Abstract: The aim of this study was to improve glyoxylic acid production in Corynebacterium glutamicum ATCC 13032 by repressing the genes on the branch metabolic pathways of glyoxylic acid biosynthesis using clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology. First, the gene lldh encoding lactate dehydrogenase, the key enzyme involved in tributary metabolism, was knocked out by homologous recombination. Then the expression levels of the key enzyme isocitrate dehydrogenase gene (icd) and malate synthase gene (ms) were down-regulated by CRISPRi. At the same time, the isocitrate lyase gene (icl) was overexpressed using a free plasmid, icl-pXMJ19GZ, which increased the synthesis of glyoxylic acid. The results of fermentation experiments showed that there was almost no significant difference in the growth status of the engineered and wild-type strains. The level of glyoxylic acid in the fermentation broth of the engineered strain was as high as 5 mg/mL, which was much higher than that of the wild-type strain (almost zero). This study provides reference for the industrial production of glyoxylic acid using C. glutamicum.

Key words: Corynebacterium glutamicum ATCC 13032; glyoxylic acid; clustered regularly interspaced short palindromic repeats interference; engineered bacteria