Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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An integrated view on the metabolic regulation in response to culture environments and the specific gene knockout in Escherichia coli

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2009.08.334 ◽

2009 ◽

Vol 108 ◽

pp. S114

Author(s):

Kazuyuki Shimizu

Keyword(s):

Escherichia Coli ◽

Metabolic Regulation ◽

Gene Knockout ◽

Specific Gene

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Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.7880-7887.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 7880-7887 ◽

Cited By ~ 228

Author(s):

Sang Jun Lee ◽

Dong-Yup Lee ◽

Tae Yong Kim ◽

Byung Hun Kim ◽

Jinwon Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Succinic Acid ◽

In Silico ◽

Acid Production ◽

Gene Knockout ◽

Fermentation Products ◽

E Coli ◽

Metabolic Analysis ◽

Enhanced Production ◽

Succinic Acid Production

ABSTRACT Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.

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