title: Product stability and sequestration mechanisms in Solanum tuberosum engineered to biosynthesize high value ketocarotenoids.
abstract: To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4' β-oxygenase (crtW) and 3, 3' β-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules.
authors.affiliation: Centre for Systems and Synthetic Biology, School of Biological Sciences Royal Holloway University of London, Egham, Surrey, UK.
authors.forename: Cara L
authors.initials: CL
authors.lastname: Mortimer
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa, Japan.
authors.forename: Norihiko
authors.initials: N
authors.lastname: Misawa
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, UK.
authors.forename: Laurence
authors.initials: L
authors.lastname: Ducreux
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, UK.
authors.forename: Raymond
authors.initials: R
authors.lastname: Campbell
authors.affiliation: Centre for Systems and Synthetic Biology, School of Biological Sciences Royal Holloway University of London, Egham, Surrey, UK.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Bramley
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Taylor
authors.affiliation: Centre for Systems and Synthetic Biology, School of Biological Sciences Royal Holloway University of London, Egham, Surrey, UK.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Fraser
mesh_terms: D053898:Biosynthetic Pathways; D002338:Carotenoids / Q000096:biosynthesis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D016002:Discriminant Analysis; D017343:Genes, Plant; D060847:Metabolic Engineering / Q000379:methods*; D010641:Phenotype; D010788:Photosynthesis; D010858:Pigmentation / Q000235:genetics; D018515:Plant Leaves / Q000378:metabolism; D035281:Plant Tubers / Q000378:metabolism; D030821:Plants, Genetically Modified; D018087:Plastids / Q000378:metabolism / Q000648:ultrastructure; D011309:Preservation, Biological; D011198:Solanum tuberosum / Q000235:genetics / Q000378:metabolism*; D013213:Starch / Q000378:metabolism; D014170:Transformation, Genetic; D024341:Xanthophylls / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D024341:Xanthophylls; D002338:Carotenoids; C005948:astaxanthine; D013213:Starch
keywords: Solanum tuberosum; carotenoid; ketocarotenoid; metabolic engineering; sequestration; starch
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