title: Development of a multi-gene expression system in Xanthophyllomyces dendrorhous.
abstract: BACKGROUND
  Red yeast, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) that is widely used in the aquaculture, food, pharmaceutical and cosmetic industries. Recently, 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. Addition of mevalonate, the common precursor for isoprenoid biosynthesis, has been shown to be critical to enhance the astaxanthin content in X. dendrorhous. However, addition of mevalonate is unrealistic during industrial isoprenoid production because it is an unstable and costly chemical. Therefore, up-regulating the intracellular mevalonate supply by enhancing the mevalonate synthetic pathway though genetic engineering is a promising strategy to improve isoprenoid production in X. dendrorhous. However, a system to strongly express multiple genes has been poorly developed for X. dendrorhous.
  
  
  RESULTS
  Here, we developed a multiple gene expression system using plasmids containing three strong promoters in X. dendrorhous (actin, alcohol dehydrogenase and triose-phosphate isomerase) and their terminators. Using this system, three mevalonate synthetic pathway genes encoding acetoacetyl-CoA thiolase, HMG-CoA synthase and HMG-CoA reductase were overexpressed at the same time. This triple overexpressing strain showed an increase in astaxanthin production compared with each single overexpressing strain. Additionally, this triple overexpression of mevalonate synthetic pathway genes together with genes involved in β-carotene and astaxanthin synthesis showed a synergetic effect on increasing astaxanthin production. Finally, astaxanthin production was enhanced by 2.1-fold compared with the parental strain without a reduction of cell growth.
  
  
  CONCLUSIONS
  We developed a system to strongly overexpress multiple genes in X. dendrorhous. Using this system, the synthetic pathway of mevalonate, a common substrate for isoprenoid biosynthesis, was enhanced, causing an increase in astaxanthin production. Combining this multiple gene overexpression system with a platform strain that overproduces mevalonate has the potential to improve industrial production of various isoprenoids in X. dendrorhous.
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. kiyotaka.hara@dolphin.kobe-u.ac.jp.
authors.forename: Kiyotaka Y
authors.initials: KY
authors.lastname: Hara
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. tshkmrt777@gmail.com.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Morita
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. m.mochizuki@dolphin.kobe-u.ac.jp.
authors.forename: Masao
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. bbk10-ttyynmk@hotmail.co.jp.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. ochiaki@port.kobe-u.ac.jp.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Ogino
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. araki@port.kobe-u.ac.jp.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Araki
authors.affiliation: 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.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D001487:Basidiomycota / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D060847:Metabolic Engineering / Q000379:methods*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism