Tryptophan synthase and production of L-tryptophan in regulatory mutants.

A 5-fluorotryptophan-resistant mutant of Brevibacterium flavum, No. 187, accumulated 2.6g of indole 3-glycerol (InG) in addition to 8.0g of L-tryptophan per liter in the culture medium. The addition of L-serine to the medium decreased the accumulation of InG and increased that of L-tryptophan up to a concentration of 10.3 g/liter, while the addition of L-tryptophan increased the InG accumulation, suggesting that InG was formed by hydrolysis of indole 3-glycerol phosphate (InGP), the substrate of tryptophan synthase (TS) which catalyzed the final step reaction of tryptophan biosynthesis. Then, in order to examine the mechanism of the InG accumulation, TS was purified from tryptophan auxotroph, TA-60. The reaction mechanism of TS was Ordered Bi Bi with Km's of 0.63 and 0.038mM for serine and InGP, respectively. Tryptophan, a product of the TS reaction, inhibited TS competitively with respect to serine and the Ki for tryptophan was estimated to be 2.0mM. On the other hand, anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, was much less sensitive to the feedback inhibition by tryptophan in strain No. 187 than in the wild strain. The tryptophan concentration giving 50% inhibition of AS in strain No. 187 was estimated to be 2.4mM, almost comparable to that of TS, 7.7mM. From these results, it was concluded that the accumulation of InG in strain No. 187 would result from the product inhibition of TS by the tryptophan accumulated.