Amino Acid Metabolism in Microorganisms

Certain Streptomyces strains were found to accumulate an unknown substance in culture broth when the microorganisms were grown in the medium containing dl-methionine. The substance was isolated from the culture broth as hydrochloride and was identified as 3-methylthiopropylamine (MTPA), decarboxylated product of methionine, from its melting point, chemical composition, infrared spectrum, and other properties. Cultural conditions for MTPA formation in Streptomyces sp. K 37 were investigated. The yield of MTPA from l-methionine reached about 90% with a culture medium containing corn steep liquor. Namely, 6.47 mg of MTPA per millilitre of culture broth was produced from 10 mg of l-methionine per millilitre of the growth medium. The transforming activity was found in the cells of the early culture period. MTPA-producing activity was induced by l- methionine in the medium. d-Methionine was not utilized as a substrate of the reaction with intact cells. Optimum pH for the reaction appeared to be 6.0~8.0.     

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.     

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.     

Studies on d-Glucose Isomerizing Activity of d-Xylose Grown Cells from Bacillus coagulans,Strain HN-68

A thermophilic spore-forming strain HN-68, only d-xylose grown cells of which have an activity of d-glucose isomerization, was isolated from soil, and identified to be similar to Bacillus coagulans Hammer. The conditions necessary for maximal production of the glucose isomerizing activity by the cells from shaken cultures in d-xylose media were studied. Much higher activities were observed with the cells grown from 14 ~ 16 hours at 40°C on d-xylose medium containing yeast extract, ammonium chloride, manganese sulfate and calcium carbonate. d-Glucose isomerizing activity was also developed inductively by exposing the washed cells grown on d-glucose to d-xylose within one hour. With the use of living cells as an enzyme source, the addition of both cobaltous ion and toluene in reaction system remarkably enhanced the reaction rate of d-glucose isomerization.     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

Purification,Crystallization and Properties of Tyrosine Phenol Lyase from Erwinia herbicola

Crystalline tyrosine phenol lyase was prepared from the cell extract of Erwinia herbicola grown in a medium supplemented with l-tyrosine. The crystalline enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 259,000. The crystalline enzyme catalyzed the conversion of l-tyrosine into phenol, pyruvate and ammonia, in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from d-tyrosine, S-methyl-l-cysteine, 3, 4-dihydroxyphenyl-l-alanine, l- and d-serine, and l- and d-cysteine, but at lower rates than from l-tyrosine. l-Phenyl-alanine, l-alanine, phenol and pyrocatechol inhibited pyruvate formation from l-tyrosine.

Crystalline tyrosine phenol lyase from Erwinia herbicola is inactive in the absence of added pyridoxal phosphate. Binding of pyridoxal phosphate to the apoenzyme is accompanied by pronounced increase in absorbance at 340 and 425 mμ. The amount of pyridoxal phosphate bound to the apoenzyme was determined by equilibrium dialysis to be 2 moles per mole of enzyme. Addition of the substrate, l-tyrosine, or the competitive inhibitors, l-alanine and l-phenyl-alanine, to the holoenzyme causes appearance of a new absorption peak near 500 mμ which disappears as the substrate is decomposed but remains unchanged in the presence of the inhibitor.     

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.     

Fermentative Production of l-Arginine

Most of the bacteria, which were examined for the sensitivity to l-arginine analogs (l-canavanine, l-homoarginine, d-arginine and arginine hydroxamate), were insensitive to the analogs at a concentration of 8 mg/ml. Corynebacterium glutamicum DSS-8 isolated as d-serine-sensitive mutant from an isoleucine auxotroph KY 10150, was found to be sensitive to d-arginine and arginine hydroxamate. Furthermore, DSS-8 produced l-arginine in a cultural medium. l-Arginine analog-resistant mutants were derived from DSS-8 by N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment. Most of them were found to produce a large amount of l-arginine. An isoleucine revertant from one of these mutants produced 19.6 mg/ml of l-arginine in the medium containing 15% (as sugar) of molasses.

The mechanism of the sensitivity to l-arginine analogs and that of the production of l-arginine in the d-serine-sensitive mutant, DSS-8, were investigated. DSS-8 seems to be a mutant having increased permeability to d- and l-arginine.     

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.     

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.     

Purification,Crystallization and Properties of Tryptophanase from Proteus rettgeri

An inducible tryptophanase was crystallized from the cell extract of Proteus rettgeri grown in a medium containing l-tryptophan. The purification procedure included ammonium sulfate fractionation, heat treatment, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystals were obtained from solutions of the purified enzyme by the addition of ammonium sulfate.

The crystalline enzyme preparation was homogeneous by the criteria of ultracentrifugation and zone electrophoresis. The molecular weight was determined to be approximately 210,000.

The crystalline enzyme catalyzed the degradation of l-tryptophan into indole, pyruvate and ammonia in the presence of added pyridoxal phosphate. The enzyme also catalyzed pyruvate formation from 5-hydroxy-l-tryptophan, 5-methyl-l-tryptophan, S-methyl-l-cysteine and l- cysteine. l-, d-Alanine, l-phenylalanine and indole inhibited pyruvate formation from these substrates.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

Effects of Glycine and d-Amino Acids on Growth of Various Microorganisms

Growth of various microorganisms in media containing high concentrations of glycine or d-amino acids was examined. Susceptibilities to glycine or d-amino acids differed among microorganisms, and the differences in susceptibility have no direct relation with Gram staining, morphological forms, and aerobic or anaerobic nature of the organisms. Certain glycine-resistant bacteria tested, which included Bacillus cereus, Staphylococcus aureus and Serratia marcescens, exhibited relatively high oxidative activities towards glycine. The inhibition of the growth of Escherichia coli by either glycine or d-amino acids, which included d-threonine, d-alanine and d-lysine, was reversed by l-alanine, partialy by l-serine, and not by l-lysine or l-threonine. These results suggest that the growth inhibition of microorganisms by d-amino acids was similar to that by glycine. The incorporation of l-alanine into E. coli cells which were preincubated with glycine was less than those of preincubated without glycine. Particularly, the incorporation into the cell wall fraction was most susceptible to glycine. An additive effect of penicillin and glycine was observed in the inhibition of cell wall biosynthesis as determined by the intracellular accumulation of N-acetylamino sugar compounds.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

S-Adenosyl- l-methionine Production by Saccharomyces sake:. Optimization of the Culture Conditions for the Production of Cells with a High S-Adenosyl-l-methionine Content

In studies on the production of S-adenosyI- l-methionine (AdoMet) by Saccharomyces sake Kyokai No. 6 in a bench-scale fermentor, the rate of growth of the organism in a medium containing sucrose and urea as carbon and nitrogen sources, respectively, increased with a higher agitation speed (500 rpm) or sucrose feeding, but the cellular content of AdoMet was lower than that with a lower agitation speed (300 rpm) or ethanol feeding. The additin of l-methionine was necessary for enhancement of both growth and AdoMet production. The l-methionine added (15g/l) was efficiently incorporated into the cells during the course of the cultivation, about 20 % of the l-methionine being found as AdoMet in the cells. The ultraviolet absorbance (258 nm) based on the extracted AdoMet comprised 73 % of the total ultraviolet absorbance of material extracted from the cells, whereas that based on S-adenosylhomocysteine was very low (1.4%).     

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.     

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.     

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.