Production by Escherichia coli W Mutants

We previously constructed an l-threonine-producing strain of E. coli W, KY8280, which is an Ile⁺ revertant of KY8279 which requires l-methionine, a,£-diaminopimelic acid and l-isoleucine [H. Kase et al., Agric. Biol. Chem., 35, 2089 (1971)]. From KY8280, another l-threonine-hyperproducing strain, KY8366, was obtained as an α-amino-β-hydroxy valeric acid (AHV, a threonine analog)-resistant mutant. Enzymatic analysis revealed that KY8280 constitutively expressed 8-fold higher l-threonine-sensitive aspartokinase I activity than KY8279. In addition, KY8366 constitutively expressed 13-fold higher l-lysine-sensitive aspartokinase III activity than KY8280. Such elevated levels of aspartokinases may contribute to the hyperproduction of l-threonine by these mutant strains. KY8366 produced 28 mg/ml of l-threonine in a culture medium fed with 12% glucose.     

Pyrolysis of Sulfur-containing Amino Acids

Sulfur-containing amino acids (l-cysteine, l-cystine and dl-methionine) were pyrolyzed. From pyrolyzed cysteine and cystine were identified 7~8 volatile compounds including 2-methylthiazolidine which is considered to be the product of the reaction of acetaldehyde with mercaptethylamine, and from pyrolyzed methionine were identified 11 volatiles. At the same time, besides these volatile compounds, alanine, cystine and isoleucine, and alanine, isoleucine and methionine were detected in the pyrolyzed products of cysteine and cystine, respectively, but no amino acid was detected from that of methionine. The mixture of seven identified volatiles generated from l-cystine developed a pop-corn like aroma with a roasted sesame like one, and methylmercaptane seemed to be the main contributor to the pickled radish like odor produced from pyrolysis of dl-methionine. Degradation schemes of cystine and methionine were proposed.     

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.     

Biological Activities of Sex Hormone Produced by Tremella mesenteriea

d-Aminoacylase was found to be produced not only by S. olivaceus 62–3 isolated from soil but also by three strains of type culture of Streptomyces species. All four of these strains produced d-aminoacylase intracellularly only when an inducer was added to the culture medium. d-Amino acids or N-acetyl-d-amino acids were effective as inducers.

As S. tuirus showed the highest d-aminoacylase activity, the enzyme extract of this strain was subjected to further investigation to determine the optimal conditions for optical resolution of N-acetyl-dl-phenylglycine. Almost all contaminating l-aminoacylase in the enzyme extract could be eliminated by DEAE-Sephadex adsorption. d-Phenylglycine of 99.9% optical purity was obtained after complete hydrolysis of d-isomer with the use of d-aminoacylase solution.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

A New Antibiotic K-52B

A new antibiotic K-52B, different from K-52A, was isolated from the culture broth of Streptoverticillium roseoverticillatum subsp. albosporum, strain No. K-52. The antibiotic K-52B was thought to be a similar saccharide to K-52A from its physicochemical properties but differed from K-52A in the presence of nitrogen content. Antibiotic K-52B inhibited the growth of Gram-positive and Gram-negative bacteria, including Pseudomonas aeruginosa on a chemically defined medium. The growth inhibition was, however, reversed by l-glutamine, l-glutamic acid, l-asparagine and l-aspartic acid.