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

枯草芽孢杆菌（纳豆）γ-聚谷氨酸生物合成的研究

• 批准号: 11660081
• 负责人: TAHARA Yasutaka
• 金额: $ 2.3万
• 依托单位: Shizuoka university
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660081/
• 关键词: Bacillus subtilis; Bacillus subtilis (natto); γ-polyglutamic acid synthetase; gene disruption; ywsC-ywtABC genes; γ-polyglutamic acid; γ-ポリグルタミン酸合成遺伝子; グルタミン酸合成酵素; 相同組換え

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.

从γ-聚谷氨酸（PGA）高产菌株B.subtilis IFO 16449中克隆了PGA合成所需的编码蛋白基因，在克隆的4.2-K B b DNA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。北方印迹分析表明这四个基因组成一个操纵子。破坏三个基因ywsC、ywtA和ywtB以研究哪些基因参与PGA生物合成。ΔywsC和ΔywtA菌株不产生PGA，而ΔywsB菌株产生少量PGA。为阐明YwsC蛋白的功能，在YwsC基因C端引入组氨酸标签密码子，并在ΔywsC菌株中表达了组氨酸标签的YwsC（YwsC-H）。用镍亲和层析法从溶菌酶处理的大肠杆菌裂解液中纯化出Yws-H蛋白，Western blot分析表明YwsC-H蛋白由44 K和33 K两个亚基组成。北方杂交分析表明，这两种蛋白在ywsC基因中是同相重叠编码的。纯化的蛋白<14>在ATP、MgCl_2和~（44）C-L-谷氨酸存在下合成~（44）C-标记的PGA<14>，并且44 K和33 K蛋白都是体外合成PGA所必需的，表明ywsC基因编码PGA合成酶的两个亚基。

项目成果

Tahara Y.et al.: "Efficient production of Efficient production of γ -polyglutamic acid by Bacillus subtilis (natto) desruptant defective in glutamate synthase"Biosci.Biotechnol.Biochem.. (発表予定).

Tahara Y.等人：“谷氨酸合酶缺陷的枯草芽孢杆菌（纳豆）破坏剂有效生产γ-聚谷氨酸”Biosci.Biotechnol.Biochem..（待提交）。

Urushibata, Y., Tokuyama, S., and Tahara, Y.: "Chracterization of ywsC gene of Bacillus subtilis involved in γ-polyglutamic acid production"J.Bacteriol.. (in preparation).

Urushibata, Y.、Tokuyama, S. 和 Tahara, Y.：“参与 γ-聚谷氨酸生产的枯草芽孢杆菌 ywsC 基因的表征”J.Bacteriol..（准备中）。

Urushibata, Y., Tokuyama, S., and Tahara, Y.: "γ-Polyglutamic acid production by a Bacillus subtilis IFO 16449 mutant defective in glutamate synthase"J.Biosci.Bioeng.. (in preparation).

Urushibata, Y.、Tokuyama, S. 和 Tahara, Y.：“谷氨酸合酶缺陷的枯草芽孢杆菌 IFO 16449 突变体产生 γ-聚谷氨酸”J.Biosci.Bioeng..（准备中）。

Tahara Y.et al.: "Construction of Bacillus subtilis ( natto) disruptant defective in genes essential for γ -polygltamic acid biosynthesis"Biosci.Biotechnol.Biochem.. (発表予定).

Tahara Y.等人：“构建γ-聚谷氨酸生物合成必需基因缺陷的枯草芽孢杆菌（纳豆）破坏体”Biosci.Biotechnol.Biochem..（待提交）。

Y.Urushibata,S.Tokuyama and Y.Tahara: "Chracterization of ywsC gene of Bacillus subtilis involved in γ-poly-glutamic acid production"J.Bacteriol.. (発表予定).

Y.Urushibata、S.Tokuyama 和 Y.Tahara：“参与 γ-聚谷氨酸生产的枯草芽孢杆菌 ywsC 基因的表征”J.Bacteriol..（待提交）。