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. Homoserine dehydrogenases of crude extracts from L-threonine producing AHV resistant mutants derived from FA–1–30 and FA–3–115 were insensitive to the inhibition by L-threonine, and those of L-threonine and L-lysine producing AHV resistant mutants from FA–1–30 were partially sensitive.

Correlation between L-threonine or L-lysine production and regulations of enzymatic activities of the mutants was discussed.     

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.     

Screening of l-Isoleucine Producers among Ethionine Resistant Mutants of l-Threonine Producing Bacteria

Two types of l-isoleucine producing mutants were derived from l-threonine producers by the supplement of the resistance to ethionine.

Main control site in l-isoleucine biosynthetic pathway after threonine is threonine dehydratase. In case of Brevibacterium flavum No. 14083, l-isoleucine production was based on the insensitiveness of this key enzyme to feedback inhibition by l-isoleucine. As regards Brevibacterium flavum No. 168, it was based on the increase in the specific activity of this enzyme.

The former produced 11.3 g/liter of l-isoleucine and the latter produced 9.92 g/liter from glucose. The former showed a vigorous ability of acetic acid assimilation, but the latter did not.     

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 the Isoleucine Fermentation

Accumulation of L-isoleucine and L-valine was studied on 14 genera, 47 species and 110 strains of aerobic bacteria using bacterial type cultures. A large amount of L-isoleucine and a small amount of L-valine accumulated when 1% of DL-α-aminobutyric acid was added to the culture medium. As a rule, facultative aerobes such as Aerobacter, Erwinia, Serratia and Bacillus showed good accumulation. In the absence of α-aminobutyric acid, powerful L-isoleucine accumulators produced a large amount of L-valine, although the accumulation of L- isoleucine was scarcely observed under that condition. In the presence of α-aminobutyric acid, the accumulation of L-valine was generally suppressed, but in several strains, on the contrary, the accumulation increased as well as that of L-isoleucine. When DL-threonine was used instead of α-aminobutyric acid, the amount of L-isoleucine accumulated was not as high as that with α-aminobutyric acid in almost all strains except Serratia marcescens. It was concluded that a distinct relationship between bacterial genera or species and accumulation of L-isoleucine did not exist, that is, powerful accumulators were limited to special strains, and that the addition of α-aminobutyric acid was necessary for the accumulation of a large amount of L-isoleucine.     

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.     

Conversion of the Extracellular Dextransucrase Isoenzymes from Leuconostoc mesenteroides NRRL B-1299

Effect of oxygen tension on l-lysine, l-threonine and l-isoleucine accumulation was investigated. Sufficient supply of oxygen to satisfy the cell’s oxygen demand was essential for the maximum production in each fermentation. The dissolved oxygen level must be controlled at greater than 0.01 atm in every fermentation, and the optimum redox potentials of culture media were above ?170 mV in l-lysine and l-threonine and above ?180 mV in l-isoleucine fermentations. The maximum concentrations of the products were 45.5 mg/ml for l-lysine, 10.3 mg/ml for l-threonine and 15.1 mg/ml for l-isoleucine. The degree of the inhibition due to oxygen limitation was slight in the fermentative production of l-lysine, l-threonine and l-isoleucine, whose biosynthesis is initiated with l-aspartic acid, in contrast to the accumulation of l-proline, l-glutamine and l-arginine, which is biosynthesized by way of l-glutamic acid.     

Mechanism of l-Threonine and l-Lysine Production by Analog-resistant Mutants of Corynebactemum glutamicum

Homoserine dehydrogenases and aspartokinases in l-threonine- or l-threonine and l-lysine-producing mutants derived from Corynebacterium glutamicum KY 9159 (Met^?) were studied with respect to the sensitivity to the inhibition by end products, l-threonine and l-lysine. The activities of homoserine dehydrogenases in the mutants which produced l-threonine or l-threonine and l-lysine were slightly less susceptible to the inhibition by l-threonine than the activity in the parent strain, KY 9159. The aspartokinases in the threonine-producing mutants, KY 10484 and KY 10230, which were resistant to α-amino-β-hydroxylvaleric acid (AHV, a threonine analog) and more sensitive to thialysine (a lysine analog) than the parent, were sensitive to the concerted feedback inhibition by l-lysine and l-threonine by about the same degree as KY 9159. The aspartokinase in an AHV- and thialysine-resistant mutant, KY 10440, which was derived from KY 10484 and produced about 14 mg/ml of l-threonine in a medium containing 10% glucose was less susceptible to the concerted feedback inhibition than KY 10484 or KY 9159, although the activity was still under the feedback control. In the parent strain, l-threonine activated aspartokinase activity in the absence of ammonium sulfate, an activator of the enzyme, but partially inhibited the activity in the presence of the salt. On the other hand, the enzyme of KY 10440 was activated by l-threonine either in the presence or in the absence of the salt. In another AHV- and thialysine-resistant mutant, KY 10251, which was derived from KY 10230 and produced both 9 mg/ml of l-threonine and 5/5 mg/ml of l-lysine, l-threonine and l-lysine simultaneously added hardly inhibited the activity of aspartokinase.

Implications of these results are discussed in relation to l-threonine or l-lysine production, AHV or thialysine resistance and regulation of l-threonine biosynthesis in these mutants.     

Effects of a Feedback-Resistant Aspartokinase III Gene on L-Isoleucine Production in Escherichia coli K-12

An L-isoleucine-overproducing recombinant strain of E. coli, TVD5, was also found to overproduce L-valine. The L-isoleucine productivity of TVD5 was markedly decreased by addition of L-lysine to the medium. Introduction of a gene encoding feedback-resistant aspartokinase III increased L-isoleucine productivity and decreased L-valine by-production. The resulting strain accumulated 12 g/l L-isoleucine from 40 g/l glucose, and suppression of L-isoleucine productivity by L-lysine was relieved.     

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.     

Stereoselective Incorporation of Isoleucine into Cypridina Luciferin in Cypridina hilgendorfii (Vargula hilgendorfii)

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four ²H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.     

Production of l-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met^?) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met^?, AHV^R, s-Lys^R) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose).

Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

L-Threonine Production by a Mutant of Arthrobactev paraffineus

An isoleucine leaky auxotroph of Arthrobacter paraffineus, which was isolated by Takayama et al.³⁾ as a mutant producing L-threonine and L-valine from n-paraffin, was subjected to further mutagenesis in an attempt to obtain better L-threonine producers. Some of the double auxotrophs derived from the isoleucine auxotroph and some of their revertants with respect to isoleucine requirement produced more L-threonine than the original isoleucine auxotroph. In contrast to the original isoleucine auxotroph, a revertant derived from a methionine plus isoleucine double auxotroph, KY7135, produced an increased amount of L-threonine and a decreased amount of L-valine. The optimum level of L-methionine for L-threonine production in KY7135 was much higher (1000 ~ 2000 μg/ml) with n-paraffin medium than with sorbitol or mannitol medium (10 ~ 50 μg/ml). L-Threonine production reached a maximum level (11.5 mg/ml) in 7 days incubation with the medium containing 10% n-paraffin (C₁₂ ~ C₁₄ rich). Several mutants which produce L-threonine more than 12 mg/ml were obtained from KY 7135 by monocolony isolation procedure.     

Mutant of Escherichia coli Tolerant to the Lysis Induced by Cephalexin

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes α,β-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5′-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the α,β-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D- and L-β-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.     

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.     

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.     

Effects of Acetylation with Acetic Anhydricle

1. L-Asparaginase (EC 3.5.1.1) from Escherichia coli A–l–3 was acetylated using acetic anhydride as a modifying chemical. The fully acetylated L-asparaginase retained 60% of the activity of the unmodified L-asparaginase.

2. The acetylated L-asparaginase hydrolyzed D-asparagine and L-glutamine as well as L-asparagine in the same ratio as the unmodified L-asparaginase did.

3. However, the effects of pH on the activity of the acetylated L-asparaginase showed very interesting differences from that of L-asparaginase. On the other hand, both L-asparaginase and the acetylated L-asparaginase exhibited similar pH activity curves on L-glutamine hydrolysis.

4. The acetylated L-asparaginase was found to become more stable against acid or heat in the presence of L-aspartate than in its absence in the same manner as L-asparaginase was.

     

Free Amino Acid Contents of Plasma and Urine,and Liver Enzyme Activities in Rats Fed High Glycine Diets

The contents of plasma free amino acids, the amounts of urinary excreted amino acids and urea, and the activities of liver serine dehydratase, glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase were determined in weanling rats fed ad libitum a 10% casein diet (control), a 10% casein diet containing 7% glycine and 10% casein diets containing 7% glycine supplemented with 1.4% L-arginine and/or 0.9% L-methionine for 14 days.

The remarkable increase of glycine and the moderate increase of serine in the plasma of animals fed excess glycine diets were observed. The amount of excreted glycine in the urine of animals fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than that of animals given the excess glycine diet. Urinary excreted urea of rats fed the excess glycine diet was a little greater and that of rats fed the excess glycine diet supplemented with L-arginine and L-methionine was much greater than the control. Liver serine dehydratase activity of animals given the excess glycine diets with or without L-arginine was higher than the control and the highest activity was observed in the liver of animals fed the excess glycine diet containing L-arginine and L-methionine. The activity of liver glutamic-oxalacetic transaminase of rats fed the excess glycine diet containing L-arginine and L-methionine was a little higher than that of rats given the other diets. Liver glutamic-pyruvic transaminase activity was a little higher in animals given the excess glycine diets with or without L-arginine and further higher in animals fed the excess glycine diet containing L-arginine and L-methionine than the control.     

An Improved Synthesis of DL-Canavanine

Excellent l-proline producers were screened for among sulfaguanidine resistant mutants derived from three typical l-glutamic acid-producing bacteria: Brevibacterium flavum, B. lactofermentum, and C. glutamicum.

The best strain, No. 199, is a sulfaguanidine resistant mutant derived from an isoleucine auxotroph of B. flavum 2247 by nitrosoguanidine. Strain No. 199 produced 35 mg/ml of l-proline after 72 hr of cultivation with 10% glucose as a carbon source. The strain also accumulated purine bases such as adenine, guanine, and hypoxanthine, i.e., degradation products of purine nucleotides. In the mutant, 1.6 ~ 2.0 fold more intracellular ATP was found than that in the parent strain; it is a substrate of glutamate kinase relating to l-proline biosynthesis.

On the contrary, the levels of intracellular glutamic acid, a substrate of glutamate kinase, were similar among these strains.

It was confirmed that the increment of internal ATP, which was important in the l-proline production mechanism, was very effective in the improvement of l-proline producers.