Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli.
Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli.
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大肠杆菌l -苯丙氨酸过量产生的转录组学和代谢组学分析。
DOI：
10.1186/s12934-023-02070-w
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发表时间：
2023-04-06
影响因子：
6.4
通讯作者：
中科院分区：
文献类型：
--
作者：
通过对莽草酸（SHIK）和L-Phe分支通路的关键基因进行多轮随机诱变和修饰，大肠杆菌高效生产l -苯丙氨酸（L-Phe）。在本研究中，我们基于时间序列对产生L-Phe的工程大肠杆菌菌株进行转录组学（16,24和48 h）和代谢组学（8,16,24,32,40和48 h）分析，旨在揭示发酵过程中代谢活性的整体变化。在发酵16 h和24 h时，生物量增幅最大，产量最高，分别达到5.9 h−1和2.76 g/L/h，而L- phe滴度在发酵48 h时达到60 g/L的最大值。在发酵的不同阶段，EMP、PP、TCA、SHIIK和l - ph分支通路中涉及的deg和代谢物存在显著差异。具体来说，编码限速酶（aroD和yidB）和关键基因（aroF、pheA和aspC）的显著上调推动了更多的碳通量流向L-Phe合成。一些重要代谢物（DAHP、DHS、DHQ、Glu和PPN）的RIA变化为高产L-Phe的生产提供了充足的前体。此外，与Glc转运和磷酸盐代谢相关的其他基因增加了Glc的吸收，并有助于将碳通量重新定向到l - ph分支。对一株l -苯丙氨酸高产菌株的转录组学和代谢组学分析证实，前体供应不是该菌株的主要限制因素，而通过碳通量的重新分配（如tyrB、aspC、aroL和aroF/G/H等基因的表达强度或一定程度上提高这些酶的活性）来实现代谢通量的合理分布，是提高l -苯丙氨酸产量的最佳策略。在线版本包含补充材料，可在10.1186/s12934-023-02070-w获得。
Highly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process. The largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h−1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch. Transcriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production. The online version contains supplementary material available at 10.1186/s12934-023-02070-w.
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