<br>通过改变大肠杆菌中苏氨酸代谢途径增强 L-异亮氨酸合成的策略,ACS Omega

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通过改变大肠杆菌中苏氨酸代谢途径增强 L-异亮氨酸合成的策略
ACS Omega ( IF 3.7 ) Pub Date : 2024-02-22 , DOI: 10.1021/acsomega.3c07619
HaoJie Zhang ₁ , Tong Ye ₁ , Liu Fengmin ₁ , Xiangjun Zhang _{1,

2} , Jipeng Wang ₁ , Xiaobo Wei ₁ , Yun Ping Neo ₃ , Huiyan Liu ₁ , Haitian Fang ₁

Affiliation

L-苏氨酸作为L-异亮氨酸的重要前体物质，提高其在大肠杆菌中的积累成为构建高产L-异亮氨酸基础菌株的重要思路。与此同时，大肠杆菌中L-苏氨酸代谢途径的破坏对于提高L-异亮氨酸产量的影响仍未被揭示。在本研究中，通过灭活与l-苏氨酸代谢相关但与l-异亮氨酸合成无关的特定代谢途径（例如，Δ tdh 、Δ ltaE和Δ yiaY ）来设计大肠杆菌突变株。这样做的目的是减少L-苏氨酸的分解，从而增加L-异亮氨酸的产量。结果表明，与原始菌株相比，突变菌株的l-异亮氨酸产量从 4.34 g·L ^–1增加了 72.3%，并且在 OD ₆₀₀处测得的细菌生长意外增加了 10.3%。本研究还对突变菌株NXU102和原始菌株NXU101进行了转录组分析，以全面了解其生理属性。研究结果显示，两个菌株之间的 1294 个基因存在显着差异，其中 658 个基因表现出上调，636 个基因表现出下调。发现三羧酸（TCA）循环相关基因的活性降低，但氧化磷酸化相关基因高度上调，这解释了突变菌株的活性增加。例如， L-赖氨酸分解代谢相关基因被发现上调，从而重新配置进入 TCA 循环的碳流。乙酸降解途径相关基因的增强有助于减少可能阻碍细胞生长的乙酸积累。值得注意的是，天冬氨酸途径中大多数基因的大幅上调可能是本研究中L-异亮氨酸产量增加的原因。本文通过切断苏氨酸代谢途径，成功构建了L-异亮氨酸产量为7.48 g·L ^–1的底盘菌株。同时，转录组分析表明，苏氨酸代谢途径的切断会引起与L-异亮氨酸合成相关途径相关的基因的扰动，例如三羧酸循环、糖酵解和天冬氨酸途径。

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Strategies to Enhance l-Isoleucine Synthesis by Modifying the Threonine Metabolism Pathway in Escherichia coli

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.

更新日期：2024-02-22

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