title: Production of β-ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants-like raspberries and roses-by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid β-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP.
  
  
  RESULTS
  Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of β-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased β-ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of β-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase.
  
  
  CONCLUSIONS
  An efficient β-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene-the highest β-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.
authors.affiliation: Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago, Chile. jclopez1@uc.cl.
authors.forename: Javiera
authors.initials: J
authors.lastname: López
authors.affiliation: Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago, Chile. kaessus@uc.cl.
authors.forename: Karen
authors.initials: K
authors.lastname: Essus
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Gothenburg, Sweden. ilkwon.kim@pkic.co.kr.|Bio R&D Center, Paikkwang Industrial Co. Ltd., 57 Oehang-4 gil, Gunsan, Korea. ilkwon.kim@pkic.co.kr.
authors.forename: Il-kwon
authors.initials: IK
authors.lastname: Kim
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Gothenburg, Sweden. ruipereira@deb.uminho.pt.|Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal. ruipereira@deb.uminho.pt.
authors.forename: Rui
authors.initials: R
authors.lastname: Pereira
authors.affiliation: Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago, Chile. jherzog@uc.cl.
authors.forename: Jan
authors.initials: J
authors.lastname: Herzog
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Gothenburg, Sweden. siewers@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden. siewers@chalmers.se.
authors.forename: Verena
authors.initials: V
authors.lastname: Siewers
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Gothenburg, Sweden. nielsenj@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden. nielsenj@chalmers.se.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2970, Hørsholm, Denmark. nielsenj@chalmers.se.
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.affiliation: Department of Chemical and Bioprocess Engineering, School of Engineering, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, Santiago, Chile. agosin@ing.puc.cl.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Agosin
mesh_terms: D001487:Basidiomycota / Q000235:genetics; D002338:Carotenoids / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D045792:Norisoprenoids / Q000096:biosynthesis*; D032306:Petunia / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
chemical_list: D045792:Norisoprenoids; D010940:Plant Proteins; D002338:Carotenoids; C008157:beta-ionone