Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering

Abstract

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 μg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, β-carotene, phytoene, α-carotene, lycopene, and β-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a β-carotene hydroxylase in addition to a β-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.

Fig 1. Carotenoid biosynthetic pathway.

Possible pathways leading to the formation of the ketocarotenoids canthaxanthin and astaxanthin, which can be formed from β-carotene through reactions catalyzed by ketolase and hydroxylase enzymes. The crtB gene encodes a phytoene synthase enzyme that catalyzes the formation of phytoene. A “*” indicates a β-carotene ketolase, such as the ketolases encoded by the crtW or bkt1 genes used in this study. This enzyme can use different substrates, so its final products depend on the substrates available.

Fig 2. Schematic representations of transformation constructs.

See Plasmid Construction for construct details. Abbreviations used: hph, hygromycin resistance gene; Bcon, β-conglycinin; StUbi, Solanum tuberosum ubiquitin; kanR, kanamycin resistance.

Fig 3. Soybean seeds from events producing canthaxanthin.

The top row in each panel is of wild-type (WT) soybean cultivar Jack. The second row is of carotene-accumulating seeds resulting from transformation with crtB. The third row is of seeds containing canthaxanthin after transformation with pCEH1B+bkt (left panel) or pCEH1B+crtW (right panel). The cotyledons from one seed have been separated in each case.

Fig 4. RT-PCR analysis of carotenoid biosynthetic pathway RNA from transgenic soybean seeds.

Panel A shows RT-PCR products for crtB, crtW, and bkt1. Lower panel (B) shows reactions without reverse transcriptase. Cv. Jack NT is a non-transgenic control sample. NTC stands for no template control.

Fig 5. Soybean somatic embryo accumulating carotenoids.

From left to right: non-transformed Jack somatic embryo, somatic embryo from Jack transformed with pCEH1B+crtW. The embryos in this picture were imaged approximately 5 months after bombardment.