Abstract
l-threonine as an important precursor substance of l-isoleucine and improving its accumulation in Escherichia coli became an important idea to construct a chassis strain with high l-isoleucine production. Meanwhile, the effect of l-threonine metabolic pathway disruption in E. coli for the improved production of l-isoleucine remains unrevealed. In the present study, a mutant strain of E. coli was engineered by inactivating specific metabolic pathways (e.g., Δtdh, ΔltaE, and ΔyiaY) that were associated with l-threonine metabolism but unrelated to l-isoleucine synthesis. This was done with the aim to reduce the breakdown of l-threonine and, thereby, increase the production of l-isoleucine. The results obtained demonstrated a 72.3% increment in l-isoleucine production from 4.34 to 7.48 g·L(-1) in the mutant strain compared with the original strain, with an unexpected 10.3% increment in bacterial growth as measured at OD(600). Transcriptome analysis was also conducted on both the mutant strain NXU102 and the original strain NXU101 in the present study to gain a comprehensive understanding of their physiological attributes. The findings revealed a notable disparity in 1294 genes between the two strains, with 658 genes exhibiting up-regulation and 636 genes displaying down-regulation. The activity of tricarboxylic acid (TCA) cycle-related genes was found to decrease, but oxidative phosphorylation-related genes were highly up-regulated, which explained the increased activity of the mutant strain. For instance, l-lysine catabolism-related genes were found to be up-regulated, which reconfigured the carbon flow into the TCA cycle. The augmentation of acetic acid degradation pathway-related genes assisted in the reduction in acetic acid accumulation that could retard cell growth. Notably, substantial up-regulation of the majority of genes within the aspartate pathway could potentially account for the increased production of l-isoleucine in the present study. In this paper, a chassis strain with an l-isoleucine yield of 7.48 g·L(-1) was successfully constructed by cutting off the threonine metabolic pathway. Meanwhile, transcriptomic analysis revealed that the cutting off of the threonine metabolic pathway induced perturbation of genes related to the pathways associated with the synthesis of l-isoleucine, such as the tricarboxylic acid cycle, glycolysis, and aspartic acid pathway.