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

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.