Whole-genome sequencing of a psychrophilic bacterium, analysis of genes involved in cold adaptation, and exploitation of cold-active enzymes

嗜冷细菌的全基因组测序、冷适应相关基因分析以及冷活性酶的开发

• 批准号: 13556014
• 负责人: ESAKI Nobuyoshi
• 金额: $ 9.02万
• 依托单位: KYOTO UNIVERSITY
• 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 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13556014/
• 关键词: psychrophilic bacteria; psychrophiles; psychrotrophs; Shewanella; Acinetobacter; genome analysis; cold adaptation; cold-active enzymes; 好冷酵素; 形質転換; 宿主-ベクター系; Shewanella sp.Ac10; Acinetobacter sp.strain no.6; 宿主ベクター系; 脂質分解酵素

1. We carried out whole-genome analysis of Shewanella sp. Ac10, a psychrophile isolated from the Antarctic Ocean. The genome size was 4.5 Mbp, and the number of ORFs was about 4, 800.2. Shewanella sp. Ac10 grown at 4℃ produced a large quantity of a protein homologous to a cold-shock protein, CspA, from Escherichia coli.3. A collagenase of Shewanella sp. Ac1O was overproduced and characterized.4. We established a transformation method for Shewanella sp. Ac1O, isolated a promoter, and constructed an expression vector.5. A psychrotrophic bacterium, Acinetobacter sp. strain no. 6, was isolated from Siberian tundra soil. The bacterium produced extracellular lipolytic enzymes, and degraded various triglycerides in the medium at low temperatures. The bacterium is expected to be useful for the waste water treatment at low temperatures.6. The gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters was cloned from Acinetobacter sp. strain no. 6. The enzyme was shown to be useful for the synthesis of esters by transesterification using vinyl esters as an acyl donor.7. We cloned the gene coding for a cold-active esterase from Acinetobacter sp. strain no. 6 that belongs to the EST group of the esterase/lipase family.8. We constructed a vector to introduce exogenous genes into Acinetobacter sp. strain no. 6 and isolated powerful promoters suitable for overexpression of exogenous genes.

1.我们对从南极海洋分离的嗜冷菌希瓦氏菌Ac 10进行了全基因组分析。基因组大小为4.5Mbp，开放阅读框数约为4，800.2。希瓦氏菌Ac 10在4℃下生长，产生大量与大肠杆菌冷休克蛋白CspA同源的蛋白.希瓦氏菌Ac 1 O的胶原酶被过量产生并被表征.建立了希瓦氏菌Ac 1 O的转化方法，分离了启动子，构建了表达载体.从西伯利亚冻原土壤中分离到一株嗜冷细菌，命名为不动杆菌6号。该细菌产生胞外脂肪分解酶，并在低温下降解培养基中的各种甘油三酯。该菌有望用于低温下的废水处理.从不动杆菌属6号菌株中克隆了编码对脂肪酸乙烯基酯具有底物偏好的冷活性脂肪分解酶的基因。该酶被证明是有用的酯的合成通过酯交换使用乙烯基酯作为酰基供体。7.我们克隆了编码来自不动杆菌属6号菌株的冷活性酯酶的基因，该酯酶属于酯酶/脂肪酶家族的EST组。我们构建了将外源基因导入不动杆菌属6号菌株的载体，并分离了适合外源基因过表达的强大启动子。

项目成果

Suzuki, Takeshi et al.: "Cold-active lipolytic activity of psychrotrophic Acinetobacter sp. strain No."J. Biosci. Bioeng.. 92(2). 144-148 (2001)

Suzuki，Takeshi 等人：“嗜冷不动杆菌菌株 No. 的冷活性脂肪分解活性”J。

Suzuki, Takeshi et al.: "Primary structure and catalytic properties of a cold-active esterase from a psychrotroph, Acinetobacter sp.strain no.6, isolated from Siberian soil"Biosci.Biotechnol.Biochem.. 66・8. 1682-1690 (2002)

Suzuki、Takeshi 等：“从西伯利亚土壤中分离出的耐冷菌 Acinetobacter sp.6 号菌株的冷活性酯酶的初级结构和催化特性”Biosci.Biotechnol.Biochem. 66・8。 (2002)

Suzuki, Takeshi et al.: "A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp.strain no.6: gene cloning, purification, and characterization"J.Mol.Catal.B : Enzym.. 16. 255-263 (2002)

Suzuki、Takeshi 等人：“一种冷活性酯酶，其底物偏好来自耐冷菌不动杆菌属 6 号菌株的乙烯基酯：基因克隆、纯化和表征”J.Mol.Catal.B：酶。

Wei, Yun-lin et al.: "A novel esterase from a psychrotrophic bacterium, Acinetobacter sp.strain no.6, that belongs to the amidase signature family"J.Mol.Catal.B : Enzym.. (In press). (2003)

Wei, Yun-lin 等人：“一种来自耐冷细菌 Acinetobacter sp.strain no.6 的新型酯酶，属于酰胺酶特征家族”J.Mol.Catal.B : Enzym..（出版中）。

Suzuki Takeshi et al.: "A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6"J. Mol. Catal. Enzym.. 16. 255-263 (2002)

Suzuki Takeshi 等人：“一种冷活性酯酶，其底物偏好来自耐冷菌不动杆菌属 6 号菌株的乙烯基酯”J。