Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity
- 698 Downloads
Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.
KeywordsRice (Oryza sativa L.) Astaxanthin Ketocarotenoids Carotenoids β-carotene ketolase Multigene transformation
This work was supported by the Ministerio de Economía y Competitividad, Spain (BIO2014-54441-P; BIO2014-54426-P; BIO2011-23324; BIO2011-22525; PIM2010PKB-00746); the Plan Nacional de I + D + I (2008-2011); the IBERCAROT network (112RT0445); PROGRAMA ESTATAL DE INVESTIGACIÓN CIENTÍFICA Y TÉCNICA DE EXCELENCIA, Spain (BIO2015-71703-REDT); COST action (EUROCAROTEN, OC-2015-1-19780); European Union Framework 7 European Research Council IDEAS Advanced Grant (to P.C.) Program-BIOFORCE and ERC Proof of Concept Grant (to P.C.), Catalan Government 2014 SGR 1296 Agricultural Biotechnology Research Group. C.B. is the recipient of a Ph.D. fellowship from the Universitat de Lleida, Spain.
T.C., G.S., P.C. and C.Z. conceived and designed research. C.B., J.B., G.F., T.C., G.S., P.C. and C.Z. conducted experiments. C.B., J.B., G.F. and C.Z. contributed new reagents or analytical tools. C.B., T.C., G.S., P.C. and C.Z. analyzed data. C.B., T.C., G.S., P.C. and C.Z. wrote the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Farré G, Naqvi S, Sanahuja G, Bai C, Zorrilla-López U, Rivera SM, Canela R, Sandman G, Twyman RM, Capell T, Zhu C, Christou P (2012) Combinatorial genetic transformation of cereals and the creation of metabolic libraries for the carotenoid pathway. Methods Mol Biol 847:419–435CrossRefPubMedGoogle Scholar
- Misawa N, Yamano S, Linden H, de Felipe MR, Lucas M, Ikenaga H, Sandmann G (1994) Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtI in transgenic plants showing an increase of β-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon. Plant J 4:833–840CrossRefGoogle Scholar
- Sambrook J, Fritschi EF, Maniatis T (1989) Molecular cloning, a laboratory manual, vol 1. Cold Spring Harbor, Cold Spring Harbor Laboratory Press, New York, pp 63–70Google Scholar
- Zhong YJ, Huang JC, Liu J, Li Y, Jiang Y, Xu ZF, Sandmann G, Chen F (2011) Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. J Exp Bot 62:3659–3669CrossRefPubMedPubMedCentralGoogle Scholar
- Zhu C, Sanahuja G, Yuan D, Farré G, Arjó G, Berman J, Zorrilla-López U, Banakar R, Bai C, Pérez-Massot E, Bassie L, Capell T, Christou P (2013) Biofortification of plants with altered antioxidant content and composition, genetic engineering strategies. Plant Biotechnol J 11:129–141CrossRefPubMedGoogle Scholar