Studies on biosynthesis of γ-polyglutamic acid in Bacillus subtilis (natto)

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The genes encoding proteins required for γ-polyglutamic acid (PGA) production were cloned from B.subtilis IFO 16449, a strain producing a high amount of PGA.Four open reading frames were found in the cloned 4.2-Kb DNA fragment and were almost identical to ywsC and ywtABC genes of B.subtlis 168. Northern blot analysis showed the four genes constitute an operon. Three genes, ywsC, ywtA and ywtB, were disrupted to investigate which genes participate in the PGA biosynthesis. No PGA was produced in ΔywsC and ΔywtA strains, however a small amount PGA was produced from ΔywsB strain. To clarify the function of YwsC protein, histidine-tag codon was introduced into the C-terminal region of ywsC gene and the histidine-tagged YwsC (YwsC-H) was expressed in ΔywsC strain. The Yws-H protein was purified from the lysozyme-treated lysate of the transformant by nickel affinity chromatography and Western blot analysis revealed YwsC-H protein consists of two subunits, 44K and 33K proteins. Northern blot analysis also indicated the two proteins was encoded by in phase overlapping in ywsC gene. The purified proteins synthesized ^<14>C-labeled PGA in the presence of ATP, MgCl_2 and ^<14>C-L-glutamate, and both 44K and 33K proteins was requisite for the in vitro PGA biosynthesis, indicating that ywsC gene codes for two subunits of PGA synthetase, a crucial enzyme in PGA production.