title: Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli.
abstract: 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 P1 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 (6mg/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.
authors.affiliation: DuPont Central Research and Development, Experimental Station, Wilmington, DE 19880, USA.
authors.forename: Luke Z
authors.initials: LZ
authors.lastname: Yuan
authors.forename: Pierre E
authors.initials: PE
authors.lastname: Rouvière
authors.forename: Robert A
authors.initials: RA
authors.lastname: Larossa
authors.forename: Wonchul
authors.initials: W
authors.lastname: Suh
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D019207:beta Carotene / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D019207:beta Carotene