Construction of Useful Temperature-dependent Gene Expresssion System in S. cerevisiae and It's Application to a Cultivation Process

酿酒酵母有用温度依赖性基因表达系统的构建及其在培养过程中的应用

• 批准号: 03650772
• 负责人: SHIOYA Suteaki
• 金额: $ 0.9万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03650772/
• 关键词: Recombinant yeast; Temperature-sensitive; Rice-alpha-amylase; Acidic phosphatase; Specific growth rate; Two-stage production; ∂ーアミラ-ゼ; 酸性ホスファタ-ゼ

The goal of this study are to construct an efficient temperature-sensitive gene expression system in Saccharomyces cerevisiae and to get an optimum solution for maximum production of a useful bio-product using this host-vector system. We got the following results.(1) Utilizing MF_<alpha>1::PHO5 vector, we constructed a temperature-sensitive gene expresssion system and used for acidec phosphatase(APase) production. Optimal strategy was a two-level control of the cultivation temperature which could be interpreted as a two-stage production. This was confirmed experimentally.(2) This gene-expression system was applied to production of a foreign protein, rice alpha-amylase and got a fairly good result.(3) PHO84 promotor was introduced to a host(SH3337-strain) which is PHO81 temperature-sensitive mutant. Using this gene expression system, rice-alpha-amylase was produced more than the former system.(4) For this gene expression system, an optimal strategy of cultivation temperature was also obtained using the relationship between the specific growth rate, specific production rate and cultivation temperature. Optimal strategy was a bang-bang control of temperature such as switching from 30 C to 25 C and it could be also iterpreted as a two-stage production having growth stage and production stage.

本研究的目的是在酿酒酵母中构建一个高效的温度敏感的基因表达系统，并获得一个最佳的解决方案，最大限度地利用这种宿主载体系统生产有用的生物产品。我们得到了以下结果。(1)利用MF_<alpha>1：：PHO 5载体，构建了温度敏感型基因表达系统，并用于酸性磷酸酶（APase）的生产。最优策略是两级控制培养温度，可以解释为两个阶段的生产。这一点在实验上得到了证实。(2)将该基因表达系统应用于水稻α-淀粉酶的生产，获得了较好的效果。(3)将PHO 84启动子导入PHO 81温敏突变株SH 3337 - 1。利用该基因表达系统，水稻α-淀粉酶的产量比原系统高。(4)利用比生长速率、比生产速率与培养温度之间的关系，得到了该基因表达系统的最佳培养温度策略。最优策略是温度的开关控制，例如从30 ℃切换到25 ℃，也可以迭代为具有生长阶段和生产阶段的两阶段生产。

项目成果

S.Shioya, K.Uchiyama T.Ohtani, A.Kanda K.Suga, S.Harashima,: "Maximum Production in Batch Culture of S.Cerevisiae.Using Temperature-depedenst gene expression system" Y,Oshima Proceeding of 12th IFAC morld congress,at Sydney. (1993)

S.Shioya，K.Uchiyama T.Ohtani，A.Kanda K.Suga，S.Harashima，：“利用温度依赖性基因表达系统实现酿酒酵母分批培养的最大产量” Y，Oshima 第 12 届 IFAC 世界会议论文集

Suteaki Shioya: Springer-Verlag. Optimization and Control in Fed-batch Bioreactors, in "Advances in Biochemical Engineering & Biotechnology Vol.46"(A. Fiechter, ed.), 30(200) (1992)

Suteaki Shioya：施普林格出版社。

S.Shioya,K.Uchiyama T.Ohtani,A.Kanda K.Suga,S.Harashima,Y.Oshima: "Maxinum Production in Batch Culture of S.cevevisial.using Temperatue-depedenst gene expression System" Proceeding of 12th IFAC world congress,at Sydney. (1993)

S.Shioya，K.Uchiyama T.Ohtani，A.Kanda K.Suga，S.Harashima，Y.Oshima：“使用依赖温度的基因表达系统批量培养 S.cevevisial 的最大产量”第 12 届 IFAC 世界会议论文集

塩谷 捨明: "流加培養系の最適化とその実現に関する研究" 醗酵工学会誌. 71. 395-404 (1992)

Suteaki Shioya：“补料分批培养系统的优化和实现的研究”发酵工程学会杂志71. 395-404（1992）。

S.SHIOYA: "Adrances in Biochem. Eng.& Biotechmol.46.(Ed;A,Fiechter)" Springer-Verlag, 200(30) (1992)

S.SHIOYA：“生物化学的进展。工程。