Engineering the Glycolytic Pathway of E. coli K12 Mutants By Gene Deletions and Introduction of a Fructose 6-Phosphate Aldolase

Wolfer M.¹, Gottlieb K. ¹, Trachtmann N. ¹, Sprenger G.A. ^1
1University of Stuttgart, Institute of Microbiology, Stuttgart, Germany

Escherichia coli K-12 is a model organism in microbiology and genetics. Its genes and enzymes which govern the central metabolic Embden-Meyerhof-Parnas (glycolysis) route and pentose phosphate pathway (PPW) are well understood [1,2]. The systematic knockout of single genes has furthermore allowed to assess essential reactions in the E. coli central metabolism [3,4]. Combination of gene knock-outs for enzymes of the upper glycolytic pathway is presented. Triple-negative mutant strains of E. coli K12 W3110 which lack both genes (pfkA, pfkB) for phosphofructokinase (PFK) and the gene pgi for phosphoglucoisomerase were created. The triple mutant strain was analyzed (in comparison with its parent strains) for its growth behaviour in LB, various mineral salt media (MM) with both, PTS- and non-PTS substrate, and on MacConkey agar plates. Whereas growth on LB and MM with fructose as C-source was nearly unaffected in the pfkA pfkB pgi triple mutant, growth on MM with C-sources which are catabolised via the intermediate, fructose 6-P, was either abolished or severely affected. Growth on glucose was still possible, presumably via the shunt to the PPW. The triple mutant was, however, severely inhibited for its growth on MacConkey agar plates containing bile acids. Efficiency of plating (eop) on MacConkey agar dropped for at least three orders of magnitude compared to the wild type strain. This reflect that the lack of PGI activity leads to an altered composition of the lipopolysaccharide (LPS) envelope of E. coli which protects these gram-negative bacteria from the effects of bile acids [5]. The most severe effect could be seen on MacConkey agar plates with 0.5% of mannitol added. The eop was reduced for more than 4 orders of magnitude in comparison to the wild type, presumably due to the combined effects of cholate sensitivity and the accumulation of fructose 6-P due to the block in PFK and PGI activities.

We transformed the triple-negative mutant strain with a plasmid-borne copy of the gene (fsaA) which encodes a fructose 6-phosphate aldolase enzyme (FSA) [6] variant with improved affinity for fructose 6-phosphate, FSA A129S [7]. Then the cells were subjected to an adaptive evolution experiment in shake flasks with minerals salts medium with fructose (0.02%) and mannitol (0.5%) as second C source. By serial transfers we could evolve strains which regained the ability to grow on mannitol as sole carbon source with a generation time of about 4 hours. Data on the in-depth analysis of this strain and its further improvement and alterations of the glycolytic bypass pathway will be presented and discussed.

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