Isamu Shiio

Microbial Production of l-Threonine: Part II. Production by α-Amino-β-hydroxyvaleric Acid Resistant Mutants of Glutamate Producing Bacteria

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 acetoaci...

Microbial Production of Long-chain Dicarboxylic Acids from n -Alkanes:Part I. Screening and Properties of Microorganisms Producing Dicarboxylic Acids

Microorganisms which produced n-alkane ω,ω′-dicarboxylic acid (DC) from n-alkane were selected from natural sources. It was found that the best three producers thus obtained belonged to yeast. All of the stock cultures which are able to assimilate n-alkane and are belonged to genus Candida and Pichia were also found to produce DC from n-alkane.Candida cloacae 310, a representative strain selected from natural source, was able to produce DCs having 5 to 16 carbon atoms from various n-alkanes. Among them, DCs with 5 to 9 carbon atoms were more heavily accumulated than those with more than 9, except those with the same number of carbon atoms as the substrates which were the main products from the substrates with less than 15 carbon atoms. It was also clearly demonstrated that DCs with odd carbons alone were produced from n-alkanes with odd carbons, while DCs with even carbons alone from n-alkanes with even carbons.Then, cultural conditions of Candida cloacae 310 were studied for the production of DC-12 from ...

Isolation and Properties of α-Ketobutyrate-resistant Lysine-producing Mutants from Brevibacterium flavum.

The growth of Brevibacterium flavum FA1-30, a L-lysine-producing mutant strain with an aspartokinase desensitized to feedback inhibition, was almost completely inhibited by a-ketobutyrate (αKB) at concentrations more than 4 mg/ml. Aspartic acid hydroxamate (Asphx) did not inhibit the growth at a concentration up to 6 mg/ml, but slightly stimulated the αKB inhibition. Mutants resistant to αKB in the presence and absence of Asphx (type-I and -II, respectively) were derived and further selceted by their lysine productivity. The best producers among the type-I and -II mutants produced 41.9 and 29.4 g/liter of L-lysine·HCl, 1.9- and 1.4-fold that by the parent, respectively. The type-II mutant was confirmed to be resistant to αKB alone, but was still sensitive to aKB in the presence of Asphx, similar to the parent. The type-I mutants were resistant to αKB in the presence and absence of Asphx. Pyruvate dehydrogenase (PD) activity or both PD and citrate synthase (CS) activities significantly decreased in the type-II or -I mutants, respectively. The apparent Km of PD with respect to pyruvate and that of CS with respect to oxaloacetate increased 1.7-fold higher than those of the parent, respectively.

Microbial Production of l-Threonine: Part I. Production by Escherichia coli Mutant Resistant to α-Amino-β-hydroxyvaleric Acid

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 f...

Multiple Interaction of Fructose 1,6-Bisphosphate and Other Effectors on Phosphoenolpyruvate Carboxylase from Brevibacterium flavum and Its Aspartate-producing Mutant

Phosphoenolpyruvate (PEP) carboxylase purified from Brevibacterium flavum was specifically activated by fructose 1,6-bisphosphate (FBP). The other intermediates of sugar metabolism or their structural analogues did not influence the activity. FBP decreased the apparent Km for PEP but did not affect that for another substrate, bicarbonate, or the apparent maximum velocity for PEP. The dissociation constants for FBP from enzyme-FBP and enzyme-PEP-FBP complex were 63 and 32 μm, respectively, being almost equivalent to those for acetyl-CoA. Synergistic activation by FBP and acetyl-CoA was not observed with the B. flavum enzyme, unlike the Escherichia coli enzyme. FBP, like acetyl-CoA, was kinetically competitive with aspartate. With respect to another feedback inhibitor, 2-oxoglutarate, acetyl-CoA was non-competitive, whereas FBP was of mixed-type, i.e., FBP but not acetyl-CoA prevented 2-oxoglutarate from binding to the enzyme to a certain extent. Homotropic cooperativity was observed only with FBP but not w...

Production of L-Tryptophan by Sulfonamide-resistant Mutants

Sulfaguanidine-resistant mutant S-225, derived from a tryptophan-producing 5-fluoro-tryptophan-resistant mutant of Brevibacterium flavum, accumulated 19 g/liter of L-tryptophan at maximum when cultured for 72 hr in a medium containing 13% glucose as carbon source, 1.8-fold higher than did the parent. Strain S-225 accumulated 17 g/liter of L-tryptophan in a medium containing 10% sucrose as carbon source (17% yield based on the sugar). It also accumulated 450 mg/liter of chorismate, an intermediate common to the biosyntheses of tryptophan and p-aminobenzoate. The accumulation was 1.7-fold higher than that by the parent, suggesting that the intracellular concentration of chorismate was increased through acquisition of the sulfaguanidine resistance. Sulfaguanidine-resistant mutants were also derived from a tryptophan-producing mutant of Corynebacterium glutamicum. The mutants showed 2.2-fold higher maximum tryptophan production than did the parent.

O-Acetylhomoserine sulfhydrylase from Brevibacterium flavum

Publisher Summary In Brevibacterium flavum, L-methionine is synthesized from L-homoserine via O-acetylhomoserine and L-homocysteine. The formation of L-homocysteine from O-acetyl-L-homoserine is catalyzed by O-acetylhomoserine sulfhydrylase, in contrast to enteric bacteria and Neurospora, in which L-homocysteine is synthesized through cystathionine formation. . The assay procedure, which uses the nitroprusside reaction to determine homocysteine in the presence of sulfide, is a slight modification of that for O-acetylserine sulfhydrylase described by Kredich and Becker. O-Acetyl-e-homoserine and sulfide are incubated with bacterial extract. The homocysteine formed is converted to a stable S-nitrosothiol derivative by adding nitrous acid, which also stops the reaction. Excess nitrous acid is removed by the addition of ammonium sulfamate, and then mercuric chloride, sulfanilamide, and N-l-naphthylethylenediamine are added. The S-nitrosothiol derivative of homocysteine decomposes in the presence of mercuric ion to give nitrous acid which diazotizes the sulfanilamide. The diazotized sufanilamide couples with naphthylethylenediamine to give the chromophore, an azo dye.

Production of L-Threonine by Mutants Resistant to Both α-Amino-β-hydroxyvaleric Acid and S-(2-Aminoethyl)-L-cysteine Derived from Brevibacterium flavum

Growth of Brevibacterium flavum FA-1-30 and FA-3-115, L-lysine producers derived from Br. flavum No. 2247 as S-(2-aminoethyl)-L-cysteine (AEC) resistant mutants, was inhibited by α-amino-β-hydroxyvaleric acid (AHV), and this inhibition was reversed by L-threonine. All the tested AHV resistant mutants derived from FA-1-30 accumulated more than 4 g/liter of L-threonine in media containing 10% glucose, and the best producer, FAB-44, selected on a medium containing 5 mg/ml of AHV produced about 15 g/liter of L-threonine. Many of AHV resistant mutants selected on a medium containing 2 mg/ml of AHV accumulated L-lysine as well as L-threonine, AHV resistant mutants derived from FA-3-115 produced 10.7 g/liter of L-threonine maximally. AEC resistant mutants derived from strains BB–82 and BB–69, which were L-threonine producers derived from Br. flavum No. 2247 as AHV resistant mutants, did not produce L-threonine more than the parental strains, and moreover, many of them did not accumulate L-threonine but L-lysine....