Biosynthesis of Carotenoids in Plants: Enzymes and Color.
The need for CRTI apparently conflicts with the presence of PDS and ZDS transcripts in wild-type endosperm, as shown by quantitative RT-PCR analyses. This could be due to low level presence of the enzyme rather than its mRNA. Due to the low-level expression, the complex reaction mechanisms of PDS and ZDS, and the unavailability of radioactive carotene substrates, the investigations were done using a transgenic approach rather than in vitro reactions. The tissue-specific expression of the PDS/ZDS system, instead of CRTI, in rice endosperm resulted in the formation of coloured carotenoids, showing that the rice endosperm provides the complex requirements for the activity of the plant desaturases.
Biosynthesis of Carotenoids in Plants: Enzymes and …
Regulation of two carotenoid biosynthesis genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis.
Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtii.
Genes and enzymes of carotenoid biosynthesis in ..
These efforts led to the development of what we could call the first generation of Golden Rice (after the proof of concept), also known as GR1. This version only contained the phytoene synthase (Psy) gene from daffodil and the carotene desaturase (CrtI) gene from the bacterium Pantoea ananatis (previously known as Erwinia uredovora). Further, in this early version both transgenes were expressed only in the rice endosperm (by placing the genes under the control of the endosperm-specific gt1 promoter). The levels of carotenoids obtained in the field amounted to an average of 6 μg/g (about 4 times higher than the prototype), probably due the availability of large numbers of transformation events to select from and the use of the tissue-specific gt1 promoter to drive CRTI expression, while the constitutive 35S promoter had been used in the proof-of-concept prototype.
Rewiring the grain Metabolic engineering
The first generation of Golden Rice showed that it was possible to produce provitamin A in rice grains, but it was recognised that to combat vitamin A deficiency more higher β-carotene levels would be required. As only two biosynthetic transgenes are required in the process, the logical approach was to identify the bottleneck of the biosynthetic pathway and fine-tune the enzymatic activities of the two gene products involved, phytoene-synthase (PSY) and carotene-desaturase (CRTI).
Acyrthosiphon pisum - Wikipedia
The primary sequence of CRTI is unrelated to the plant-type desaturases. Its structure has been partially resolved and the reaction mechanism investigated (Schaub et al., 2012). Clearly, CRTI is simpler than plant-type desaturases. CRTI employs molecular oxygen directly as an electron acceptor, while the plant enzyme utilizes plastoquinone for this purpose, and is thus linked to and dependent on complex redox-chains (Beyer et al., 1989; Mayer et al., 1990; Nievelstein et al., 1995). This electron path is also indirectly linked to molecular oxygen as the terminal electron acceptor via an oxidase identified through the immutans mutation of Arabidopsis (for review, see Kuntz, 2004). This redox pathway is especially important in non-green carotenoid-bearing tissues, while the photosynthetic electron transport is thought to play an analogous role in chloroplasts. Moreover, CRTI does not form poly-cis-configured intermediates, as plant desaturases do (Bartley et al., 1999), and therefore, cis-trans isomerases (cf Fig. 2) are not required. CRTI is also capable of introducing all four double bonds in one step.
Phytohormones and their metabolic engineering for …
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.