Addition of L-proline to the production medium for cultivation of strain No. 14-5, an isoleucine auxotrophic mutant of Brevibacterium flavum 2247, reduced L-proline accumulation by about 40%. The formation, but not the activity, of glutamate kinase, the first enzyme required for L-proline biosynthesis from L-glutamate, seems to be controlled by L-proline. No difference was observed in specific activities of this enzyme assayed in vitro in either strain No. 14-5 or the parent strain.
During the growth of strain No. 14-5 (lacking in threonine dehydratase activity), the increase of intracellular glycine, methionine, aspartate, and threonine was considerably greater than in the parent strain. Intracellular valine, leucine, and lysine also increased. L-Proline produced was reduced to 42.9% without changing the level of intracellular glutamate by the addition of L-threonine and L-lysine to the medium before cultivation, but L- proline production was increased by nearly 20% when both amino acids were added near before the growth of this strain arrived at the stationary phase. Glutamate kinase formation was repressed by nearly 20% by the addition of both amino acids, whereas the activity was not inhibited.
A cell homogenate of the parent strain, which was not able to produce L-proline in the medium, did produce L-proline from L-glutamate in the presence of ATP. From these findings, it was concluded that in strain No. 14-5 both the higher level of available ATP resulting from the inhibition of aspartate and homoserine kinase and the accumulation of intracellular L-glutamate promoted by biotin-rich condition contributed to the abundant production of L-proline through stimulation of the glutamate kinase reaction.