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扩大CRISPR工具箱,用于在Corynebacterium glutamicum中进行烯生物合成工程

Zhimin Zhan¹, Xiong Chen¹, Zhifang Ye¹

¹Key Laboratory of Fermentation Engineering (Ministry of Education), National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China.

|April 27, 2024

概括

研究人员开发了一种新的CRISPR/MAD7遗传工具,用于对Corynebacterium glutamicum进行高产量利科生产的工程. 这使得利科产量增加了102倍,达到创纪录的405.02毫克/升.

科学领域:

微生物生物技术微生物生物技术
代谢工程是代谢工程.
合成生物学合成生物学

背景情况:

利科是一种强大的抗氧化类胡卜素,在医学,食品和化品中具有重要的应用.
微生物对烯的生产越来越感兴趣,Corynebacterium glutamicum (C. glutamicum) 是一个有前途的宿主.
现有的挑战包括有限的遗传工具和难以识别C. glutamicum中高产量利科生产的关键代谢基因.

研究的目的:

开发一种新的,高效的基因编辑工具包,用于C. glutamicum.
通过代谢工程来改造C. glutamicum,通过代谢工程来增强利科的生产.
为了在C. glutamicum中实现破纪录的烯产量.

主要方法:

开发和优化CRISPR/MAD7系统,以便在C. glutamicum中有效编辑基因.
使用CRISPR/MAD7同时淘汰crtEb和crtR基因,以阻止利科副产品通路.
识别和过度表达关键的胡卜素基因 (crtE,crtB,crti,idsA,idi,cg0722) 以优化甘生物合成途径.

主要成果:

克里斯普尔/MAD7系统实现了高效的基因删除,包括25kb的基因组DNA删除.
同时淘汰crtEb和crtR基因成功增强了白甘的积累.
改造后的CBIEbR菌株实现了405.02毫克/升 (9.52毫克/克DCW) 的创纪录的烯产量,提高了102倍.
编码膜蛋白的基因cg0722,被确定为生产甘烯的关键.

结论:

CRISPR/MAD7工具箱为C. glutamicum的基因工程提供了一种强大而精确的方法.
代谢工程策略,包括基因淘汰和过度表达,显著提高了柳科的产量.
这项研究证明了先进的遗传工具的成功应用,用于工业微生物生产有价值的化合物,如利科.

关键词:

在CRISPR/MAD7中使用.在Corynebacterium glutamicum的研究中,我们发现柳科的生产生产.代谢工程是代谢工程.

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