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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation
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Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation

机译：基于转录组分析和计算机基因敲除模拟的大肠埃希氏菌代谢工程技术，用于生产L-缬氨酸

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The L-valine production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement based on transcriptome analysis and gene knockout simulation of the in silico genome-scale metabolic network. Feedback inhibition of acetohydroxy acid synthase isoenzyme Ⅲ by L-valine was removed by site-directed mutagenesis, and the native promoter containing the transcriptional attenuator leader regions of the HvGMEDA and ilvBN operon was replaced with the tac promoter. The ilvA, leuA, and panB genes were deleted to make more precursors available for L-valine biosynthesis. This engineered Val strain harboring a plasmid overexpressing the ilvBN genes produced 1.31 g/liter L-valine. Comparative transcriptome profiling was performed during batch fermentation of the engineered and control strains. Among the down-regulated genes, the lrp and ygaZH genes, which encode a global regulator Lrp and L-valine exporter, respectively, were overexpressed. Amplification of the Irp, ygaZH, and lrp-ygaZH genes led to the enhanced production of L-valine by 21.6%, 47.1 %, and 113%, respectively. Further improvement was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and pfkA genes as knockout targets. The VAMF strain (Val ΔaceF Δmdh ΔpfkA) overexpressing the ilvBN, ilvCED, ygaZH, and lrp genes was able to produce 7.55 g/liter L-valine from 20 g/liter glucose in batch culture, resulting in a high yield of 0.378 g of L-valine per gram of glucose. These results suggest that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification, transcriptome profiling, and systems-level in silico analysis.
机译：通过计算机基因组规模代谢网络的转录组分析和基因敲除模拟，通过合理的代谢工程和逐步改进，构建了大肠杆菌的L-缬氨酸生产菌株。通过定点诱变去除了L-缬氨酸对乙酰羟酸合酶同工酶Ⅲ的反馈抑制作用，并用tac启动子替换了含有HvGMEDA和ilvBN操纵子转录减弱前导区的天然启动子。删除了ilvA，leuA和panB基因，以使更多前体可用于L-缬氨酸的生物合成。带有过量表达ilvBN基因的质粒的这种工程Val菌株产生了1.31 g /升的L-缬氨酸。在工程菌株和对照菌株的分批发酵过程中进行了比较转录组分析。在下调的基因中，分别编码全局调节子Lrp和L-缬氨酸输出物的lrp和ygaZH基因过表达。 Irp，ygaZH和lrp-ygaZH基因的扩增分别导致L-缬氨酸的产量增加21.6％，47.1％和113％。通过使用计算机模拟基因敲除模拟可以实现进一步的改进，该模拟将aceF，mdh和pfkA基因鉴定为敲除靶标。过量表达ilvBN，ilvCED，ygaZH和lrp基因的VAMF菌株（ValΔaceFΔmdhΔpfkA）能够从20 g /升的葡萄糖中分批培养产生7.55 g /升的L-缬氨酸，导致0.378 g的高产量L-缬氨酸每克葡萄糖。这些结果表明，基于合理的修饰，转录组分析和系统级计算机模拟分析，可以通过代谢工程有效地开发出具有工业竞争力的菌株。

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