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Phytochemistry xxx (2014) xxx–xxx

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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,⇑
Molecular Biosciences, J.W. Goethe Universität Frankfurt, Max von Laue Str. 9, D-60438 Frankfurt am Main, Germany
Departament de Producció Vegetal i Ciència Forestal, Universitat de Lleida-Agrotecnio Center, Avenida Alcalde Rovira Roure, 191, Lleida E-25198, Spain
Departament de Química, Universitat de Lleida, Avenida Alcalde Rovira Roure, 191, Lleida E-25198, Spain
Institucio Catalana de Recerca i Estudis Avancats, Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early
Received 3 October 2013 carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus
Received in revised form 29 November 2013 with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as
Available online xxxx
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-
Keywords: 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-
Carotenoid biosynthesis
Genetic engineering
xanthin. This was subsequently identified as 4-keto-a-carotene by HPLC co-chromatography, chemical
Rice endosperm modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli.
4-keto-a-carotene 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 accumulation of lutein, zeaxanthin, and 5,6-epoxy derivatives
Quackenbush et al. (1963)), rice endosperm is colourless. Never-
Genetic engineering is a powerful tool to modulate existing bio- theless, rice endosperm possesses a hidden potential for carotenoid
synthesis pathway or establish novel routes in microbes and biosynthesis even though the initial steps in the carotenoid path-
plants. Carotenogenesis is a key target for genetic engineering of way are absent. The endogenous levels of phytoene synthase and
staple crops due to the many nutritional and other health benefits phytoene desaturase in wild type endosperm are below the thresh-
of a number of such molecules for humans and animals (Fraser old level for carotenoid biosynthesis (Schaub et al., 2005). It has
et al., 2009; Misawa, 2011). Successful examples are increase of been demonstrated that the limitation of carotenogenesis can be
the carotenoid yield, e.g., lycopene in tomato (Fraser et al., 2002), overcome in rice endosperm by expressing genes encoding a phy-
the accumulation of intermediates to higher levels, e.g., zeaxanthin toene synthase and a bacterial phytoene desaturase able to replace
in potato (Römer et al., 2002) or extension of an existing pathway all plant desaturation and isomerisation reactions (Ye et al., 2000).
to a novel end product, e.g., astaxanthin in maize (Zhu et al., 2008). The expected product of these reactions is lycopene. Interestingly
In addition, maize was used to explore interactions between an in- biosynthesis proceeded beyond lycopene by cyclization to a- and
duced transgenic carotenoid pathway and the endogenous path- b-carotene and the hydroxylation of both carotenes to lutein and
way (Naqvi et al., 2011). On a number of occasions there have zeaxanthin, respectively (Ye et al., 2000). Thus intrinsic rice cyclas-
been examples of novel unexpected phenotypes due to unknown es and hydroxylases are expressed in the endosperm. A survey of
regulatory mechanisms and unpredicted enzyme interactions phytoene synthase genes from different plant species indicated
(see Sandmann et al. (2006) for review). Rice provides an interest- that the maize enzyme is the most effective in rice (Paine et al.,
ing example of carotenogenesis by genetic engineering. In contrast 2005). Its use led to the generation of a rice line rich in a- and b-
to the endosperm of yellow maize which is pigmented due to the 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
⇑ Corresponding author.
the pathway beyond carotenes to astaxanthin. Astaxanthin is a
E-mail address: (G. Sandmann).
J.B. and C.B. contributed equally to this work.
high priced carotenoid which is beneficial for human health

0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.

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),

Its identification is the scope of this oid... its maximum of 452 nm is 14 nm lower than that of echine- work. 2009). hydroxylase which is active and specific enough for astaxanthin ene synthase. successful in terms of engineering a ketolation pathway leading One compound which does not fit into this pathway is the to astaxanthin as the end product.doi. 1. we have chosen rice as an alternative crop for 6).. This en- and labour intensive regeneration of intact plants. The endosperm-based rice cal. nous lycopene cyclases.phytochem. in Fig. 2006) and is used as an essential Results and discussion feed additive in salmon farming (Bjerkeng. 2001). Breitenbach et al.008 . 1) modulation on carotenoid composition before the time consuming which was initiated by the transgenic carotenoid ketolase. Although it has a very similar shape to the spectrum we detected a novel carotenoid which did not fit directly into of echinenone (Fig. 2012). 2008). 2A). / Phytochemistry xxx (2014) xxx–xxx Fig. Our strategy has been to engineer suitable bination with a bacterial carotenoid ketolase (Fig. Two other peaks resemble intermediates of this path- b-carotene ketolase gene which in combination with the endoge..2013). In previous viously (Bai et al.. maximum at 465 nm (Fig. 3B) which may indicate a mono-keto caroten- the pathway to astaxanthin. Therefore. Expression of plant material such as rice grains to synthesize astaxanthin for carotenoid ketolase and all other transgenes has been shown pre- directly feeding or consumption (Sandmann. A novel carotenoid. as an unexpected by-product during genetic engineering of carotenogenesis in rice callus. The biosynthesis pathway of astaxanthin formation in transgenic endosperm-derived rice callus.7 min with the typical the engineering of carotenogenesis using combinatorial genetic bell shaped optical absorbance spectrum with its maximum at transformation (Farre et al. Transformation zyme works by interaction with the endogenous b-carotene was carried out simultaneously with three genes. Fig. 3B). solid arrows indicate maize-specific carotenogenic reactions. et al. carotenoid represented by peak 3 (Fig. (Guerin et al. Considerable effort has been invested to achieve sustainable The initial synthesis of phytoene and its 4-step desaturation and and economic production of astaxanthin in microorganisms and isomerization to lycopene was engineered into rice callus in com- plants (Zhu et al.2013. open arrows indicate the reactions catalaysed by the transgenes psy (phytoene synthase). way. However.12. Dotted arrows indicate pathway limitations in rice. The prominent carotenoid peak 1 at 6. none. Two carotenes were synthesized by the and an additional hydroxylase gene resulted in the accumulation interaction of phytoene synthase and desaturase and the endoge- of astaxanthin (Zhu et al. both with nous endosperm expressed b-carotene hydroxylase should be able an asymmetrical bell shaped optical absorbance spectrum with a to synthesize astaxanthin. 2A shows the HPLC carotenoid profile work. a-carotene (peak 5) and b-carotene (peak isfactory.. Hussein et al.1016/j. a bacterial phytoene desaturase and a bacterial synthesis. the other by ketolation of zeaxanthin. The latter originated directly from Engineering of carotenoid biosynthesis in rice endosperm was ketolation of b-carotene. a maize phyto. The only known carotenoid with the same absorbance and Please cite this article in press as: Breitenbach.2 J. we reported that transformation of maize with a ketolase of a typical transgenic line. This is the end product of the ket- lus system allowed us to investigate the impact of genetic pathway olation pathway starting from b-carotene or zeaxanthin (Fig. 3A. 4-keto-a-carotene. Phytochemistry (2014). the yields were not sat. 2003. 4-keto-zeaxanthin peak 2 and echinenone peak 4. http://dx. However in addition to the ex. Its spectrum is shown pected hydroxy and keto intermediates derived from b-carotene. 1). 475 nm resembles astaxanthin. crtI (phytoene desaturase) and crtW (carotene ketolase). 2008).

(G) 4-hydroxy-a-carotene (HOaC) standard. (C) 4-keto-b-zeacarotene (KbZc) standard.w-carotene-4- one) and 4-keto-a-carotene (b.. From the spectrum genetically engineered rice callus. 2012).2013.e-carotene-4-one) are the most Conclusion likely candidates for the novel carotenoid molecule. In was identified as 4-keto-a-carotene. Several carotenoid masses fit this value includ- ing the one for echinenone with 550.008 . a specific side reaction occurred in rice ence compounds by combinatorial biosynthesis in Escherichia coli callus..5 min primary reason for the formation of 4-keto-a-carotene in the compared to 12. Starting from [M+H]+ ion of 552. tene and zeaxanthin to astaxanthin but also accepted the b-ionone bance maximum was the same as that of the novel compound ring of a-carotene as substrate for ketolation. Phytochemistry (2014). Obviously. 4A). 8. 2. Its formation branches off addition. KL. the carotenoid extract from rice was reduced by sodium borohydride. (F) carotenoids from transgenic rice callus polyene chain which influences the optical absorbance spectrum after reduction with NaBH4. In the case of 4-keto-b-zeacarotene the absor. 2004). J. 4-hydroxy-a-carotene.6 > 93 by in-chain elimination of toluene and transi- tion 551.6 > 203.1 resulting from the cleavage at C-10. 3C). as an unexpected by-product during genetic engineering of carotenogenesis in rice callus. this carotenoid did not co-chromatograph with the desired b-carotene to astaxanthin route. novel unknown carotenoid. The resulting spec- trum resembles that of a-carotene (Britton et al. it is also able to ketolate the b-ionone end of the a-caro- from transgenic Nicotiana tabaccum nectary tissue transformed tene molecule. b-carotene. HPLC separation of carotenoids from transgenic rice callus expressing a insect Ectatosoma tiaratum (Kayser. 2D) and co-chromatographied with peak 3 (Fig. A novel carotenoid. lutein. This was extracted addition. echinenone. 1981).6 > 69 previously reported for echinenone (Enzell et al. it does not contribute to the conjugated extract with 4-keto-a-carotene standard. a more polar compound was formed (Fig. 4B were those with characteristic signals for identification.1. The spec- trum of this hydroxy derivative shows the typical three maxima of non-ketolated carotenoids exhibiting a hypsochromic shift to 442 nm of the main central maximum (Fig. 7. However. In shape of the spectrum is 4-ketolutein (Fig. This is a unique carotenoid not reported thus far (Britton et al. 6. with three pronounced peaks at 423. 3. (E) co-chromatography of rice the keto group at C-2. http://dx. from the spectrum in Fig..6 > 123. 4-keto-lutein. 4-keto-zeaxanthin.. This loss for the Please cite this article in press as: Breitenbach. 2B). / Phytochemistry xxx (2014) xxx–xxx 3 compound 3 due to its longer retention time (Fig. This may resemble the protonated molecular ion of compound 3 with a molecular mass of 551 g/mol. This chemical modifica- tion of the keto group was also carried out with the 4-keto-a-car- otene reference compound resulting in 4-HO-a-carotene. 2002). and the basic carotenoid pathway to a. Breitenbach et al.1016/j. 1969). This is consistent with 4-keto-a-carotene which has the same molecu- lar mass as echinenone. Peak 1. 2D and F). it exhibited the same retention time in HPLC (Fig.doi. 446 and 475 nm.8-dihydro-b. 1995). it is more polar with a retention time of 7. (D) 4-keto-a-carotene standard (KaC). the a-carotene concentrations as substrate for the ketolase is the 2007). transitions related to functional groups in the carotenoid structures were built. L. The resulting product but the shape of the absorbance peak did not match (Fig. The dominating mass peak in the spectrum is the ion at m/z 552. Due to the position of ketolase gene (A). we generated these mono keto carotenoids which should exhibit Although genetic engineering of the carotenoid pathway to not only a similar absorbance but also a similar polarity as refer. (B) isolate with 4-keto-lutein. et al. Only 4- keto-a-carotene matched exactly the spectrum of compound 3 (Fig. Formation of 4-keto-a-carotene is a-carotene.. 5.phytochem. A similar but different carotenoid is 2-keto-a-carotene from the stick Fig. One of the most intense transitions is 551. This transition is a specific indicator of the existence of an e-ring in the structure of compound 3 (Enzell and Back. The expressed bacterial ketolase not only converted b-caro- (Sandmann. possesses broad substrate specificity. a mass spectrum was determined by UHPLC-MS with atmospheric pressure chemi- cal ionisation identifying the [M+H]+ ions (Fig. For further confir- mation of the structure of compound 3.12. the carotene ketolase from Brevundimonas sp. Taken to- gether results from mass spectrometry suggest that compound 3 is a carotenoid combining the ketolated half of echinenone and the other half with the e-ring of a-carotene (boxed in Fig. 2. KZ.. transition 551. This enzyme typically keto- lates both b-ionone rings in b-carotene (Nishida et al. 4-hydroxy echinenone. astaxanthin was successful. chemical modification and mass spectrometry all identified compound 3 as 4-keto-a-carotene. 3E. 2005). due to the relatively high a-carotene content in the transgenic rice callus. For further structure elucidation of peak 3. 2004). In each case. HPLC co-chromatography. 2E). 4C).2 min for peak 3 (Fig. 3D). 3E). J. 4-keto-zeaxanthin. distinct astxanthin.86 g/mol (Rivera et al..and b-carotene related compounds. The ones shown in Fig. 4. 4-keto-b-zeacarotene (7. 2011). 4C (Van Breemen et al. The ones to identify an echinenone-related structure are the tran- sitions 551. Therefore.11 of a 4- keto fragment as indicated in Fig. 2C).. This broad substrate specificity in combination with with a cyanobacterial b-carotene ketolase gene (Gerjets et al. 3F) for reduction of a keto group conjugated with the polyene chain.

1994) mediates the formation of a-carotene.. 2013). http://dx... et al.phytochem. The maize 4-Keto-b-zeacarotene is synthesized from neurosporene generated psy1 cDNA (Buckner et al. 2008). 4-keto-b-zeacarotene (D). 4-keto-a-carotene (E) and 4-hydroxy-a-carotene (F).. coli transformant generating 4-keto-a-carotene was grown at transit peptide signal from the Phaseolus vulgaris small subunit of 37 °C in the presence of ampicillin (100 lg/ml). Their maps are shown in Fig... A novel carotenoid. amplified by PCR and then transferred to pHorp-P expression of rice specific lycopene e-cyclase depleting the 4-keto-lutein (C). Breitenbach et al.doi.4 J. astaxanthin pathway may be avoided by further genetic engineer. The three plasmids contained phytoene pathways for 4-keto-a-carotene and 4-keto-b-zeacarotene the fol- synthase Zmpsy1. phytoene desaturase Pacrtl and b-carotene keto.2013. Plasmid pACCAR16DcrtX (Misawa et al.. form E.. 2008) cloned into pBBR1-MCS1 with kanamycin as selection marker and to the transit peptide sequence from pea ribulose-1. coli strain DH5a.. 2000) and fi- 1999) and nos terminator.. 1985). 2011). maize c-zein gene promoter and the nos terminator.. 3. 1996) was transferred into plasmid by pACCRT-EBIRC (Linden et al. The latter gene was chemically synthesized according to 1995) in combination with pBBRK-ara-epsilon with the lycopene the codon usage of Brassica napus and fused to the full-length rice e-cyclase from Arabidopsis thaliana (Cunningham et al. 5-bis. echinenone (=4-keto-b-carotene) (B). / Phytochemistry xxx (2014) xxx–xxx Fig. nally ketolated by expression of pCRBKT (Zhong et al. 1996) alcohol dehydrogenase 5́-untranslated region (Sugio et al. Transgenic rice Combinatorial carotenoid synthesis in E.. 1994). This phospate carboxylase small subunit (Schreier et al.. The merly known as Erwinia uredovora) was fused in frame with the E.. 1993) which is cyclised by expres- p326 containing the wheat LWM glutenin promoter (Stoger et al. 2005). Optical spectra of the novel unknown carotenoid (A).12. lowing combinations of compatible plasmids were used to trans- lase CrtW from Brevundimonas sp. containing the barley D-hordein promoter (Sørensen et al.1016/j. Trans- genic coloured calli were selected and cultured on MS selection Experimental medium (Farre et al. This carotene is then ketolated at the b-ionone ring to 4-keto-a-caro- DNA fragment was inserted into plasmid GZ63 containing the tene by expression of pPEU30crtO (Breitenbach et al. sion of plasmid pRK-crtY (Hausmann and Sandmann. J. as an unexpected by-product during genetic engineering of carotenogenesis in rice callus. and the rice ADPGPP terminator. (Kovach et al.008 . coli The transgenic endosperm-derived rice callus was generated by combinatorial nuclear transformation which has been reported For the synthesis of reference compounds the biosynthesis earlier (Zhu et al. 4-keto-a-carotene. All transformation constructs are based on pUC8 plasmids. chloramphenicol Please cite this article in press as: Breitenbach.. Strain SD212 (Nishida et al. 5. 1996) tion of a-carotene. ribulose bisphosphate carboxylase in plasmid pYPIET4 (Misawa ing of carotenogenesis by antisense or RNAi down-regulation of the et al. 2012). Phytochemistry (2014). The crtI gene from Pantoea ananatis (for.

Phytochemistry (2014).008 .doi. A novel carotenoid.phytochem. Boxes indicate the two halves of the identified molecule. 4-keto-a-carotene. (B) transitions from the [M+H]+ ion of 552.1016/j. 4. as an unexpected by-product during genetic engineering of carotenogenesis in rice callus.2013. 4-keto-a-carotene.12. J.. / Phytochemistry xxx (2014) xxx–xxx 5 Fig. Mass spectroscropy of novel unknown compound. J. http://dx. Breitenbach et al. et al. (A) Atmospheric pressure chemical ionisation mass spectrum. Please cite this article in press as: Breitenbach. (C) fragmentation pattern of echinenone and

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