The Formation and Sequestration of Nonendogenous Ketocarotenoids in TransgenicNicotiana glauca.
Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4' β-oxygenase (crtW) and 3,3' β-hydroxylase (crtZ) genes fromBrevundimonassp. SD-212 were expressed under constitutive transcriptional control inNicotiana glauca, which has an emerging potential as a biofuel and biorefining feedstock. The transgenic lines produced significant levels of nonendogenous carotenoids in all tissues. In leaf and flower, the carotenoids (∼0.5% dry weight) included 0.3% and 0.48%, respectively, of nonendogenous ketolated and hydroxylated carotenoids. These were 4-ketolutein, echinenone (and its 3-hydroxy derivatives), canthaxanthin, phoenicoxanthin, 4-ketozeaxanthin, and astaxanthin. Stable, homozygous genotypes expressing both transgenes inherited the chemotype. Subcellular fractionation of vegetative tissues and microscopic analysis revealed the presence of ketocarotenoids in thylakoid membranes, not predominantly in the photosynthetic complexes but in plastoglobules. Despite ketocarotenoid production and changes in cellular ultrastructure, intermediary metabolite levels were not dramatically affected. The study illustrates the utility ofBrevundimonassp. SD-212 CRTZ and CRTW to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester nonendogenous carotenoids, and the robustness of plant metabolism to these changes.
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.).
Cara L
0000-0003-2170-9556
CL
Mortimer
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.).
Norihiko
N
Misawa
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.).
Laura
0000-0002-8567-7074
L
Perez-Fons
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.).
Francesca P
0000-0001-7157-5053
FP
Robertson
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.).
Hisashi
0000-0001-9294-6968
H
Harada
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.).
Peter M
0000-0001-8204-1073
PM
Bramley
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom (C.L.M., L.P.-F., F.P.R., P.M.B., P.D.F.); and p.fraser@rhul.ac.uk.|Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan (N.M., H.H.) p.fraser@rhul.ac.uk.
Paul D
0000-0002-5953-8900
PD
Fraser
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism*; D042063:Caulobacteraceae / Q000201:enzymology / Q000235:genetics; D035264:Flowers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D046529:Microscopy, Electron, Transmission; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D018087:Plastids / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020524:Thylakoids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024341:Xanthophylls / Q000737:chemistry / Q000378:metabolism; D019207:beta Carotene / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D024341:Xanthophylls; D019207:beta Carotene; D002338:Carotenoids; C005948:astaxanthine; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C093259:beta-carotene ketolase