A Toolkit for Metabolic Engineering of Bacteria

Abstract

Dark, anaerobic hydrogen fermentations of starches and sugars have generally produced less than 20% of the stoichiometrically possible yield, compared to 80–90% yields in the case of ethanol and methane fermentations. Indeed, a 33% yield from glucose, with the remaining electrons secreted as acetate, is generally considered the maximum achievable hydrogen production. However, this restriction would not apply to non-growing cultures; in principle, non-growing cells could recycle much of the metabolic energy derived from the initial hydrogen/acetate fermentation into driving additional hydrogen production. Such improvements in hydrogen production may be possible using directed strategies to manipulate cellular metabolism—metabolic engineering.

Metabolic engineering for hydrogen production may necessitate the introduction of novel genes and coordinated expression of those genes. Once introduced into the cell, it will be necessary to optimize expression of these genes during the hydrogen production (slow- or non-growth) stage of the culture. To enable controlled and reproducible expression of the genes for a redirected metabolic pathway, a metabolic engineering toolkit has been developed for Escherichia coli. The first tool is a low-copy plasmid that exists at 1–2 copies/cell and is segregationally stable. The second tool is a strategy to introduce hairpins into the 5′-end of mRNA to control its stability. The third tool is a host organism that is unable to metabolize intracellular polyphosphates and, as a result, has improved control over gene expression from phosphate-starvation promoters. The fourth tool is a mathematical method to predict the fluxes through the metabolic pathways and to tune the expression level of the genes in the introduced pathways. Such tools should find important application in metabolic engineering for high-yield hydrogen production from organic substrates.