Development of Rhodococcus promoter for utilization in actinomycetes

开发用于放线菌的红球菌启动子

• 批准号: 13556011
• 负责人: KOBAYASHI Michihiko
• 金额: $ 8.9万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13556011/
• 关键词: Promoter; Gene; Expression; Rhodococcus

The microbial degradation of nitrites proceeds through two enzymatic pathways : nitrite hydratase (NHase} catalyzes hydration of nitrite to amide, whereas nitrilase catalyzes hydrolysis of nitrite to acid and ammonium. Nitrilase is strongly induced by the addition of ε-caprolactam or isovaleronitrile to the culture medium ; the formed nitrilase corresponds to 35ーIo of aft soluble protein in Rhodococcus rhodochrous J1. On the other hand, NHase is strongly induced by the addition of urea to the culture medium ; the formed NHase corresponds to 50% of all soluble protein in this strain. At first, we trimmed the DNA region containing the nitrilase gene cluster, and constructed several plasmids where the upstream or downstream region of the gene cluster was deleted. We transformed each plasmid into Escherichia coli, and examined the nitrilase formation in the resultant transformants. SDS-PAGE and enzyme assay by HPLC revealed that the Rhodococcus nitrilase expression system was not able to function in E. coli. Next, we tried to investigate whether the nitrilase regulatory system is active or not in other Rhodococcus actinomycetes, and found that the nitrilase expression system functioned, indicating the development of a hyper-expression vector. We also obtained similar results for the NHase expression system. Furtheremore, we tried to construct a Rhodococcus host-vector system, considering the useful characteristics of this genus. We bought more than 100 Rhodococcus strains from microorganism stock centers, and searched for Rhodococcus strains carrying cryptic plasmids. We found three strains. with a cryptic plasmid, and succeeded in the construction of a Rhodococcus-E.coli shuttle-vector using one of their plasmids

亚硝酸盐的微生物降解两种酶促途径：亚硝酸盐水合物（Nhase} e，而硝酸盐硝酸盐催化剂至酸氨。培养基培养基；硝酸含量；硝酸酶corressponds in Rhododocccccus rhoodoccus Rhea的溶解性蛋白质的35-IO。在培养基中；在该菌株中，形成的Nhase Corressponds到所有可溶性蛋白的50％。 HPLC中的形成。高表达载体也为e xpression系统获得了相似的结果，我们试图构建犀牛寄宿体矢量系统，考虑到该属的有用特征。对于犀牛菌株菌株菌株隐性质粒。

项目成果

Kojima, Y., Kobayashi, M., Shimizu, S.: "A navel lipase from Pseudomonas fluorescens HU380 : Gene cloning, overproduction, renaturation-activation, two-step purification and characterization"J.Biosci.Bioeng.. 96. 242-249 (2003)

小岛 Y.、小林 M.、清水 S.：“来自荧光假单胞菌 HU380 的脐脂肪酶：基因克隆、过量生产、复性激活、两步纯化和表征”J.Biosci.Bioeng.. 96. 242

Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H., Kobayashi, M.: "Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 : Sequencing, gene expression, purification and

Oinuma, K-I.、Hashimoto, Y.、Konishi, K.、Goda, M.、Noguchi, T.、Higashibata, H.、Kobayashi, M.：“参与绿针假单胞菌碳氮三键合成的新型醛肟脱水酶

Goda, M. et al.: "Discovery of a novel enzyme, isonitrile hydratase, invdved in nitrogen-carbon triple bond cleavage"J. Biol. Chem.. 276. 23480-23485 (2001)

Goda, M. 等人：“发现一种新型酶，异腈水合酶，参与氮碳三键裂解”J.

Yamada, H., Shimizu, S., Kobayashi, M.: "Hydratases involved in nitrile conversion : Screening, characterization and application"Chemical Records. 1. 152-161 (2001)

Yamada, H.、Shimizu, S.、Kobayashi, M.：“参与腈转化的水合酶：筛选、表征和应用”化学记录。

Hashimuto, Y., Sasaki, S., Herai.S., Oinuma, K., Shimizu, S., Kubayastii, M.: "Site-directed mutagenesis for cysteine residues, of cobalt-containing nitrile hydratase"J.Inorg.Biochem.. 91. 70-77 (2002)

Hashimuto，Y.，Sasaki，S.，Herai.S.，Oinuma，K.，Shimizu，S.，Kubayastii，M.：“含钴腈水合酶的半胱氨酸残基的定点诱变”J.Inorg。