Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, People's Republic of China.
J Ind Microbiol Biotechnol. 2013 Dec;40(12):1461-75. doi: 10.1007/s10295-013-1342-y. Epub 2013 Oct 2.
α-Ketoglutarate is accumulated as the main byproduct during the aerobic succinate production from glycerol by Escherichia coli BL21(DE3) in minimal medium. To address this issue, here a strategy of directed pathway evolution was developed to enhance the alternative succinate production route-the glyoxylate shunt. Via the directed pathway evolution, the glyoxylate shunt was recruited as the primary anaplerotic pathway in a ppc mutant, which restored its viability in glycerol minimal medium. Subsequently, the operon sdhCDAB was deleted and the gene ppc was reverted in the evolved strain for succinate production. The resulting strain E2-Δsdh-ppc produced 30 % more succinate and 46 % less α-ketoglutarate than the control strain. A G583T mutation in gene icdA, which significantly decreased the activity of isocitrate dehydrogenase, was identified in the evolved strain as the main mutation responsible for the observed phenotype. Overexpression of α-ketoglutarate dehydrogenase complex in E2-Δsdh-ppc further reduced the amount of byproduct and improved succinate production. The final strain E2-Δsdh-ppc-sucAB produced 366 mM succinate from 1.3 M glycerol in minimal medium in fed-batch fermentation. The maximum and average succinate volumetric productivities were 19.2 and 6.55 mM h(-1), respectively, exhibiting potential industrial production capacity from the low-priced substrate.
在最小培养基中，大肠杆菌 BL21(DE3) 有氧发酵甘油生产琥珀酸时，α-酮戊二酸积累为主要副产物。为了解决这个问题，我们开发了一种定向途径进化策略，以增强替代琥珀酸生产途径——乙醛酸支路。通过定向途径进化，乙醛酸支路被招募为 ppc 突变体中的主要氨同化途径，这恢复了其在甘油最小培养基中的生存能力。随后，删除了 operon sdhCDAB 并在进化菌株中恢复了基因 ppc 用于琥珀酸生产。与对照菌株相比，所得菌株 E2-Δsdh-ppc 产生的琥珀酸增加了 30%，α-酮戊二酸减少了 46%。在进化菌株中发现了基因 icdA 中的 G583T 突变，该突变显著降低了异柠檬酸脱氢酶的活性，是导致观察到的表型的主要突变。在 E2-Δsdh-ppc 中过表达α-酮戊二酸脱氢酶复合物进一步减少了副产物的量并提高了琥珀酸的产量。最终的菌株 E2-Δsdh-ppc-sucAB 在分批补料发酵中从 1.3 M 甘油的最小培养基中生产 366 mM 琥珀酸。最大和平均琥珀酸比生产率分别为 19.2 和 6.55 mM·h(-1)，展示了从低价底物生产的潜在工业生产能力。
