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

从大量产γ-聚谷氨酸（PGA）的枯草芽孢杆菌IFO 16449中克隆到了γ-聚谷氨酸（γ-polyglutamic acid， PGA）所需蛋白的编码基因。在克隆的4.2 kb DNA片段中发现4个开放阅读框，与B.subtlis 168的ywsC和ywtABC基因几乎相同。Northern blot分析表明，这4个基因组成一个操纵子。通过对ywsC、ywtA和ywtB三个基因的干扰，研究哪些基因参与了PGA的生物合成。ΔywsC和ΔywtA菌株不产PGA，而ΔywsB菌株产少量PGA。为了明确YwsC蛋白的功能，我们在YwsC基因的c端区引入组氨酸标签密码子，并在ΔywsC菌株中表达了组氨酸标记的YwsC （YwsC- h）。通过镍亲和层析和Western blot分析，Yws-H蛋白由44K和33K两个亚基组成。Northern blot分析还表明，这两个蛋白在ywsC基因中通过相重叠编码。在ATP、MgCl_2和^<14> c -l -谷氨酸存在的情况下，纯化的蛋白合成了^<14> c -l -谷氨酸标记的PGA，并且44K和33K蛋白是体外合成PGA所必需的，这表明ywsC基因编码PGA合成酶的两个亚基，PGA合成酶是PGA生产的关键酶。

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.