Directed pathway evolution of the glyoxylate shunt in Escherichia coli for improved aerobic succinate production from glycerol
- 905 Downloads
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.
KeywordsEscherichia coli Glycerol Succinate Glyoxylate shunt Isocitrate dehydrogenase
This work was supported by National 973 Project (2011CBA00804, 2012CB725203), National Natural Science Foundation of China (NSFC-21176182, NSFC-21206112) and National High-tech R&D Program of China (2012AA02A702, 2012AA022103).
- 19.Lee PC, Lee WG, Lee SY, Chang HN (2001) Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. Biotechnol Bioeng 72(1):41–48. doi: 10.1002/1097-0290(20010105)72:1<41:aid-bit6>3.0.co;2-n PubMedCrossRefGoogle Scholar
- 20.Lin H, Bennett GN, San K-Y (2005) Chemostat culture characterization of Escherichia coli mutant strains metabolically engineered for aerobic succinate production: a study of the modified metabolic network based on metabolite profile, enzyme activity, and gene expression profile. Metab Eng 7(5–6):337–352. doi: 10.1016/j.ymben.2005.06.002 PubMedCrossRefGoogle Scholar
- 29.Noronha SB, Yeh HJC, Spande TF, Shiloach J (2000) Investigation of the TCA cycle and the glyoxylate shunt in Escherichia coli BL21 and JM109 using 13C-NMR/MS. Biotechnol Bioeng 68(3):316–327. doi: 10.1002/(sici)1097-0290(20000505)68:3<316:aid-bit10>3.0.co;2-2 PubMedCrossRefGoogle Scholar
- 40.Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, Anthony LC, Regentin R, Keasling JD, Renninger NS, Newman JD (2009) High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin Escherichia coli. PLoS One 4(2):e4489. doi: 10.1371/journal.pone.0004489 PubMedCrossRefGoogle Scholar
- 45.Yuzbashev TV, Yuzbasheva EY, Sobolevskaya TI, Laptev IA, Vybornaya TV, Larina AS, Matsui K, Fukui K, Sineoky SP (2010) Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica. Biotechnol Bioeng 107(4):673–682. doi: 10.1002/bit.22859 PubMedCrossRefGoogle Scholar