Canthaxanthin has significant commercial value due to its use as an antioxidant and feed additive in aquaculture and poultry farms. The biological production of high-value carotenoids, such as canthaxanthin, represents a significant challenge. Presented here is an Escherichia coli bioprocess, which includes the recombinant production of a strain, a novel medium and a fermentation process, for the efficient biosynthesis of canthaxanthin. The chromosomal overexpression of the E. coli genes Idi, Dxs and Dxr increased the carbon flux into the plasmid-based canthaxanthin biosynthetic pathway, achieving a total carotenoid productivity of 11.7 mg/g (91% canthaxanthin). The development of a novel culture medium increased the productivity 2.9-fold to 78.2 mg/L total carotenoids. This high-titer platform was subsequently employed for the analysis of mutations known to be advantageous to carotenoid biosynthesis, providing the first such evaluation in a high titer background. This demonstrated that thee increased expression of IspB or the deletion of PykF, PykA or YtjC improved the total carotenoid yield, whereas the increased expression of Pps and Ppc, was not improve productivity. This analysis resulted in an increase in the carotenoid yield to 16.1 mg/g. The use of 1 L lab-scale fermentations allowed for further improvements in productivity, and 170 mg/L total carotenoids was obtained. (C) 2012 Elsevier Ltd. All rights reserved.