As an alternative carotenoid producer, non-carotenogenic Pichia pastoris was chosen for a reddish carotenoid lycopene production because it can grow to high cell density without accumulation of ethanol and utilize various classes of organic materials such as methanol as carbon sources. Two synthetic lycopene-pathway plasmids, pGAPZB-EBI* and pGAPZB-EpBpl*p, were designed and constructed. The pGAPZB-EpBpl*p plasmid encoded three carotenogenic enzymes that were engineered to be targeted into peroxisomes of P. pastonis whereas the pGAPZB-EBI* plasmid encoded non-targeted enzymes. After both plasmids were transformed into P. pastoris, the lycopene-producing clone containing the pGAPZB-EpBpl*p plasmid, referred to as Omega, was selected and used for further optimization study. Of the carbon sources tested, glucose resulted in the highest level of lycopene production in complex and minimal media. Batch fermentation of the Omega clone resulted in the production of 4.6 mg-lycopene/g-DCW, with a concentration of 73.9 mg/l of lycopene in minimal medium. For the first time non-carotenogenic yeast P. pastoris was metabolically engineered by heterologously expressing lycopene-pathway enzymes and the lycopene concentration of 73.9 mg/l was obtained. This serves as a basis for the development of biological process for carotenoids using P. pastoris at a commercial production level. (C) 2009 Elsevier Ltd. All rights reserved.