Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli

For metabolic engineering it is advantageous in terms of stability, genetic regulation, and metabolic burden to modulate expression of relevant genes on the chromosome rather than relying on over-expression of the genes on multi-copy vectors. Here we have increased the production of β-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (PT5). We recombined PCR fragments with the λ-Red recombinase to effect chromosomal promoter replacement, which allows direct integration of a promoter along with a selectable marker that can subsequently be excised by the Flp/FRT site-specific recombination system. The resulting promoter-engineered isoprenoid genes were combined by serial P1 transductions into a host strain harboring a reporter plasmid containing β-carotene biosynthesis genes allowing a visual screen for yellow color indicative of β-carotene accumulation. Construction of an E. coli PT5-dxs PT5-ispDispF PT5-idi PT5-ispB strain resulted in producing high titers (6 mg/g dry cell weight) of β-carotene. Surprisingly, over-expression of the ispB gene, which was expected to divert carbon flow from the isoprenoid pathway to quinone biosynthesis, resulted in increased β-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of β-carotene in a non-carotenogenic E. coli. The high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes.