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.     

l-Threonine Production by Auxotrophs of E. coli

Methionine auxotrophs were derived by the treatment with ultraviolet ray or N-methylN′-nitro-N-nitrosoguanidine from five strains of Escherichia coli. One of the methionine auxotrophs of E. coli C-6, strain No. 15, produced maximum amount of l-threonine (4.3 mg/ml) with the medium containing 5 % cane-molasses (as sugars). Double auxotrophs were derived with further mutational treatment from strain No. 15. It was found that l-threonine production was greatly enhanced by cultivating methionine-valine auxotrophs in the presence of l-valine and methionine. o.ne of the methionine-valine auxotroph, strain No. 234, produced maximum amount of l-threonine (10.5 mg/ml) from cane-molasses.

The requirement of l-valine for the growth of the strain No. 234 was found to be leaky, and it was suggested that some enzymes relating to l-valine metabolism were mutationally altered to temperature-sensitive.     

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.     

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.     

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.     

Production of L-Isoleucine by AHV Resistant Mutants of Brevibacterium flavum

The growth of Brevibacterium flavum No. 2247A was inhibited by α-amino-β-hydroxy-valeric acid (AHV), and the inhibition was partially reversed by L-isoleucine.

AHV resistant strain ARI-129, which was isolated on a medium supplemented with 2 mg/ml of AHV, produced 11 g/liter of L-isoleucine.

No difference was observed in threonine dehydratase between No. 2247A and ARI–129. Homoserine dehydrogenase from ARI–129 was insensitive to the feedback inhibition by L-isoleucine and L-threonine.

O-Methyl-L-threonine resistant mutant, strain AORI–126, which was derived from ARI–129, produced 14.5 g/liter of L-isoleucine. Specific activity of threonine dehydratase from AORI–126 increased about two-fold higher than those from No. 2247A and ARI–129, whereas degree of inhibition of the enzyme by L-isoleucine was the same among three strains.

Among auxotrophic mutants derived from ARI–129, adenine and lysine auxotrophs produced more L-isoleucine than the parent did.

In the adenine auxotroph, L-isoleucine production was markedly reduced by the addition of excess adenine.     

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.     

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.     

L-Tyrosine Production by Analog-resistant Mutants Derived from a Phenylalanine Auxotroph of Corynebacterium glutamicum

Mutants resistant to various phenylalanine- or tyrosine-analogs were isolated from a phenylalanine auxotroph of Corynebacterium glutamicum KY 10233 by treatment with N- methyl-N′-nitro-N-nitrose guanidine (NTG) and screened for L-tyrosine production. A mutant, 98–Tx–71, which is resistant to 3-aminotyrosine, p-aminophenylalanine, p-fluoro-phenylalanine, and tyrosine hydroxamate was found to produce L-tyrosine at a concentration of 13.5 mg/ml in the cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine-sensitive mutant, pr–20 which was derived from 98–Tx–71 produced L-tyrosine at a concentration of 17.6 mg/ml. L-Tyrosine formation in the strain pr–20 was found to be still inhibited by L-phenylalanine though it was not inhibited by L-tyrosine. The L-tyrosine formation in the mutant was repressed neither by L-phenylalanine nor by L-tyrosine.     

Studies on l-Threonine Fermentation

Fifteen strains of bacteria were treated with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine to derive auxotrophic mutants, which were screened for their ability to produce l-threonine. A number of auxotrophs were derived from each strain. Among them, those which produced a large amount of l-threonine were found in Aerobacter aerogenes, Serratia marcescens and Escherichia coli, the members of the family Enterobacteriaceae. Nutritional requirements of these threonine producers were proved to be methionine, lysine, or α, ε-diaminopimelic acid (DAP).

In A. aerogenes and E. coli, double and triple auxotrophs were derived with futher mutational treatment. As a, rule, imposition of additional block led to the increase of l-threonine production. In E. coli, many triple auxotrophs (DAP^?, Met^?, He^?) and their isoleucine revertants were screened for their ability to produce l-threonine. Enhancement of l-threonine production was achieved with these mutants.

One of the isoleucine revertants, KY8280, was used to investigate some cultural conditions. As a result, l-threonine accumulation reached to a level of 13.8 mg/ml with the medium containing 7.5% fructose.     

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.     

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.     

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.     

Bacterial Degradation of N-Lauroyl-L-valine

Studies were conducted on the degradation of N-lauroyl-L-valine by type cultured bacteria. Many strains could utilize sodium N-lauroyl-L-valinate as carbon and nitrogen sources for their growth. Metabolism of N-lauroyl-L-valine was investigated in detail using Ps. aeruginosa AJ2116. Laurie acid was identified by gas chromatography suggesting cleavage of N-acyl linkage in N-lauroyl-L-valine.

Laurie acid might be metabolized to capric acid (C₁₀) and caprylic acid (C₈) becuase the accumulated substances gave nearly identical peaks with those of authentic fatty acids on gas chromatograms. The experiment using N-lauroyl-L-valine (¹⁴C) indicated that ¹⁴CO₂ was produced as a final product. Valine was not detected because it might be metabolized very rapidly immediately after its release.

It was supposed that the enzymes or enzyme systems degrading N-lauroyl-L-valine might be constitutive from the experiment using two kinds of cells grown in the medium containing N-lauroyl-L-valine or nutrient broth.     

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.     

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.     

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.     

Fermentative Production of l-Proline with Auxotrophs of Corynebacterium glutamicum

Two auxotrophic mutants of Corynebacterium glutamicum were found to produce a large amount of l-proline in the culture medium. High concentration of MgSO₄ or MnSO₄ in the medium stimulated the l-proline production by an isoleucine auxotroph. Optimum concentration of l-isoleucine was 200 μg/ml, and the higher concentration of l-isoleucine reduced the l-proline production. The auxotroph produced 14.8 mg/ml of l-proline when cultured in a medium containing 12% glucose, 1.7% NH₄C1,0.6% MgSO₄·7H₂O, 0.06% MnSO₄·4H₂O and 200 μg/ml of l-isoleucine. The other mutant, whose growth responds to the bases of nucleic acids, produced 7 to 13 mg/ml of l-proline in a cane molasses (15%, as glucose concentration)-medium containing 2% of the acid-hydrolyzate of soybean meal. The l-proline production by this mutant increased to a level of 27 to 31 mg/ml when the growth was suppressed by the addition of 4% NH₄C1 to the medium, or by the addition of 2 mg/ml of polyoxyethylenestearylamine, a surfactant, to a culture at an appropriate stage of the fermentation.     

Fermentative Production of Nδ-Acetyl-L-ornithine with an Arginine and Pyrimidine Auxotroph of Paracolobactrum coliforme

In the course of selecting a useful mutant strain for a fermentative production of L-valine, it was found that an arginine-pyrimidine auxotroph of Paracolobactrum coliforme accumulated N^δ-acetyl-L-ornithine (δ-AO) in the culture medium. The accumulation of it reached a level of 16 mg/ml with medium containing 12.5 % glucose, 2.2% (NH₄)₂SO₄, 0.5% peptone and 300 μg/ml of uracil. The wild strain 775 also accumulated 1.4 mg/ml of δ-AO in the medium supplemented with a high level (300μg/ml) of uracil when L-ornithine (10 mg/ml) was added in the middle phase of fermentation. The mutant cells elongated under the condition with limited supply of uracil.

The mechanism of the accumulation of δ-AO was discussed from the information of relevant biosynthetic regulation in other organisms.     

New Resolving Agents

Seven optical active 2-benzylamino alcohols were synthesized by reduction of N-benzoyl derivatives of L-alanine, L-valine, L-leucine, L-phenylalanine, L-aspartic acid, L-glutamic acid and L-lysine and applied for the resolution of (±)-trans-chrysanthemic acid. d-trans-Chrys-anthemic acid was obtained by resolution via the salts of 2-benzylamino alcohols derived from L-valine and L-leucine, while (?)-trans-chrysanthemic acid was prepared through the salts of the amino alcohols derived from L-alanine and L-phenylalanine.