利用CRISPR-Cas9和Cre/loxP系统删除34个非必需基因构建L-苏氨酸生产菌

Construction of L-Threonine-Producing Escherichia coli by Deleting 34 Non-Essential Genes Using CRISPR-Cas9 and Cre/loxP System

摘要:

L-苏氨酸是一种被广泛应用于食品、饲料及医药等领域的必需氨基酸。大肠杆菌以葡萄糖为碳源合成L-苏氨酸的过程中，一些非必需基因的转录和翻译会消耗碳源。利用CRISPR-Cas9和位点特异性重组系统Cre/loxP，敲除了大肠杆菌MG1655基因组中puuE和ynaI区间的34个非必需基因（共30.372 kb），获得了突变菌MG003，然后通过高表达L-苏氨酸合成途径中关键基因thrA*、thrB和thrC以及L-苏氨酸转运酶编码基因rhtA和rhtC提高L-苏氨酸产量。与对照菌MG1655/pFW01-thrA*BC相比，MG003/pFW01-thrA*BC生长加快，L-苏氨酸产量提高25.5%；MG003/pFW01-thrA*BC-rhtA的L-苏氨酸产量提高了43.3%；MG003/pFW01-thrA*BC-rhtC的L-苏氨酸产量提高了74.5%。研究结果表明，大肠杆菌基因组中34个非必需基因的删除有利于其提高L-苏氨酸合成能力。

Abstract:

L-threonine is an essential amino acid widely used in food, feed and medicine. In the process of L-threonine synthesis from glucose, transcription and translation of some non-essential genes consume carbon source. In this study, CRISPR-Cas9 and site-specific recombination system Cre/loxP were used to delete 34 non-essential genes (30.372 kb) between puuE and ynaI genes in the genome of E.coli MG1655, resulting the mutant strain MG003. The genes thrA*, thrB and thrC, the key genes in the L-threonine biosynthetic pathway, and the genes rhtA and rhtC encoding the L-threonine transporters, were then overexpressed in MG003 to increase the L-threonine production. Compared to the control strain MG1655/pFW01-thrA*BC, MG003/pFW01-thrA*BC grew faster and produced 25.5% more L-threonine, MG003/pFW01-thrA*BC-rhtA produced 43.3% more L-threonine, and MG003/pFW01-thrA*BC-rhtC produced 74.5%more L-threonine. The results indicate that deletion of the 34 non-essential genes in E. coli could improve L-threonine production.