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Engineering of tomato for the sustainable production of ketocarotenoids and its evaluation in aquaculture feed

Significance Ketocarotenoids are high-value pigments used in the food and feed industry to confer color. Aquaculture is a good example, where the addition of carotenoids to the feed is essential for the coloration of trout or salmon flesh, and thus product viability. In this study, complex engineering has been carried out to produce a renewable source of ketocarotenoids for use as feed additives. Production in tomato fruit has enabled the testing of this “generally recognized as safe” material with low-energy minimal bioprocessing in aquaculture trials to demonstrate production, technical, and economic feasibility of the system. This achievement represents a potential paradigm in the bioproduction of specialty and bulk chemicals without our reliance on fossil fuel-derived chemical processes. Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond β-carotene through the expression of the β-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by β-carotene enhancement through the introgression of a lycopene β-cyclase (β-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this “generally recognized as safe” material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.