Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies
Optimization of hydrogenobyrinic acid biosynthesis in Escherichia coli using multi-level metabolic engineering strategies
复制标题
使用多级代谢工程策略优化大肠杆菌中的氢木酸生物合成
DOI：
10.1186/s12934-020-01377-2
复制
发表时间：
2020-06
影响因子：
6.4
通讯作者：
Zhang D.
中科院分区：
文献类型：
--
作者：
Jiang P.;Fang H.;Zhao J.;Dong H.;Jin Z.;Zhang D.
背景
氢比林酸是维生素B12从头合成需氧生物合成途径的关键中间体。在大肠杆菌中引入异源从头合成维生素B12的途径为维生素B12的生产提供了另一种途径。虽然大肠杆菌避免了目前维生素B12工业生产者面临的主要限制，如生长周期长，但氢比林酸供应不足限制了工业维生素B12的生产。
结果
通过设计体内hemABCD基因的核糖体结合位点组合文库，我们发现它们的最佳相对翻译起始率为10：1：1：5。尿卟啉原III生物合成模块的转录协调是通过hemABCD操纵子的启动子工程实现的。通过RBS工程抑制竞争性的血红素和铁血红素的生物合成途径，使氢比林酸的滴度分别提高到20.54mgL−1和15.85mgmgBB1。通过对血红素和铁血红素生物合成途径的联合微调，使氢化比林酸滴度提高到22.57mgFH_215-−1，较出发菌株FH_215-HBA显著提高了1356.13%。
结论
通过多水平的代谢工程策略，实现了尿卟啉原Ⅲ生物合成途径的代谢平衡，消除了副产物积累带来的毒性，最终在大肠杆菌中获得了高达22.57mgHbA的L−1。这为在大肠杆菌中高产生产维生素B12奠定了基础，并有望加速其工业化生产。
Background
Hydrogenobyrinic acid is a key intermediate of the de-novo aerobic biosynthesis pathway of vitamin B12. The introduction of a heterologous de novo vitamin B12 biosynthesis pathway in Escherichia coli offers an alternative approach for its production. Although E. coli avoids major limitations that currently faced by industrial producers of vitamin B12, such as long growth cycles, the insufficient supply of hydrogenobyrinic acid restricts industrial vitamin B12 production.
Results
By designing combinatorial ribosomal binding site libraries of the hemABCD genes in vivo, we found that their optimal relative translational initiation rates are 10:1:1:5. The transcriptional coordination of the uroporphyrinogen III biosynthetic module was realized by promoter engineering of the hemABCD operon. Knockdown of competitive heme and siroheme biosynthesis pathways by RBS engineering enhanced the hydrogenobyrinic acid titer to 20.54 and 15.85 mg L−1, respectively. Combined fine-tuning of the heme and siroheme biosynthetic pathways enhanced the hydrogenobyrinic acid titer to 22.57 mg L−1, representing a remarkable increase of 1356.13% compared with the original strain FH215-HBA.
Conclusions
Through multi-level metabolic engineering strategies, we achieved the metabolic balance of the uroporphyrinogen III biosynthesis pathway, eliminated toxicity due to by-product accumulation, and finally achieved a high HBA titer of 22.57 mg L−1 in E. coli. This lays the foundation for high-yield production of vitamin B12 in E. coli and will hopefully accelerate its industrial production.
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DOI：
10.1051/lait:2004035
发表时间：
2005-01-01
期刊：
LAIT
影响因子：
--
作者：
Murooka, Y;Piao, Y;Yamashita, M
通讯作者：
Yamashita, M
影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
6.4
作者：
Zhang B;Zhou N;Liu YM;Liu C;Lou CB;Jiang CY;Liu SJ
通讯作者：
Liu SJ
影响因子：
5.3
作者：
通讯作者：
--
影响因子：
13.8
作者：
Martin J. Warren;A. Scott
通讯作者：
Martin J. Warren;A. Scott