l-Glutamic Acid Fermentation

It is well known that biotin has a marked effect on l-glutamic acid fermentation.

The authors have intended to find strains which are independent of the amounts of biotin in the culture medium. As a result, oleic acid-requiring mutants were obtained from a strain of Brevibacterium thiogenitalis which is an auxotroph for biotin. The growth of the mutant was remarkably stimulated by Tween 20, 40, 60, Ca ions and a small amount of corn steep liquor. And also, the mutant was found to have lost its requirement for biotin and showed growth response only to oleic acid or unsaturated fatty acids.

The effect of biotin, oleic acid and other unsaturated fatty acids on the production of l-glutamic acid was investigated by using an oleic acid-requiring mutant of Brevibacterium thiogenitalis No. 653. The results described in the present paper showed that the oleic acid-requiring mutant D-248 produced a large amount of l-glutamic acid in the excess biotin-contaming media, and that oleic acid seemed to be completely replaced by other unsaturated fatty acids such as palmitoleic acid and linoleic acid.     

l-Glutamic Acid Fermentation

Structure of a sugar lipid produced by an oleic acid-requiring mutant of Brevibacterium thiogenitalis was studied and established as (I).

Relation between biotin and oleic acid was studied using a biotin-requiring organism accumulating l-glutamic acid and its blocked mutants lacking the biosynthetic system of biotin or/and oleic acid. The results support the following considerations. Biotin is not formed from oleic acid and does not substantially affect the growth of l-glutamic acid-accumulating bacteria and their productivity of l-glutamic acid.

Consequently, biotin serves only for the synthesis of fatty acids in the present organisms. The essential factor for their growth and metabolism is an unsaturated fatty acid like oleic acid and not biotin. And also, saturated fatty acids have substantially no relation with their growth and metabolism like accumulation of l-glutamic acid.     

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.     

Relation between Fatty Acid Composition of Cellular Phospholipids and the Excretion of L-Glutamic Acid by a Glycerol Auxotroph of Corynebacterium alkanolyticum

Relation between fatty acid composition of cellular phospholipids and the excretion of L-glutamic acid was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).

When grown on n-hexadecane, the proportion of unsaturated fatty acids was higher in L-glutamic acid-accumulating cells than in L-glutamic acid-nonaccumulating cells. When grown on fructose or acetic acid, the reverse relation was observed. Moreover, cells containing no oleic acid produced L-glutamic acid from n-pentadecane.

These results suggest that the membrane permeability to L-glutamic acid is not always controlled by the cellular content of unsaturated fatty acids.     

Change in Permeability to l-Glutamic Acid in the Glycerol Auxotroph GL-21

In this study, the mechanism of the extracellular accumulation of l-glutamic acid by the glycerol auxotroph was partially clarified. Whenever Corynebacterium alkanolyticum GL–21 (glycerol auxotroph) accumulated a large amount of l-glutamic acid in the fermentation broth, the content of its cellular phospholipids was not more than 50% of that of C. alkanolyticum No. 314 (prototroph).

Moreover, biotin, oleic acid or thiamine had no influence on the cellular phospholipid content of the auxotroph.

Under limited supply of glycerol, the efflux of l-glutamic acid in the auxotroph was extremely enhanced, but its enzyme activities participating in l-glutamic acid biosynthesis remained at the same level as those of the prototroph.

From the results, it is considered that the regulation of phospholipid content gave rise to the destruction of the permeability barrier to l-glutamic acid in the cell membrane.     

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.     

Culture Conditions for the Production of l-Glutamic Acid from n-Paraffins by Glycerol Auxotroph GL-21

A novel process for the microbial production of l-glutamic acid on an industrial scale was successfully established by using a glycerol auxotroph.

The most suitable carbon source for producing L-glutamic acid was n-paraffins (C₁₃–C₁₅). The production of L-glutamic acid was not affected by a large amount of biotin or oleic acid in the absence of penicillin, and occurred maximally at the glycerol concentration of 0.02% at pH 6.6. The most effective temperature was 28°C.

Under optimal conditions in a 200 liter fermentor, the mutant produced 72 g/liter of L-glutamic acid. On the other hand, the parent produced 53 g/liter of L-glutamic acid in the presence of penicillin.

It is believed that the low productivity of L-glutamic acid by the parent strain was mainly due to the occurrence of the marked decrease in the viable cell counts at the later phase of the fermentation caused by the action of penicillin added.     

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

Brev. lactofermentum rapidly took up biotin from culture medium and stored it in the cells. The saturation level of the stored biotin (3.8 × 10⁴ molecules/cell) exceeded the level required for the maximum growth by ten times, and the minimum level (1.3 × 10³ molecules/cell) was the most adequate to the accumulation of l-glutamic acid. The stored cellular biotin over the minimum level was metabolically available in the subsequent culture lacking in supplemented biotin. The cellular biotin was gradually reduced to the minimum level with the multiplication of the cells, and them the accumulation of l-glutamic acid was observed. This relation between the level of cellular biotin and the accumulation of l-glutamic acid was impaired by the addition of Tween 60 or some saturated fatty acid. In the presence of biotin and Tween 60 the biotin-saturated cells turned into cells capable of accumulating l-glutamic acid keeping the maximum level; and in the same medium the cells having the minimum amount of biotin took up biotin and then were saturated with it, and yet the cells preserved the acid-accumulating property. It was confirmed with the use of bioautographic technique and avidin test that the biotin released from the cells by acid hydrolysis was identical with authentic d-biotin.     

Microbial Production of l-Glutamic Acid by Glycerol Auxotrophs

To establish a novel process for the production of l-glutamic acid from n-paraffins, a glycerol auxotroph GL-21, a new type mutant, was successfully obtained from Corynebacterium alkanolyticum No. 314 by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. This auxotroph required glycerol for its growth regardless of the carbon source used.

At 72 hr, this mutant GL-21 produced about 40 mg/ml of l-glutamic acid from n-paraffins in the culture broth at 0.01 per cent addition of glycerol in the absence of penicillin.

A thiamine auxotroph, a biotin auxotroph and an oleic acid auxotroph were also obtained by a similar technique, but these auxotrophs were found to be inapplicable for the production of l-glutamic acid from n-paraffins.     

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

In the preceding paper on the interrelation between sucrose ester of fatty acid and biotin, the fatty acid being a mixture of C₁₀ to C₁₈ acid, it was described that carbon chain length of fatty acid has a great influence on the accumulation of l-glutamic acid. Fatty acids with C₁₂ to C₁₈ chain length, particularly myristic, palmitic and margaric acids were effective on the accumulation of l-glutamic acid in the culture medium containing sufficient biotin, whereas lower and higher length acids were ineffective. In the form of polyoxyethylene sorbitan or polyethylene glycol ester, C₁₆ and C₁₈ acids were remarkably effective. However, the ester of C₁₂ acid and polyoxyethylene ethers of C₁₂ to C₁₈ alcohols had little or no effect.     

Studies on l-Glutamic Acid Fermentation

The authors have carried out a series of studies on l-glutamic acid fermentation with a strain of Brevibacterium divaricatum nov. sp. in the previous papers.

In this paper, some metabolism of l-glutamic acid and oxidative decomposition of several organic acids concerning the tricarboxylic acid cycle by the resting cells have been studied. The results suggest that l-glutamic acid is one of the final fermentative products of this bacterium, and the tricarboxylic acid cycle is working as a glutamic acid forming cycle.

The presence of glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, DPN-linked glyceraldehyde-3-phosphate dehydrogenase, and TPN-linked glucose-6-phosphate dehydrogenase in cell-free extracts of this bacterium was also demonstrated.     

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

Tween 60 (polyoxyethylene sorbitan monostearate) has been found to be the most effective derivative of fatty acid in accumulating l-glutamic acid in biotin-sufficient medium. The effect was exceedingly subject to the influence of the addition time of the ester, and this was observed also on the growth curve of Brev. lactofermentum. Changes of the growth curve caused by the varied addition time of the ester corresponded to those by the concentration of biotin in the medium that did not contain Tween 60. The patterns of fermentation course in the two corresponding conditions, such as biotin 3 μg/l and biotin 20 μg/l-Tween 60 mg/ml, agreed closely with each other. It seemed that identical cells were grown on the conditions. The only difference between the cells was observed as to the contents of intracellular biotin. Although l-glutamic acid was not accumulated by biotin-sufficient cells, cells with sufficient biotin and capable of accumulating l-glutamic acid were obtained in the presence of Tween 60, in which case the ester neither prevented the cells from taking up biotin nor controlled the level of intracellular biotin.     

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures

At maximum production of l-glutamic acid, the oxidation-reduction potential of the culture broth in l-glutamic acid fermentation showed a stable value of 9.0 to 9.6 as rH value. When biotin concentration in the medium was high (40γ/liter), the production of l-glutamic acid decreased, and the rH was 8.0 and it was out of accordance with that of the control (biotin-poor; 2γ/liter). Under “less-aerobic” conditions, its rH rose to 10.4.

From these results, it was concluded that the rH during maximum production of l-glutamic acid showed a stable value affected actively by the redox system, l-glutamic acid/α-ketoglutaric acid and      

Fermentative Production of l-Glutamine by Sulfaguanidine Resistant Mutants Derived from l-Glutamate Producing Bacteria

Excellent l-glutamine producers were screened for among sulfaguanidine resistant mutants derived from the wild type l-glutamic acid-producing bacteria, Brevibacterium flavum, Brevibacterium lac to fermentum, Corynebacterium glutamicum and Microbacterium ammoniaphilum.

The best strain, No. 1~60, was a sulfaguanidine resistant mutant derived from B. flavum 2247 by mutation. Strain No. 1~60 accumulated 41.0 mg/ml of l-glutamine after 48 hr of cultivation from 10% glucose as a carbon source. This yield was the highest among those so far reported.

The addition of Mn^{2 +} (2 ppm) to the standard medium for B. flavum 2247 decreased the l- glutamine production and increased the l-glutamic acid excretion markedly. On the contrary, strain 1 —60 was not affected the Mn²⁺ (2 ppm) addition at all.

Glutamate kinase activity and the intracellular content of ATP in sulfaguanidine resistant mutant No. 1~60 were higher than those in the parent strain, B. flavum 2247.

It was confirmed that the increase in glutamate kinase and the increase in internal ATP, which were important for the l-glutamine synthesis, were very effective for the improvement of l-glutamine-producing mutants.     

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.     

Isolation and Identification of O-Acetyl and 12-Hydroxyradiclonic Acids

An N-acetylglutamate-acetylornithine acetyltransferase-deficient arginine-requiring mutant AA–1, was derived from an l-arginine producer of Corynebacterium glutamicum. It accumulated a large amount (30 mg per ml) of l-glutamic acid and a small amount (1.2 mg per ml) of N^α-acetylornithine, an intermediate of arginine biosynthesis, in the culture medium.

The production of N^α-acetylornithine by AA–1 was not affected by the concentration of l-arginine in the medium, whereas that of l-glutamic acid was inhibited by a high concentration of l-arginine in the medium containing excess biotin.     

Relationship among Cellular Fatty Acid Composition,Amino Acid Uptake and Neomycin Formation in a Mutant Strain of Streptomyces fradiae

Neomycin-producing strains of Streptomyces fradiae, whose cellular fatty acid spectra are of iso 16: 0-type or normal 16: 0-type, had about two to six times larger amino acid and hexosamine pools than a neomycin-nonproducing strain which has the anteiso 15: 0-type cellular fatty acid spectrum. About 50 to 80 percent of the amount of extractable free amino acids were L-glutamic acid in either type of cells. The difference of pool size in these strains seems to be explained by the difference in ability for amino acid uptake. That is, the ability for L-glutamate uptake of anteiso 15:0-type cells was markedly reduced and accumulated glutamate was easily washed out by buffer. Glucose, magnesium ions and L-glutamate were essential for the formation of neomycin by washed cells and, therefore, even the mutant ST–5B of anteiso 15: 0-type could accumulate a large amount of glutamate and produce neomycin as far as it was grown in a medium containing a high concentration of glutamate. These results indicate that a large pool of glutamate is essential for the formation of neomycin and the fatty acid spectrum is a factor governing the capacity to accumulate L-glutamic acid.     

Relation between the Extracellular Accumulation of L-Glutamic Acid and the Excretion of Phospholipids by Penicillin-treated Corynebacterium alkanolyticum

The addition of penicillin (50 u/ml) to the cells of Corynebacterium alkanolyticum No. 314 growing on n-paraffins medium at the logarithmic growth phase was the most suitable for the extracellular accumulation of L-glutamic acid and the excretion of phospholipids.

The relation between the extracellular accumulation L-glutamic acid and the excretion of phospholipids in the presence of penicillin was very close and specific. The kinds of phospholipids excreted and their fatty acid components were the same as those of intracellular phospholipids.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

Extracellular Accumulation of Phospholipids,UDP-N-Acetylhexosamine Derivatives and L-Glutamic Acid by Penicillin-treated Corynebacterium alkanolyticum

The addition of penicillin to cells of Corynebacterium alkanolyticum No. 314 growing on n-paraffins medium caused the simultaneous excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid.

Among many antibiotics which inhibit cell wall synthesis, only the inhibitors of peptideglycan transpeptidase such as penicillin G and cephaloridine were effective for inducing the excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid, while the others promoted only the excretion of UDP-N-acetylhexosamine derivatives.

From the close relationship between the excretion of L-glutamic acid and the excretion of phospholipids, it was suggested that the action of penicillins and cephalosporins on the cell membrane resulted in the excretion of L-glutamic acid.