Phytoene is a colorless carotenoid with increasing economic potential for skin care but with limited availability. The red yeast Xanthophyllomyces dendrorhous which has previously been used as a production platform for carotenoids was engineered as a prototype for the yield of this carotene. Phytoene was accumulated by prevention of its metabolization by desaturation in the carotenoid pathway. In a first step, the phytoene desaturase gene crtl was disrupted by insertion of a hygromycin-resistance gene. Most of the resulting transformants were heterozygote for intact and inactivated crtl. Upon re-cultivation of this orange transformants under selection pressure, white colonies homozygote for disrupted crtl were obtained. In contrast to reddish wild-type, the orange transformants contained colored carotenoids together with phytoene whereas the homozygote transformant synthesized phytoene exclusively. This targeted mutagenesis approach was first tested with the wild type and then applied to a high-yield carotenoid synthesizing X. dendrorhous mutant. In a second step, precursor supply for phytoene synthesis was enhanced by over-expression of the genes HMGR, crtE and crtYB which encode limiting enzymes of the pathway. The combination of this engineering approaches resulted in a phytoene producing X dendrorhous strain which accumulated 7.5 mg/g dw in shaking cultures. Finally, experimental small scale fermenter studies demonstrated continuous growth of this strain during fermentation and stable phytoene production without selection pressure. This fermenter culture contained the highest phytoene content ever reached by any organism with more than 10 mg/g dw.(C) 2017 Elsevier B.V. All rights reserved.