A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of carotenogenesis in rice callus Jürgen Breitenbach a,1 , Chao Bai b,1 , Sol M. Rivera c , Ramon Canela c , Teresa Capell b , Paul Christou b,d , Changfu Zhu b , Gerhard Sandmann a,⇑ a Molecular Biosciences, J.W. Goethe Universität Frankfurt, Max von Laue Str. 9, D-60438 Frankfurt am Main, Germany b Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida-Agrotecnio Center, Avenida Alcalde Rovira Roure, 191, Lleida E-25198, Spain c Departament de Química, Universitat de Lleida, Avenida Alcalde Rovira Roure, 191, Lleida E-25198, Spain d Institucio Catalana de Recerca i Estudis Avancats, Barcelona, Spain article info Article history: Received 3 October 2013 Received in revised form 29 November 2013 Available online xxxx Keywords: Astaxanthin Carotenoid biosynthesis Genetic engineering Rice endosperm 4-keto-a-carotene abstract Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as cyclization and hydroxylation reactions were catalyzed by endogenous rice enzymes in the endosperm. The carotenoid pathway was extended further by including a bacterial ketolase gene able to form asta- xanthin, a high value carotenoid which is not a typical plant carotenoid. In addition to astaxanthin and precursors, a carotenoid accumulated in the transgenic callus which did not fit into the pathway to asta- xanthin. This was subsequently identified as 4-keto-a-carotene by HPLC co-chromatography, chemical modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli. We postulate that this keto carotenoid is formed from a-carotene which accumulates by combined reac- tions of the heterologous gene products and endogenous rice endosperm cyclization reactions. Ó 2013 Elsevier Ltd. All rights reserved. Introduction Genetic engineering is a powerful tool to modulate existing bio- synthesis pathway or establish novel routes in microbes and plants. Carotenogenesis is a key target for genetic engineering of staple crops due to the many nutritional and other health benefits of a number of such molecules for humans and animals (Fraser et al., 2009; Misawa, 2011). Successful examples are increase of the carotenoid yield, e.g., lycopene in tomato (Fraser et al., 2002), the accumulation of intermediates to higher levels, e.g., zeaxanthin in potato (Römer et al., 2002) or extension of an existing pathway to a novel end product, e.g., astaxanthin in maize (Zhu et al., 2008). In addition, maize was used to explore interactions between an in- duced transgenic carotenoid pathway and the endogenous path- way (Naqvi et al., 2011). On a number of occasions there have been examples of novel unexpected phenotypes due to unknown regulatory mechanisms and unpredicted enzyme interactions (see Sandmann et al. (2006) for review). Rice provides an interest- ing example of carotenogenesis by genetic engineering. In contrast to the endosperm of yellow maize which is pigmented due to the accumulation of lutein, zeaxanthin, and 5,6-epoxy derivatives Quackenbush et al. (1963)), rice endosperm is colourless. Never- theless, rice endosperm possesses a hidden potential for carotenoid biosynthesis even though the initial steps in the carotenoid path- way are absent. The endogenous levels of phytoene synthase and phytoene desaturase in wild type endosperm are below the thresh- old level for carotenoid biosynthesis (Schaub et al., 2005). It has been demonstrated that the limitation of carotenogenesis can be overcome in rice endosperm by expressing genes encoding a phy- toene synthase and a bacterial phytoene desaturase able to replace all plant desaturation and isomerisation reactions (Ye et al., 2000). The expected product of these reactions is lycopene. Interestingly biosynthesis proceeded beyond lycopene by cyclization to a- and b-carotene and the hydroxylation of both carotenes to lutein and zeaxanthin, respectively (Ye et al., 2000). Thus intrinsic rice cyclas- es and hydroxylases are expressed in the endosperm. A survey of phytoene synthase genes from different plant species indicated that the maize enzyme is the most effective in rice (Paine et al., 2005). Its use led to the generation of a rice line rich in a- and b- carotene which both exhibit provitamin A activity, in addition to lutein and zeaxanthin (structures shown in Fig. 1). In our current carotenogenesis engineering experiments we attempted to extend the pathway beyond carotenes to astaxanthin. Astaxanthin is a high priced carotenoid which is beneficial for human health 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.12.008 ⇑ Corresponding author. E-mail address: sandman@bio.uni-frankfurt.de (G. Sandmann). 1 J.B. and C.B. contributed equally to this work. Phytochemistry xxx (2014) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Breitenbach, J., et al. A novel carotenoid, 4-keto-a-carotene, as an unexpected by-product during genetic engineering of carotenogenesis in rice callus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2013.12.008