The physiology of carotenogenesis

30. Sedkova N, Tao L, Rouvière PE, Cheng Q. Diversity of carotenoid synthesis gene clusters from environmental strains. 2005;17:8141-8146

A higher-plant type zeta-carotene desaturase in the cyanobacterium Synechocystis PCC6803.

13. Linden H, Misawa N, Saito T, Sandmann G. A novel carotenoid biosynthesis gene coding for ζ-carotene desaturase: functional expression, sequence and phylogenetic origin. 1994;24:369-379

Commercial production of β-carotene from Dunaliella salina

Analysis of dry weight (DW), titratable acidity (acidity), soluble solids and conductivity were performed on fruit homogenate obtained by blending the fruits in a food processor. To measure DW, a sub-sample of the homogenate was weighed and dried in an oven set at 70°C until the weight was constant. Values were expressed as grams per 100 g of fresh weight (FW). The other analytical measurements were carried out on the liquid portion obtained after centrifuging the homogenate. Acidity was determined by titration with a Metrohm 654 pH-meter (Metrohm Ltd, Switzerland) and values were expressed in mEq of citric acid per 100 g. Soluble solids were determined using the digital refractometer ATAGO DBX-55 (ATAGO CO. LTD, Japan), which provides values as Brix degrees (°B). Conductivity was measured with the Metrohm Conductometer 660 (Metrohm Ltd, Switzerland) and expressed as μS/ cm. Carotenoids were extracted from ripe tomato fruits and analysed by HPLC, as described previously (D’Ambrosio et al. ).

Carotenoid biosynthesis in the primitive red alga Cyanidioschyzon ..

All plant tissues that accumulate high levels of carotenoids have mechanisms for carotenoid sequestration, including crystallisation, oil deposition, membrane proliferation or protein-lipid sequestration. It has been shown that lipid accumulation can be a driver of carotenoid formation by acting as a lipophilic sink (Rabbani et al., 1998). The non-carotenogenic starchy rice endosperm, on the other hand, is very low in lipid and apparently lacks any such means for carotenoid deposition. It was also doubtful whether would have the necessary precursors for carotene biosynthesis present and available in the grains, with many believing that the whole, multi-step carotenoid biosynthetic pathway was completely absent from the endosperm.

the production of beta-carotene and xanthophylls in plants

This explains why a long research phase preceded the achievement of the proof-of-concept for Golden Rice. By the early 1990s, the data accumulated became encouraging enough for Profs Peter Beyer and Ingo Potrykus to gather forces and dare to tackle this feat. Their breakthrough showed that only two transgenes were required to turn Golden Rice into a reality (Ye et al., 2000). The first transgene encodes a plant phytoene synthase (PSY), which utilises the endogenously synthesised geranylgeranyl-diphosphate (GGPP) to form phytoene, a colourless carotene with a triene chromophore (Burkhardt et al., 1997). The second gene encodes a bacterial carotene desaturase (CRTI) that introduces conjugation by adding four double bonds. Between 1993 and 1999, collaborative research between Peter Beyer and Prof Peter Bramley (Royal Holloway College, UK), was funded through EU networks B102-CT-930400, B104-CT97-2077 and FAIR CT96 1633. Working with genetically modified tomatoes, Peter Bramley established the advantage of using a single phytoene desaturase gene (bacterial CRTI), rather than introducing multiple plant desaturases (Romer et al., 2000). The combined activity of PSY and CRTI leads to the formation of lycopene, which is a red compound, its colour stemming from its undecaene chromophore, as is well established in tomato fruit. However, lycopene has never been observed in any rice transformant and different genetic backgrounds. Instead, α- and β-carotene are found together with variable amounts of oxygenated carotenoids (xanthophylls), such as lutein and zeaxanthin. The carotenoid pattern observed in the grain's endosperm revealed that the pathway proceeded beyond the end point expected from the enzymatic action of the two transgenes alone. A detailed analysis of the underlying mechanism has been published (Schaub et al., 2005). The findings are explained in some detail below (Figure 2).

Metabolic engineering of carotenoid biosynthesis in ..

The explanation is that enzymes further down the pathway, such as lycopene cyclases (LCYs) and α- and β-carotene hydroxylases (HYDs), are still being produced in wild-type rice endosperm, while PSY and one or both of the plant carotene desaturases —phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS)— as well as the cis-trans isomerases, namely ζ-carotene cis-trans Isomerase (Z-ISO; Chen et al., 2010) and carotene cis-trans isomerase (CRTISO; Isaacson et al., 2002; Park et al., 2002; Yu et al., 2011) are not. Synthesis of lycopene by PSY and CRTI in transgenic plants provides the substrate for these downstream enzymes and consequently enables the formation of the observed products.