Functional analysis of green algal {221}-carotene ketolases and metabolic engineering of astaxanthin biosynthesis in higher plants

Abstract

Astaxanthin (3,3'-dihydroxy-β-carotene-4,4'-dione) is a ketocarotenoid that is

beneficial for human health due to its ability of boosting immune function and

preventing tumor formation. The biosynthesis of astaxanthin is, however, limited only to a few organisms. The burgeoning demand for natural astaxanthin has attracted much recent interest in extending the carotenoid pathway of higher plants to astaxanthin by expressing a microbial β-carotene ketolase (BKT). One major challenge of engineering an astaxanthin pathway in plants is the low astaxanthin content achieved.

Five green microalgae including Chlamydomonas reinhardtii,

Chlorococcum sp., Neochloris wimmeri, Protosiphon botryoides and Scotiellopsis oocystiformis were selected with enhanced function for astaxanthin biosynthesis. The products of the BKT cDNAs from the algae are similar in sequence to the BKT from Haematococcus pluvialis (ca 70% amino acid identity). Based on an Escherichia coli system, the BKT enzymes were shown to exhibit various efficacies in converting zeaxanthin into astaxanthin with Chlamydomonas BKT exhibiting the highest conversion rate (ca 85%).

To investigate if the function-enhanced Chlamdomonas BKT (CRBKT) has

advantages over other algal BKTs in triggering astaxanthin biosynthesis in higher plants, the CrBKT, together with the BKTs from Chlorella zofingiensis (CzBKT) and H. pluvialis (HpBKT3) was expressed in Arabidopsis thaliana. Transgenic Arabidopsis expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g-1 dry weight. In contrast, the expression of CzBKT resulted in much lower content of astaxanthin (0.24 mg g-1 dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in Arabidopsis. Similarly, overexpression of CrBKT in tobacco also resulted in the massive accumulation of astaxanthin in leaves (1.60 mg g-1 DW). Taken all together, it can be concluded that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic plants.

Tomato is an important food crop with high amounts of carotenoids in its fruit. To investigate if tomato fruit can serve as a bio-factory for astaxanthin production, the CrBKT was overexpressed in three genotypes of tomato. All transgenic tomato plants developed brown red leaves that accumulated canthaxanthin rather than astaxanthin as a major carotenoid, resulting from the poor catalytic activity of the endogenous BHY1 toward canthaxanthin. To overcome this problem, CrBKT and HpBHY, the best pair of genes catalyzing the formation of astaxanthin in β- carotene-producing E. coli, were coexpressed in tomato. Canthaxanthin was efficiently converted to astaxanthin, resulting in a massive accumulation of astaxanthin in leaf (3.12 mg g-1) and fruit (16.1 mg g-1) with enhanced total carotenoid capacities of 1.7-fold in leaf and 16.6-fold in fruit. Moreover, the over-production of astaxanthin in fruit enhanced its antioxidant capacity 3-5-fold and vitamin C 2-fold, although it did not affect growth and development.

In summary, the Chlamydomonas BKT is proven to be superior to other

sources of BKT/CrtW enzymes in triggering astaxanthin biosynthesis in plants. By coexpressing a pair of well-cooperating BKT and BHY genes, the transgenic B-type tomato could accumulate commercially attractive amounts of the high-value astaxanthin in its fruit. This study highlights the potential of higher plants to be engineered as cell factories for producing the high-value astaxanthin.