Escherichia coli is a non-carotenogenic bacterium that could synthesize farnesyl pyrophosphate precursor through the isoprenoid pathway. Carotenoid production in E. coli requires heterologous expression of carotenoid synthesis genes. The carotenoid synthesis operons are assembled from genes isolated from carotenogenic bacterial sources. Expression of the different operons yields different carotenoid titers. The operons containing the idi gene give more than fivefold higher carotenoid titers than the operons lacking the idi gene. The carotenoid modification genes encoding ketolases and hydroxylases are incorporated into the operons for canthaxanthin and astaxanthin production. The ketolases and hydroxylases from different bacterial sources produce astaxanthin of different purity relative to the total carotenoids. Expression of the ketolases and hydroxylases closer to the promoter appears to give higher astaxanthin purity than expression farther from the promoter at the end of the operons. Balanced expression of ketolases and hydroxylases is critical to achieve high astaxanthin purity. Here, we describe methods to assemble carotenoid biosynthesis operons from carotenogenic gene clusters isolated from different bacterial sources and evaluate canthaxanthin or astaxanthin production in E. coli.