Case study of xylose conversion to glycolate in Corynebacterium glutamicum: Current limitation and future perspective of the CRISPR-Cas systems

Abstract RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance the production of value-added chemicals in Corynebacterium glutamicum as an industrial host. Here, we described the biotransformation of xylose to glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic pathway involving heterologous D-tagatose 3-epimerase and L-fuculose kinase/aldolase reactions was introduced in C. glutamicum, resulting in 9.9 ± 0.01 g/L glycolate from 20 g/L xylose at a yield of 0.51 g/g (equal to 1.0 mol/mol). Additional glyoxylate reduction pathway developed by CRISPR-Cas12a recombineering has been introduced and attempted to increase the maximum theoretical molar yield of 2.0 (mol/mol). Due to the limitation of the CRISPR-Cas12a recombineering with TTTV PAM sites, advanced CRISPR-Cas systems were suggested for the next-round metabolic engineering for improving the glycolate yield to overcome the current genome-editing tool for metabolic engineering in C. glutamicum.