Enhancement of astaxanthin production in Xanthophyllomyces dendrorhous by efficient method for the complete deletion of genes.
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.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan.
Keisuke
K
Yamamoto
Division of Environmental and Life Sciences, Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526, Japan.|Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan.
Kiyotaka Y
KY
Hara
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan.
Toshihiko
T
Morita
Medical Device Development Laboratories, Kaneka Corporation, Takasago, Hyogo, Japan.
Akira
A
Nishimura
Graduate School of Science, Technology, and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
Daisuke
D
Sasaki
Graduate School of Science, Technology, and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
Jun
J
Ishii
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan.
Chiaki
C
Ogino
Biotechnology Development Laboratories, Kaneka Corporation, Takasago, Hyogo, Japan.
Noriyuki
N
Kizaki
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan. akondo@kobe-u.ac.jp.|Graduate School of Science, Technology, and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan. akondo@kobe-u.ac.jp.
Akihiko
A
Kondo
D001483:Base Sequence; D001487:Basidiomycota / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics*; D002338:Carotenoids / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics; D004875:Ergosterol / Q000096:biosynthesis; D005285:Fermentation; D017353:Gene Deletion; D015870:Gene Expression; D005800:Genes, Fungal; D060847:Metabolic Engineering / Q000379:methods*; D008798:Mevalonic Acid / Q000378:metabolism; D016133:Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000378:metabolism; D024341:Xanthophylls / Q000096:biosynthesis / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D013729:Terpenes; D024341:Xanthophylls; D002338:Carotenoids; C005948:astaxanthine; D003577:Cytochrome P-450 Enzyme System; C066577:ERG5 protein, S cerevisiae; D008798:Mevalonic Acid; D004875:Ergosterol
Astaxanthin production; Gene deletion; Metabolic engineering; Xanthophyllomyces dendrorhous; Yeast