Metabolic engineering in Escherichia coli: lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects the growth rate and yield.

Isogenic strains of Escherichia coli W3110 containing pyruvate dehydrogenase complexes with three (wild-type), two or one lipoyl domains per lipoate acetyltransferase (E2p) chain, were constructed. The maximum growth rates (μmax) for batch cultures growing in minimal medium containing different carbon sources showed that reducing the number of lipoyl domains adversely affects cell growth. The lower μmax value of the mutant containing one lipoyl domain per E2p chain was restored by the presence of compatible multicopy plasmids encoding PDH complexes with either one or three lipoyl domains per E2p chain. In glucose-limited chemostat cultures the protein contents of all strains were similar and substrate carbon was totally accounted for in the biomass and CO2 produced. However, the carbon efficiencies (percentage carbon conversion to biomass) were significantly lower when the lipoyl domain content of the E2p subunit was reduced from three to one. Similarly, the cellular maintenance energy (m e) and the maximum growth yield (Y max) were lower in bacteria containing PDH complexes with fewer than three lipoyl domains per E2p chain. Wild-type values were restored by supplementing the medium with either casamino acids (0-01%) or acetate (up to 0-1 mM). The lower growth efficiencies of the mutants were further confirmed in competition experiments where equal numbers of genetically marked (NalR) mutant and wild-type bacteria were used to inoculate glucose-limited chemostat cultures (dilution rate 0-075 h-1). The mutants with one or two lipoyl domains per E2p chain were washed out, whereas in controls, the initial ratio of wild-type (Nal) to reconstructed wild-type (NalR) bacteria was maintained over 50 generations.