Highly Efficient Production of Recombinant Gene by Repeated Fed-Batch Culture

通过重复补料分批培养高效生产重组基因

• 批准号: 60470118
• 负责人: KOBAYASHI Takeshi
• 金额: $ 1.15万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60470118/
• 关键词: Copy number; Transcription efficiency; Plasmid; Stability; Nutrient concentration; Fed-batch culture; Tryptophan promoter; グルコースセンサー

For overproduction of useful proteins, control of gene-copy number, stability of plasmid, control of transcription efficiency and control of nutrients are important in fed-batch culture of gene-engineered microorganisms. The recombinant E. coli harboring a runaway-replication plasmid was cultivated at 25゜C and then the temperature was shifted to 37゜C. The copy number increased drastically and the gene product was amplified almost 40-fold.Effects of nutrients on <alpha> -amylase production and plasmid stability were studied in fed-batch culture of recombinant E. coli. Plasmid was maintained stably in a semi-synthetic medium but plasmid-free segregants were observed in a synthetic medium.To control transcriptional efficiency, fundamental properties of the trp promoter were also investigated in fed-batch culture using a recombinant containing the lacZ gene controlled by this promoter. On-off regulation of the promoter was achieved by controlling the tryptophan level.

在基因工程菌的流加培养过程中，控制基因拷贝数、质粒稳定性、转录效率和营养物质的控制对于过量生产有用蛋白是非常重要的。重组大肠将携带失控复制质粒的大肠杆菌在25 ℃下培养，然后将温度转移到37 ℃。研究<alpha>了营养物质对重组E.杆菌质粒在半合成培养基中保持稳定，但在合成medium.To控制转录效率，色氨酸启动子的基本特性进行了研究，在流加培养中使用重组含有lacZ基因控制的这个启动子。启动子的开关调节通过控制色氨酸水平来实现。

项目成果

Shinji Iijima: J.Chem.Tech.Biotechnol.36(11). 539-546 (1986)

饭岛真司：J.Chem.Tech.Biotechnol.36(11)。

Satoru Mizutani: J.Ferment.Technol.

水谷悟：J.Ferment.Technol。

Satoru Mizutani: Biotech.Progress.

水谷悟：生物技术进展。

Shuji Kawai, Satoru Mizutani, Shinji Iijima and Takeshi Kobayashi: "On-Off Regulation of the Tryptophan Promoter in Fed-Batch Culture" J. Ferment. Technol.64(6). 503-510 (1986)

Shuji Kawai、Satoru Mizutani、Shinji Iijima 和 Takeshi Kobayashi：“补料分批培养中色氨酸启动子的开关调节”J. Ferment。

Satoru Mizutani, Shinji Iijima, Makoto Morikawa, Kazuyuki Shimizu, Masakazu Matsubara, Yasumasa Ogawa, Rokuro Izumi, Kuniko Matsumoto and Takeshi Kobayashi: "On-Line Control of Glucose Concentration Using an Automatic Glucose Analyzer" J. Ferment. Technol

Satoru Mizutani、Shinji Iijima、Makoto Morikawa、Kazuyuki Shimizu、Masakazu Matsubara、Yasumasa Okawa、Rokuro Izumi、Kuniko Matsumoto 和 Takeshi Kobayashi：“使用自动葡萄糖分析仪在线控制葡萄糖浓度”J. Ferment。