Robustness of fermenting strains to lignocellulose derived inhibitors is critical for efficient biofuel and biochemical productions. In this study, the industrial fermenting strain Corynebacterium glutamicum S9114 was evolved for improved inhibitor tolerance using long-term adaptive evolution by continuously transferring into the inhibitors containing corn stover hydrolysate every 24 h, and finally a stably evolved C. glutamicum was obtained after 128 days of serial transfers. The evolved strain exhibited the highly increased conversion rate to the typical lignocellulose derived inhibitors including furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid. Glucose consumption was obviously accelerated, and 22.4 g/L of glutamic acid was achieved in the corn stover hydrolysate, approximately 68.4% greater than that by the original strain. Whole genome re-sequencing revealed various mutations with the potential connection to the improved performance of the evolved strain. Transcriptional analysis further demonstrated that the glucose-PTS transport and the pentose phosphate pathway were significantly upregulated in the evolved strain, which very likely contributed to the accelerated glucose consumption, as well as sufficient NAD(P)H supply for aldehyde inhibitors reduction conversion and thus enhanced the inhibitor tolerance. This study provided important experimental evidences and valuable genetic information for robust strain construction and modification in lignocellulose biorefining processes.
Keywords: Adaptive evolution; Corynebacterium glutamicum; Inhibitor tolerance; Lignocellulose; Transcriptional analysis; Whole genome re-sequencing.