posted on 2024-02-22, 17:05authored byHaoJie Zhang, Tong Ye, Liu Fengmin, Xiangjun Zhang, Jipeng Wang, Xiaobo Wei, Yun Ping Neo, Huiyan Liu, Haitian Fang
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 OD600. 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.