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 beta-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (P-T5). We recombined PCR fragments with the lambda-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 PI transductions into a host strain harboring a reporter plasmid containing beta-carotene biosynthesis genes allowing a visual screen for yellow color indicative of beta-carotene accumulation. Construction of an E.Coli P-T5-dxs P-T5-ispDispF P-T5-idi P-T5-ispB strain resulted in producing high titers (6 mg/g dry cell weight) of beta-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 beta-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of beta-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. (c) 2005 Elsevier Inc. All rights reserved.