Properties of Revertants Appearing in l-Leucine Fermentation Culture Broth

The l-leucine productivity of an l-leucine producing strain, H-1204, of Corynebacterium glutamicum substantially decreased during a large-scale culture or repetitive subculturing. This instability was found to be due to the appearance of revertants with lower or no l-leucine productivity. Strains in the culture broth could be roughly classified into three types on the basis of their phenotypes: l-type, original l-leucine producing strain, Val^L Leu⁺ (valine leaky); M-type, Val⁺ Leu⁺ (prototroph); V-type, Val⁺ Leu^- (leucine auxotroph). The appearance of these revertants was determined to be caused by the distribution imbalance of α-ketoisovaleric acid, the common precursor for l-leucine and l-valine biosynthesis.     

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived from Brevibacterium lactofermentum 2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256.

In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered.

The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.     

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.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Cultural Conditions for l-Leucine Production by Strain No. 218, a Mutant of Brevibacterium lactofermentum 2256

The excellent l-leucine producing mutant No. 218, derived from a biotin requiring glutamic acid producing strain, is methionine and isoleucine auxotrophic. A suboptimum growth condition made by adding a limiting amount of isoleucine was necessary for the maximum production of l-leucine. On the other hand, methionine was indifferent to the productivity if sufficiently supplied for growth.

Biotin of more than 50 μg/liter caused the accumulation of l-leucine; less than 50 μg/liter, however, gave a drastic change in accumulation pattern from l-leucine to l-glutamic acid. Strain No. 218 produced 28 mg/ml of l-leucine after 72 hr cultivation when 13 % glucose was supplied as a carbon source, thus giving the yield of 21.6%.

Effects on l-leucine production of concentrations of inorganic salts, pH, temperature and aeration were also investigated.     

Fermentative Production of l-Leucine with Auxotrophic Mutants of Corynebaeterium glutamicum

An auxotrophic mutant of Corynebaeterium glutamicum was found to accumulate a large amount of l-leucine in the culture medium. The nutritional requirement of the mutant is rather complex but it’s growth was most remarkably stimulated by l-phenylalanine. Acetate (1.5~3.0%) or pyruvate (3%) stimulated the l-leucine production. By a further mutagenic treatment, 329 mutants earring some defect in addition to phenylalanine auxotrophy were derived from the mutant No. 190. Among them, a histidine auxotrophic derivative produced twice as much l-leucine as the parent strain, i.e., the level of l-leucine production by this derivative reached 16 mg/ml in a medium containing 12% glucose, 1 % (NH₄)₂SO₄ and 2.5% CH₃COONH₄ as carbon and nitrogen sources. Some other auxotrophic markers such as isoleucine- (or threonine-), threonine-, purine(s)-, homoserine-, or methionine- auxotrophy also improved the L-leucine production by No, 190.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Production of l-Valine by 2-Thiazolealanine Resistant Mutants Derived from Glutamic Acid Producing Bacteria

Potent l-valine producers were screened among 2-thiazolealanine resistant mutants derived from three typical l-glutamic acid producing bacteria: Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Arthrobacter citreus. By strain No. 487, the best producer derived from Brevibacterium, 31 mg/ml of l-valine was produced after 72 hr when 10% glucose was supplied as a carbon source, thus giving the yield of 31% from glucose. Accumulation of the other amino acids was negligible. The addition of l-isoleucine and l-leucine in the culture medium did not reduce the l-valine production, indicating that the l-valine biosynthesis is insensitive to these end products in the l-valine producer.     

L-Tyrosine production by Polyauxotrophic Mutants of Corynebacterium glutamicum

Polyauxotrophic mutants of Corynebacterium glutamicum which have additional requirements to L-phenylalanine were derived from L-tyrosine producing strains of phenylalanine auxotrophs, C. glutamicum KY 9189 and C. glutamicum KY 10233, and screened for L-tyrosine production. The increase of L-tyrosine production was noted in many auxotrophic mutants derived from both strains. Especially some double auxotrophs which require phenylalanine and purine, phenylalanine and histidine, or phenylalanine and cysteine produced significantly higher amounts of L-tyrosine compared to the parents, A phenylalanine and purine double auxotrophic strain LM–96 produced L-tyrosine at a concentration of 15.1 mg per ml in the medium containing 20% sucrose. L-Tyrosine production by the strain decreased at high concentrations of L-phenylalanine.     

Metabolic regulatory mechanisms and physiological roles of functional amino acids and their applications in yeast

ABSTRACT

In yeast, amino acid metabolism and its regulatory mechanisms vary under different growth environments by regulating anabolic and catabolic processes, including uptake and export, and the metabolic styles form a complicated but robust network. There is also crosstalk with various metabolic pathways, products and signal molecules. The elucidation of metabolic regulatory mechanisms and physiological roles is important fundamental research for understanding life phenomenon. In terms of industrial application, the control of amino acid composition and content is expected to contribute to an improvement in productivity, and to add to the value of fermented foods, alcoholic beverages, bioethanol, and other valuable compounds (proteins and amino acids, etc.). This review article mainly describes our research in constructing yeast strains with high functionality, focused on the metabolic regulatory mechanisms and physiological roles of “functional amino acids”, such as l-proline, l-arginine, l-leucine, l-valine, l-cysteine, and l-methionine, found in yeast.     

Synthesis of (±)-Methyl 5-(1′,2′-Epoxy-2′,6′-dimethylcyclohexyl)-3-methyl-(2Z,4E)-2,4-pentadienoate

The effect of tripropyltin chloride (TPT) on transport systems in E. coli was investigated. The inhibition on uptakes of ¹⁴C-l-leucine, l-proline, adenine and methyl-(α-d-gluco)pyrano-side (α-methylglucoside) by TPT was examined. The active uptake of l-leucine which utilized ATP molecule as an energy source was 100% inhibited at the concentration of 10 µg/ml TPT. On the other hand, the uptake of l-proline which was generated by an “energied” membrane state of the cells was inhibited only 40% at the same concentration of TPT. α-Methylglucoside uptake was scarcely inhibited. Adenine uptake was intensely inhibited at 20 µg/ml TPT. The effect of the delayed addition of TPT on transport systems was also examined. l-Leucine incorporated into cells was completely released from cells by TPT. Leucine binding protein (LBP) was prepared from E. coli cells and the effect of TPT on LBP activity was examined. TPT scarcely inhibited LBP activity.     

2-Keto-l-gulonic Acid Fermentation

A number of bacterial strains from type culture collections and natural sources were examined in their metabolic characteristics toward sorbitol and l-sorbose.

Paper chromatographic analyses of sorbitol and l-sorbose metabolites obtained from the cultures of various bacteria revealed that the organisms producing 2-keto-l-gulonic acid from sorbitol were merely found in the genera Acetobacter, Gluconobacter and Pseudomonas, whereas those producing the acid from l-sorbose were distributed in the twelve genera of bacteria: Acetobacter, Alcaligenes, Aerobacter, Azotobacter, Bacillus, Escherichia, Gluconobacter, Klebsiella, Micrococcus, Pseudomonas, Serratia and Xanthomonas.

G. melanogenus, which was characterized by excellent production of 2-keto-l-gulonic acid from sorbitol, also formed several other sugars and sugar acids as the sorbitol metabolites. These compounds were identified to be d-fructose, l-sorbose, d-mannonic acid, L-idonic acid, 2-keto-d-gluconic acid and 5-keto-d-mannonic acid, respectively, by means of two-dimensional paper chromatography.

Bacteria producing 2-keto-l-gulonic acid from sorbitol were usually isolated from fruits but not from soil.     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

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.     

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.     

Microbial Production of l-Threonine

The growth of Brevibacterium flavum No. 2247 was inhibited over 90% at a concentration above 1 mg/ml of α-amino-β-hydroxyvaleric acid, a threonine analogue, and the inhibition was reversed by the addition of l-threonine, and to lesser extent by l-leucine, l-isoleucine, l-valine and l-homoserine. l-Methionine stimulated the inhibition. Several mutants resistant to the analogue produced l-threonine in the growing cultures. The percentage of l-threonine producer in the resistant mutants depended on the concentration of the analogue, to which they were resistant. The best producer, strain B-183, was isolated from resistant strains selected on a medium containing 5 mg/ml of the analogue. Mutants resistant to 8 mg/ml of the analogue was derived from strain B-183 by the treatment with mutagen, N-methyl-N’-nitro-N-nitrosoguanidine. Among the mutants obtained, strain BB-82 produced 13.5 g/liter of l-threonine, 30% more than did the parental strain. Among the resistant mutants obtained from Corynebacterium acetoacidophilum No. 410, strain C-553 produced 6.1 g/liter of l-threonine. Several amino acids other than l-threonine were also accumulated, and these accumulations of amino acids were discussed from the view of regulation mechanism of l-threonine biosynthesis.     

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.     

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.     

l-Isoleucine Production by Analog-resistant Mutants Derived from Threonine-producing Strain of Corynebacterium glutamicum

A thiaisoleucine-resistant mutant, ASAT–372, derived from a threonine producer of Corynebacterium glutamicum, KY 10501, produced 5 mg/ml each of l-isoleucine and l-threonine. l-Isoleucine productivity of ASAT–372 was improved stepwise, with concurrent decrease in threonine production, by successively endowing it with resistivity to such substances as ethionine, 4-azaleucine and α-aminobutyric acid. The mutant strain finally selected, RAM–83, produced 9.7 mg/ml of l-isoleucine with a medium containing 10% (as sugar) molasses.

l-Isoleucine production was significantly affected by the concentration of ammonium sulfate in the fermentation medium. At 4% ammonium sulfate l-isoleucine production was enhanced whereas l-threonine production was suppressed. At 2% ammonium sulfate l-threonine production was stimulated while l-isoleucine production decreased.