title: Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma.
abstract: BACKGROUND
  A moderate-temperature, astaxanthin-overproducing mutant strain (termed MK19) of Phaffia rhodozyma was generated in our laboratory. The intracellular astaxanthin content of MK19 was 17-fold higher than that of wild-type. The TLC profile of MK19 showed a band for an unknown carotenoid pigment between those of β-carotene and astaxanthin. In the present study, we attempted to identify the unknown pigment and to enhance astaxanthin synthesis in MK19 by overexpression of the crtS gene that encodes astaxanthin synthase (CrtS).
  
  
  RESULTS
  A crtS-overexpressing strain was constructed without antibiotic marker. A recombinant plasmid with lower copy numbers was shown to be stable in MK19. In the positive recombinant strain (termed CSR19), maximal astaxanthin yield was 33.5% higher than MK19, and the proportion of astaxanthin as a percentage of total carotenoids was 84%. The unknown carotenoid was identified as 3-hydroxy-3',4'-didehydro-β,Ψ-carotene-4-one (HDCO) by HPLC, mass spectrometry, and NMR spectroscopy. CrtS was found to be a bifunctional enzyme that helped convert HDCO to astaxanthin. Enhancement of crtS transcriptional level increased transcription levels of related genes (crtE, crtYB, crtI) in the astaxanthin synthesis pathway. A scheme of carotenoid biosynthesis in P. rhodozyma involving alternative bicyclic and monocyclic pathways is proposed.
  
  
  CONCLUSIONS
  CrtS overexpression leads to up-regulation of synthesis-related genes and increased astaxanthin production. The transformant CSR19 is a stable, secure strain suitable for feed additive production. The present findings help clarify the regulatory mechanisms that underlie metabolic fluxes in P. rhodozyma carotenoid biosynthesis pathways.
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. 724697350@qq.com.
authors.forename: Shuang
authors.initials: S
authors.lastname: Chi
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. heyanfeng127@qq.com.
authors.forename: Yanfeng
authors.initials: Y
authors.lastname: He
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. 1030462776@qq.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Ren
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. liz.su0920@gmail.com.
authors.forename: Qian
authors.initials: Q
authors.lastname: Su
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. liuxingchaode2008@163.com.
authors.forename: Xingchao
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. chenzhi@cau.edu.cn.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Applied Chemistry, College of Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. wangma@cau.edu.cn.
authors.forename: Mingan
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. yingli528@vip.sina.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratories for Agro-biotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, People's Republic of China. yingli902@163.com.
authors.forename: Jilun
authors.initials: J
authors.lastname: Li
mesh_terms: D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways; D002338:Carotenoids / Q000096:biosynthesis; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D060847:Metabolic Engineering; D024341:Xanthophylls / Q000096:biosynthesis
chemical_list: D005656:Fungal Proteins; D024341:Xanthophylls; D002338:Carotenoids; C005948:astaxanthine