l-Glutamic Acid Fermentation with Molasses

An l-glutamic acid (l-GA)-forming bacterium. Microbacterium ammoniaphium was cultured in the molasses medium with or without poiyoxyethylene fatty acid esters to obtain l-GA-accumulating cells or non-accumulating cells, respectively.

Then protoplast-like bodies (PLB) were prepared from each group of cells by reacting them with egg white lysozyme.

l-GA-accumulating reaction by the PLB was carried out under high and low osmotic pressures.

From the results of the experiment, it was shown that the difference in the ability of l-GA accumulation between l-GA-accumulating cells and non-accumulating cells was attributed mainly to the difference in the nature of the cell membrane.

Further, the relationship between the molar ratio of saturated fatty acids/unsaturated fatty acids which was reported previously and the nature of the membrane was discussed.

The lipid composition of the cell membrane from Microbacterium ammoniaphilum was determined by thin-layer and column chromatographies to make clear the relation between the extracellular accumulation of l-glutamic acid and the lipid in the cell membrane. When polyoxyethylene fatty acid ester was added to the beet medium and a large amount of l-glutamic acid was accumulated, the increase of the saturated fatty acid (C₁₆, C₁₈) in the neutural lipid fraction and the decreases of the phospholipid fraction and the unsaturated fatty acid $({\text{C}}_{18}^{1=})$ in the neutral lipid fraction were recognized.     

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.     

Studies on l-Glutamic Acid Fermentation

Micrococcus glutamicus, a glutamate-produeing bacterium, is known to have strong activity of l-glutamic acid dehydrogenase which requires NADP as co-enzyme. In this paper, the NADP-speeifie l-glutamic acid dehydrogenase was purified from M. glutamicus by means of heat treatment with sodium sulfate, precipitation with acetic acid and diethyl-amino-ethyl (DEAE) cellulose column chromatography. The activity of the purified enzyme preparation reached 200-fold as high as that of the crude extract. Some properties of the purified enzyme were investigated. As a result, it was found that the highly purified enzyme preparation acted not only on l-glutamic acid (l-GA) but also on α, ε-diaminopimelic acid (α, ε-DAP) in the presence of NADP. Some of the probable consideration for the dehydrogenation of l-GA and α, ε-DAP are noted.     

Induction of Antibiotic Formation in Streptomyces sp. No. 362 by the Change of Cellular Fatty Acid Spectrum

Microorganisms which require oleic acid for the formation of antibiotics were screened. Streptomyces sp. No. 362, one of the selected organisms, produced antimicrobial substances only when oleic acid, palmitic acid or the high concentration of l-glutamic acid (or l-glutamine) was supplemented to the medium. The cellular fatty acid composition was changed by the supplement of these fatty acids, but not by l-glutamic acid (or l-glutamine). Antibiotic-producing cells had about 4 to 10 times larger amino acid pools, especially l-glutamic acid pool, and hexosamine pools. The ability for l-glutamate uptake of cells grown in the oleic or palmitic acid supplemented medium was markedly enhanced and the efflux of the accumulated l-glutamate was reduced. The antibiotic produced by this strain was identified as one of the streptothricin-group antibiotics and the role of these additives in the antibiotic formation is discussed.     

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.     

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.     

2-Keto-l-gulonic Acid Fermentation

l-Sorbose metabolism in Pseudomonas aeruginosa IFO 3898 was studied. When the strain was cultivated in l-sorbose medium, l-idonic and 2-keto-l-gulonic acids were detected in the culture broth.

From the results on the metabolism of various sugars and sugar acids with the cell suspension and the metabolites accumulated, the following pathway was proposed for the l-sorbose metabolism in Ps. aeruginosa IFO 3898.

l-Sorbose → l-idose → l-idonic acid → 2-keto-l-gulonic acid.     

l-Glutamic Acid Fermentation with Molasses

Conditions suitable for the cell wall lysis of a l-glutamate-producing bacterium, Microbacterium ammoniaphilum, by egg white lysozyme were studied, in order to make clear the correlation of the fatty acid composition of the cellular fractions and the extracellular accumulation of l-glutamate,

The cell wall of a phage-resistant strain was recognized to be almost completely lyzed by the lysozyme.

Using this result, the relationship between the fatty acid composition of each fraction and extracellular accumulation of l-glutamate was investigated, and the following thesis was proposed: The extracellular accumulation of l-glutamate in large quantity took place when the molar ratio of saturated/unsaturated fatty acid in the cell membrane fraction was above 1.     

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.     

Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

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.     

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.     

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.     

Studies on the Synthesis of l-Amino Acids

l-Homoserine was prepared by the reduction of l-aspartic acid β-methyl ester with sodium borohydride in water solution without any racemization. The yield of l-homoserine was about 25% of the theoretical amount, and no product other than l-homoserine, l-aspartic acid and l-aspartic acid β-methyl ester was present in the reaction mixture. The low yield of l-homoserine was ascribed to the hydrolysis of the ester.

l-Azetidine-2-carboxylic acid could not be detected in the reaction mixture. In contrast with the reduction of l-glutamic acid γ-esters, the reduction of l-aspartic acid β-ester was not accompanied by the cyclization.     

Forced evolution of Escherichia coli cells with the ability to effectively utilize non-natural amino acids l-tert-leucine,l-norleucine and γ-methyl-l-leucine

Abstract

In an attempt to develop a novel biocatalyst able to efficiently catalyse the synthesis of non-natural amino acids, Escherichia coli TG1 was treated with 10 mM NaNO₂ and then cultured in selective medium supplemented with 20 mM l-tert-leucine. Each culture was grown for 2 weeks and then subcultured into fresh medium with successive decreases of l-tert-leucine concentration at each transfer to a final value of 0.5 mM. The adapted cells resulting from this forced evolution procedure were able to grow in minimal medium with 0.1 mM l-tert-leucine as sole nitrogen source. Both HPLC and TLC verified progressive removal of l-tert-leucine from the medium during bacterial growth. Further studies revealed that the adapted cells metabolized l-tert-leucine by transamination, removing the amino group but leaving the carbon skeleton of the corresponding 2-oxoacid intact. Despite the mutagenesis, when the four obvious candidate amino acid aminotransferase genes were cloned and sequenced, there was no change in these structural genes. The activity of the adapted cells with l-tert-leucine is apparently attributable to the wild-type branched-chain amino acid aminotransferase (IlvAT), presumably expressed at higher levels as a result of a regulatory mutation. With the isolate I-4, the resting cells transaminate l-tert-leucine, l-norleucine, l-norvaline, γ-methyl-l-leucine and dl-homophenylalanine as effectively as does the crude extract. These evolved cells may be useful for synthesizing non-natural amino acids for the pharmaceutical industry. In addition, the adapted cells can also catalyse transamination of naturally occurring hydrophobic amino acids.     

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.     

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.     

Biochemical Effects of Fatty Acid and its Derivatives on l-Glutamic Acid Fermentation

It has been found that although Brevibacterium lactofermentum No. 2256 is incapable of accumulating l-glutamic acid in a biotin sufficient medium, it produces a large quantity of the acid in the presence of sucrose fatty acid ester. In a biotin deficient medium, however, the ester brought the unfavorable diminution of l-glutamic acid accumulation caused by the decrease of glucose consumption in an incubation period. The undesirable effects were practically lost when the ester was added to the culture medium after more than eight hours in the course of incubation. This fact suggests that the ester is concerned with the growth of microorganism. It is very interesting to elucidate the interrelation between sucrose fatty acid ester and biotin. For the maximum accumulation of l-glutamic acid corresponding increase in amount of the ester to the increasing concentration of biotin was necessary. The proportional relation did not extend to excedingly high levels of the two implicating factors. The further observations concerning the effects of the individual fatty acid esters such as sucrose stearate remain unsatisfactory.     

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.     

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.