The Formation and Sequestration of Nonendogenous Ketocarotenoids in Transgenic Nicotiana glauca

Abstract

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 () and 3,3' β-hydroxylase (c) genes from sp. SD-212 were expressed under constitutive transcriptional control in, 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 of sp. 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.

The road to astaxanthin production in tomato fruit reveals plastid and metabolic adaptation resulting in an unintended high lycopene genotype with delayed over‐ripening properties

“…Criteria for identification have been summarized in Table S2 and further details were presented in Mortimer et al . (, ), Nogueira et al . ().…”

“…Agrobacterium ‐mediated transformation was used to introduce the crt Z and crt W genes from Brevundimonas sp. (Mortimer et al ., ) into Money Maker (MM) tomato plants. Twelve kanamycin resistant Money Maker (MM) plants were selected and the presence of the crt Z and crt W genes in the independent lines, designated MM: Crt ZW1 to 12, confirmed by PCR.…”

“…Whilst In planta, the presence of ketocarotenoids, has only been reported in the flower petals of the Adonis species (Cunningham and Gantt, 2005), which is not amenable to agricultural production. Although these natural sources do not represent viable industrial production platforms in their own right, biosynthetic genes isolated from them have been exploited in the metabolic engineering of heterologous hosts and the production of ketocarotenoids in crop plants has been reported on numerous occasions previously (Mortimer et al, 2016(Mortimer et al, , 2017Zhu et al, 2009). More recently the technical, production, and economic feasibility of engineered tomato fruit producing ketocarotenoids has been demonstrated as a new renewable feedstock for aquaculture .…”

Non‐endogenous ketocarotenoid accumulation in engineeredSynechocystissp. PCC 6803

“…SD212 have been chosen (Choi et al , ). This approach has been adopted on the basis of previous reports, which demonstrated the efficacy of Brevundimonas CrtW and CrtZ in enabling transgenic higher plants to produce large quantities of Asx (Hasunuma et al , Harada et al , , Mortimer et al , Nogueira et al ). By contrast, previous attempts at engineering ketocarotenoid biosynthesis in cyanobacteria by introducing carotenogenic enzymes from different organisms (Harker and Hirschberg ) and/or additional copies of the endogenous ones (Albers ) have not been as successful.…”

“…Moreover, it would also be interesting to determine the cellular localisation of the non‐endogenous ketocarotenoids in the engineered strains. In transgenic Asx‐accumulating tree tobacco plants, ketocarotenoids were shown to be present both in the chloroplast envelope as well as in the thylakoid membranes, predominantly as membrane components and only partially associated with photosynthetic complexes (Mortimer et al ). Replacement of endogenous carotenoids with Asx has been shown to occur in the main light‐harvesting complex of higher plants (Liguori et al ) and, similarly, protein–carotenoids binding redistribution occurred in the natural Asx‐producer H. pluvialis (Mascia et al ).…”