Fermentative production of L-homoserine was achieved by using a threonine requiring mutant of Micrococcus glutamicus. This strain produced a large quantity of L-homoserine as well as L-lysine in the culture broths if it was cultivated under appropriate conditions. During the studies on fermentative production of L-homoserine, it was found that L-threonine concentration affected the formation of L-homoserine and L-lysine to a great extent. Maximum productions of L-homoserine and L-lysine were attained when L-threonine concentrations were 400μg/ml and 300 μg/ml, respectively. And an excess amount of threonine caused a sharp decrease in both amino acid productions. The effects of various amino acids on this fermentation were also examined. As the results, two "negative feed-back control" phenomena caused by threonine and methionine were observed. Further investigations using intact cell suspension revealed the mode of action of the two amino acids in the "negative feed-back" mechanisms. Experimental results showed that threonine inhibited a certain enzyme activity involved in the biosynthetic path of homoserine, and methionine repressed the formation of a certain enzyme on the same path. Based on these experimental results the sites of these "negative feedback" mechanisms were discussed.
Transformation of REICHSTEIN'S compound S or progesterone by various microorganisms such as fungi, bacteria and streptomycetes of about 700 strains has been studied (Table 1). The actions of several strains upon various corticoid steroids were investigated and the transformed steroids were isolated and their structures determined. From these results it was revealed that almost all of the microorganisms tested have a certain kind of substrate specificity in their actions as shown in Table 2.
1. During the processes of cell division of Chlorella as controlled by the limited supply of sulfur and nitrogen sources, the fate of free and combined pantothenic acid was investigated in connection with the accumulation and disappearance of surplus lipid investigated in the preceding work. 2. When the young and small algal cells (D-cells) were grown synchronously in an S-free medium, the cells grew to some extent, without, however, being able to perform cell division. In the cells thus rendered S-deficient, the formation of pantothenic acid in both free and combined forms was profoundly suppressed, whereas the lipid content increased to an abnormally high level. 3. When the S-starved cells were provided with sulfate alone under photosynthesizing conditions, the cells still remained unable to divide. During this period no significant increase in the amount of combined pantothenic acid occurred, although its free form increased up to the normal level. In this case, the accumulated lipid did not disappear. 4. When the S-starved cells were made capable of performing cell division on being provided with sulfate under non-photosynthesizing conditions, or with sulfate plus nitrate under photosynthesizing conditions, the combined pantothenic acid increased remarkably and the surplus lipid disappeared. 5. Discussions were made on the possible role of the combined pantothenic acid in the algal cell division and on the competitive relation between the processes of photosynthetic growth and cell division in respect to the formation of the factor (probably CoA) containing pantothenic acid as an essential component.
To produce D-glutamic acid-requiring mutants, spores of Bacillus subtilis K were exposed to X-rays. The mutants were selected by the replica plating method following the penicillin screening. Using a medium enriched with D-glutamate in stead of the so-called complete medium, five D-glutamate dependent mutants were isolated. They were classified into three groups in terms of the growth response pattern (Table 2). Mutant 3d grows in minimal medium when it is supplemented with either D-glutamic acid, D-aspartic acid, or D-alanine. Meanwhile the mutant does not grow in the media when L-aspartic acid or L-glutamic acid coexists.
When mutant 3d is incubated in a medium devoid of D-glutamic acid, the number of viable cells increases for a while and after the lapse of seven hours begins to decrease abruptly. Because D-glutamic acid is a major component of cell wall and a minor component of cytoplasm, and because the strain cannot synthesize D-glutamic acid although it can synthesize L-glutamic acid and other L-amino acids, it is concluded that the phenomenon mentioned above is caused by the unbalanced synthesis between cell wall and cytoplasm.
To look for the site of genetic block in the mutants of Bacillus subtilis K requring D-glutamic acid, several enzymes that were considered important for the biosynthesis of D- and L-glutamic acid were investigated. In mutant 3d, alanine racemase activity was about one tenth of that in the wild-type strain. It appears therefore that the block site in this mutant is between L-alanine and D-alanine. The mutant requires D-alanine, D-aspartic acid, or D-glutamic acid for growth. L-Alanine dehydrogenase activity was exceptionally low in mutant 2a. Because the mutant is able to synthesize L-aspartic acid and L-glutamic acid from fumarate too, the requirement of L-alanine, D-alanine, D-aspartic acid, or D-glutamic acid may be due to the slow reaction between pyruvate and L-alanine. Mutant 4a possesses a low activity of D-alanine-D-glutamic acid transaminase and responds to L-aspartic acid, L-glutamic acid, D-aspartic acid, or D-glutamic acid. The dependency on D-aspartic acid or D-glutamic acid is reasonable if these D-amino acids are produced from D-alanine by transamination, while the dependency on L-aspartic acid or L-glutamic acid remains unexplained. The correlation between a genetic damage and an enzyme formation was discussed in this connection.
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