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

从枯草杆菌IFO 16449中克隆了γ-聚谷氨酸(PGA)产生所需蛋白的编码基因，在4.2kb的片段中发现了4个开放阅读框，与B.subtlis 168的ywsC和ywtABC基因基本一致。Northern杂交分析表明，这四个基因构成了一个操纵子。为了研究哪些基因参与了PGA的生物合成，对ywsC、ywtA和ywtB三个基因进行了干扰。ΔywsC和ΔywtA株不产生PgA，ΔywsB株产生少量PgA。为了阐明YwsC蛋白的功能，将组氨酸标签密码子引入到YwsC基因的C末端，并在ΔYwsC株中表达了组氨酸标签的YwsC(YwsC-H)。经镍亲和层析和Western印迹分析表明，YwsC-H蛋白由44K和33K两个亚基组成。Northern印迹分析也表明这两个蛋白在ywsC基因上是以同相重叠的方式编码的。纯化的蛋白在三磷酸腺苷、氯化镁和谷氨酸存在下合成了β&lt；14&gt；C标记的PGA，44K和33K蛋白是PGA体外生物合成所必需的，表明ywsC基因编码PGA合成酶的两个亚基，PGA合成酶是PGA合成的关键酶。

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.