Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food and feed applications. Chemical synthesis is presently the production method of choice for these compounds, with no amenable plant sources readily available. In the present study, the 4, 4' β-oxygenase (crtW) and 3, 3' β-hydroxylase (crtZ) genes from Brevundimonas sp SD-212. have been expressed under constitutive transcriptional control in Nicotiana glauca, which has an emerging potential as biofuel and biorefining feedstock. The transgenic lines produced significant levels of non-endogenous carotenoids in all tissues. In leaf and flower the carotenoids (ca. 0.5% dry weight) included 0.3 and 0.48 %, respectively, of non-endogenous keto 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 of Brevundimonas CRTZ and W to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester non-endogenous carotenoids and the robustness of plant metabolism to these changes.