Metabolic engineering of novel ketocarotenoid production in carrot plants

Abstract

Carotenoids constitute a vast group of pigments that are ubiquitous throughout nature. Carrot (Daucus carota L.) roots provide an important source of dietary beta-carotene (provitamin A), alpha-carotene and lutein. Ketocarotenoids, such as canthaxanthin and astaxanthin, are produced by some algae and cyanobacteria but are rare in plants. Ketocarotenoids are strong antioxidants that are chemically synthesized and used as dietary supplements and pigments in the aquaculture and neutraceutical industries. We engineered the ketocarotenoid biosynthetic pathway in carrot tissues by introducing a beta-carotene ketolase gene isolated from the alga Haematococcus pluvialis. Gene constructs were made with three promoters (double CaMV 35S, Arabidopsis-ubiquitin, and RolD from Agrobacterium rhizogenes). The pea Rubisco small sub-unit transit peptide was used to target the enzyme to plastids in leaf and root tissues. The phosphinothricin acetyl transferase (bar) gene was used as a selectable marker. Following Agrobacterium-mediated transformation, 150 plants were regenerated and grown in a glasshouse. All three promoters provided strong root expression, while the double CaMV 35S and Ubiquitin promoters also had strong leaf expression. The recombinant ketolase protein was successfully targeted to the chloroplasts and chromoplasts. Endogenous expression of carrot beta-carotene hydroxylases was up-regulated in transgenic leaves and roots, and up to 70% of total carotenoids was converted to novel ketocarotenoids, with accumulation up to 2,400 microg/g root dry weight. Astaxanthin, adonirubin, and canthaxanthin were most prevalent, followed by echinenone, adonixanthin and beta-cryptoxanthin. Our results show that carrots are suitable for biopharming ketocarotenoid production for applications to the functional food, neutraceutical and aquaculture industries.

Astaxanthin in microalgae: pathways, functions and biotechnological implications

“…A similar effort was made with a carrot plant, resulting in a comparable amount (ca. 916 μg g -1 dwt) of astaxanthin in root tissues (Jayaraj et al 2008). Expression of a Chlamydomonas BKT into Arabidopsis led to the accumulation of greater amounts of astaxanthin (up to 2,000 μg g -1 dwt) in leaves of the transgenic plant, whereas the expression of C. zofingiensis BKT only resulted in 240 μg astaxanthin g -1 dwt .…”

Carotenoids in nature: insights from plants and beyond

“…Underground food crops, such as potato and carrot have also been engineered to have increased carotenoid content, in particular b-carotene and ketocarotenoids (e.g. astaxanthin), which produce a deep yellow ('golden') potato tuber phenotype and a distinct pink to deep red colour compared with the typical orange colour in wild-type carrot roots (Taylor and Ramsay 2005;Diretto et al 2007;Jayaraj et al 2008). Finally, the overexpression of PSY in Arabidopsis increased carotenoid content by 10-to 100-fold in nonphotosynthetic calli and roots, predominantly b-carotene and its derivatives, which were deposited as crystals in storage plastids (Maass et al 2009).…”

Pathway engineering of plants toward astaxanthin production

“…This finding coincides with our results of the complementation experiments using E. coli transformants. As for practical plants, several ketolase genes were tried to be expressed in several crops, i.e., tomato, potato and carrot plants (Rally et al 2004;Morris et al 2006;Gerjets and Sandmann 2006;Jayaraj et al 2008). The bkt1 and crtO genes were individually introduced into potato plants, and resultant transgenic plants were found to produce ketocarotenoids including astaxanthin in the tuber tissues (Morris et al 2006;Gerjets and Sandmann 2006), as shown in Table 1.…”

“…The bkt1 and crtO genes were individually introduced into potato plants, and resultant transgenic plants were found to produce ketocarotenoids including astaxanthin in the tuber tissues (Morris et al 2006;Gerjets and Sandmann 2006), as shown in Table 1. Jayaraj et al (2008) expressed the bkt1 gene in carrot roots using the double CaMV 35S promoter, and found that the transgenic roots were able to accumulate large amounts of ketocarotenoids (236 mg g Ϫ1 fresh weight; 68% of total carotenoids) containing 91.6 mg g Ϫ1 fresh weight of astaxanthin, 57.0 mg g Ϫ1 fresh weight of adonirubin and 50.1 mg g Ϫ1 fresh weight of canthaxanthin (Table 1). Recently, Hasunuma et al (2008) introduced the genes [crtW (SD212) and crtZ (SD212)] encoding the CrtW and CrtZ proteins of Asterisks indicate data for dry weight of tissue.…”

“…The high expression of only the ketolase gene may seem enough for producing large amounts of astaxanthin, judging from the successful results with bkt1 reported by Jayaraj et al (2008). Whereas, the simultaneous high expression of the carotenoid 3,3Ј-hydroxylase gene crtZ (SD212) in addition to the ketolase gene was found to be useful for increasing astaxanthin levels .…”