Biosynthesis of acyclic and cyclic carotenoids
Phytoene is synthesized due to crtE and crtB and acts as a substrate for desaturase enzyme crtI to produce, on successive desaturation  the following six compounds shown in. (i) Phytofluene; (ii) x carotene; (iii) neurosporene; (vi) lycopene; (v) 3,4, didehydrolycopene, and (vi) 3,4,3’4’- tetradehydrolycopene. The phytoene desaturase from Erwinia sp. though synthesized small amounts of 3,4,3’4’ tetradehydrolycopene (having six now double bonds), in the above study published in 2000, an efficient six-step desaturase was evolved to generate this major carotenoid in E. coli. For this purpose a library of desturases was generated by in vitro homologous recombination (DNA shuffling) of genes from E. uredovora and E. heribicola. The pathway was also extended with a second library of shuffled genes for lycopene cyclases (crtY) to produce a variety of carotenoid products.

A new branching pathway generating a variety of cyclic carotenoids in E. coli was also developed. One clone Y2, obtained by DNA shuffling, gave a new product torulene. This represents an example, where in vitro evolution extended the pathway with a catalytic function that is not available from a natural source.