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Background  Red yeast,  Xanthophyllomyces dendrorhous  is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in  X. dendrorhous  and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in  X .  dendrorhous , however, has not yet been developed.    Results   Xanthophyllomyces dendrorhous  was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double  CYP61  genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double  CYP61  deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single  CYP61  disrupted strain. Additionally, this double deletion of  CYP61  genes showed increased astaxanthin production compared with the parental strain and the single  CYP61  knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single  CYP61  knockout strain.    Conclusions  In this study, we developed a system to completely delete target diploid genes in  X. dendrorhous . Using this method, we deleted diploid  CYP61  genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in  X. dendrorhous .    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-016-0556-x) contains supplementary material, which is available to authorized users.

Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes