ALE for improving ethanol utilization by Corynebacterium glutamicum

Adaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Here, ALE in a well-controlled microbioreactor environment using a microtiter plate recycling approach was used to evolve and improveethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT). The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 mmolETH/gCDW/h and a growth rate of 0.15/h in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH).The mutated region was previously predicted to be a binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of the repressor GlxR leads to increased expression of the rate-limiting enzyme ALDH.