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从 Corynebacterium crenatum 中异源和同源表达精氨酸生物合成 argC~H 簇以提高 (L)-精氨酸的产量。

Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of (L) -arginine production.

机构信息

The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, People's Republic of China.

出版信息

J Ind Microbiol Biotechnol. 2012 Mar;39(3):495-502. doi: 10.1007/s10295-011-1042-4. Epub 2011 Oct 19.

Abstract

The genes involved in L: -arginine biosynthesis in Corynebacterium crenatum are organized as the argCJBDFRGH cluster like in Corynebacterium glutamicum. However, the argCH cluster of the C. crenatum SYPA 5-5, which is an industrialized L: -arginine producer, had a lethal mutation occurring in the ArgR repressor encoding gene. The argCH cluster with an inactive argR was overexpressed in E. coli and C. crenatum. In the recombinant E. coli JM109 enzyme activities were increased, and more L: -arginine was found in the supernatants from L: -glutamine. When the argCH cluster was overexpressed in C. crenatum under its native promoter Parg, L: -arginine production was increased by 24.9%, but the presence of the recombinant plasmid pJC-9039 had a negative effect on cell growth. Surprisingly, the DO value of the recombinant strain dropped gently and stayed at a lower level from 24 h to the end of fermentation. The results demonstrated an increasing utilization of oxygen and the distinct enhancement of unit cell L: -arginine yields with the cluster argCH-bearing in C. crenatum SYPA-9039. This study provides a kind of Corynebacteria with improved L: -arginine-producing ability and an efficient elevation for producing amino acid. Moreover, the promoter Parg would be used as a valid promoter to express objective genes for metabolic engineering in Corynebacteria.

摘要

克雷伯氏菌中 L: -精氨酸生物合成相关基因像谷氨酸棒杆菌一样组织成 argCJBDFRGH 簇。然而,工业化生产 L: -精氨酸的克雷伯氏菌 SYPA 5-5 的 argCH 簇在 ArgR 阻遏物编码基因中发生了致死突变。在大肠杆菌和克雷伯氏菌中过表达无活性的 argR 的 argCH 簇。在重组大肠杆菌 JM109 中,酶活性增加,从 L: -谷氨酰胺上清液中发现更多的 L: -精氨酸。当 argCH 簇在其天然启动子 Parg 下在克雷伯氏菌中过表达时,L: -精氨酸的产量增加了 24.9%,但重组质粒 pJC-9039 的存在对细胞生长有负面影响。令人惊讶的是,重组菌株的 DO 值从 24 小时开始缓慢下降,并在发酵结束时保持在较低水平。结果表明,克雷伯氏菌 SYPA-9039 中带有簇 argCH 的菌株对氧气的利用不断增加,单位细胞 L: -精氨酸的产量明显提高。本研究为具有改良 L: -精氨酸生产能力的棒状杆菌提供了一种方法,为氨基酸的高效生产提供了一种方法。此外,启动子 Parg 将被用作表达目的基因的有效启动子,用于棒状杆菌的代谢工程。

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