Combinatorial genetic transformation generates a library of metabolic phenotypes for the pathway in maize

Changfu Zhua,1, Shaista Naqvia,1,Ju¨ rgen Breitenbachb, Gerhard Sandmannb, Paul Christoua,c,2, and Teresa Capella,2

aDepartament de Produccio´Vegetal i Cie`ncia Forestal, Universitat de Lleida, Avenue Alcalde Rovira Roure, 191, 25198 Lleida, Spain; bBiosynthesis Group, Department of Molecular Biosciences, J. W. Goethe Universitaet, Biocampus 213, P. O. Box 111932, D-60054 Frankfurt, Germany; and cInstitucio Catalana de Recerca i Estudis Avancats, Passeig Lluís Companys, 23, 08010 Barcelona, Spain

Communicated by M. S. Swaminathan, Center for Research on Sustainable Agricultural and Rural Development, Madras, India, October 2, 2008 (received for review July 22, 2008) Combinatorial nuclear transformation is a novel method for the rapid pathway analysis because of the diminishing rate of return as more production of multiplex-transgenic plants, which we have used to transgenes are introduced simultaneously (14). We have addressed dissect and modify a complex metabolic pathway. To demonstrate this challenge by developing a combinatorial nuclear transforma- the principle, we transferred 5 carotenogenic genes controlled by tion strategy in maize, allowing us to generate a metabolic library different endosperm-specific promoters into a white maize variety for the investigation of carotenoid biosynthesis and the synthesis of deficient for endosperm carotenoid synthesis. We recovered a diverse specific combinations of . population of transgenic plants expressing different enzyme combi- It has been acknowledged that ‘‘rational engineering of compli- nations and showing distinct metabolic phenotypes that allowed us cated metabolic networks involved in the production of biologically to identify and complement rate-limiting steps in the pathway and to active plant compounds has been greatly impeded by our poor demonstrate competition between ␤- hydroxylase and bac- understanding of the regulatory and metabolic pathways underlying terial ␤-carotene ketolase for substrates in 4 sequential steps of the the biosynthesis of these compounds’’ (15). Targeted metabolite extended pathway. Importantly, this process allowed us to generate analysis combined with cDNA-amplified fragment-length polymor- plants with extraordinary levels of ␤-carotene and other carotenoids, phism-based transcript profiling is emerging as a useful strategy for including complex mixtures of hydroxycarotenoids and ketocarote- the creation of novel tools for metabolic engineering (16). In this noids. Combinatorial transformation is a versatile approach that could context a population of transgenic plants expressing a combinato- be used to modify any metabolic pathway and pathways controlling rial transgene complement provides an invaluable resource for other biochemical, physiological, or developmental processes. targeted metabolic engineering. We used as a model system the South African elite white maize induced mutation ͉ metabolic engineering ͉ transgenic plant ͉ provitamin A variety M37W, which lacks carotenoids in the endosperm because of the absence of the enzyme synthase (PSY1) [support- lants produce a vast array of secondary metabolites that have ing information (SI) Fig. S1] (17, 18). This system allowed us to Padvantageous ecological functions, and many of these mole- carry out a preliminary screen for transgenic plants accumulating cules also have nutritional and/or pharmacological properties in different carotenoids based on endosperm color. After transform- humans (1). Carotenoids are secondary metabolites that play a role ing white maize embryos with 5 carotenogenic transgenes, we in light perception and photoprotection (2), but they are also recovered plants carrying all combinations of the input genes. This required by animals as metabolic precursors and antioxidants, and combinatorial population was mined for phenotypes corresponding some have specific health benefits such as the prevention of cancer to the production of specific carotenoids, which in turn correlated (3, 4), maintenance of the immune system (4), and the prevention with specific transgene expression and metabolic profiles. of blindness (5, 6). Animals are unable to synthesize carotenoids Our approach provides a unique and surprisingly straightforward directly and must obtain them from their diets. Although fruits and strategy for metabolic pathway analysis and multigene metabolic vegetables are particularly good sources of certain carotenoids, engineering in plants. It involves the introduction and coordinated cereal grains generally lack these compounds, leading to deficiency expression of multiple transgenes followed by the selection of stable diseases in countries where cereals are the staple diet. Also, with the lines expressing the specific combination of transgenes required for exception of Adonis aestivalis flowers, and other keto- particular metabolic outputs (Table S1). Individual lines, producing carotenoids are scarce in plants, although they are abundant in fish specific metabolites, can be goals in themselves if the aim is to and shellfish and are used in aquaculture to enhance the aesthetic engineer particular molecules. However, by examining the entire qualities of salmon (7, 8). For these reasons there is much interest diverse population of plants, it becomes possible to dissect the in studying carotenoid biosynthesis pathways in plants and modi- pathway and subsequently reconstruct it either in its original form fying cereal crops to enhance the carotenoid content (6, 9, 10). or with modifications, thus providing a basis for understanding and Studying and engineering secondary metabolism in plants can be subsequently engineering the synthesis of novel metabolites. The compromised by the sheer complexity of the pathways, which may broad significance of this approach is that it considerably simplifies have multiple branches, multifunctional enzymes, cell type-specific and compartmentalized enzymes, and complex feedback mecha- Author contributions: G.S. and P.C. designed research; C.Z., S.N., J.B., G.S., and T.C. performed nisms (11). One approach to overcome this challenge is to clone research; C.Z., S.N., J.B., G.S., and T.C. contributed new reagents/analytic tools; C.Z., S.N., J.B., genes encoding pathway enzymes and modify their expression, but G.S., P.C., and T.C. analyzed data; and C.Z., S.N., G.S., P.C., and T.C. wrote the paper. modulating single enzymes is often unhelpful because pathways are The authors declare no conflict of interest. regulated at multiple points. It is becoming increasingly apparent 1C.Z. and S.N. contributed equally to this work. that multistep engineering, where partial or complete pathways are 2To whom correspondence may be addressed. E-mail: [email protected] or capell@ reconstructed or extended by the expression of 2 or more enzymes pvcf.udl.cat. simultaneously, is the most desirable way to study and modulate This article contains supporting information online at www.pnas.org/cgi/content/full/ complex pathways such as carotenoid biosynthesis (12, 13). How- 0809737105/DCSupplemental. ever, multigene engineering is a significant hurdle in complex © 2008 by The National Academy of Sciences of the USA

18232–18237 ͉ PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809737105 Downloaded by guest on October 1, 2021 the process of carotenoid metabolic engineering by making it analogous to screening a library of metabolic variants for the correct functional combination. This approach could be applied to any pathway (metabolic or otherwise) given a suitable template for combinatorial transformation. Results Combinatorial Nuclear Transformation Generates a Diverse Library of Plants with Distinct and Stable Phenotypes. We transformed 13-day- old immature zygotic embryos of South African elite white maize variety M37W by bombarding them with metal particles coated with 6 constructs (Fig. S2), the selectable marker bar and 5 carotenogenic genes: Zmpsy1 (Zea mays phytoene synthase 1), PacrtI (Pantoea ananatis phytoene desaturase), Gllycb (Gentiana lutea ␤-cyclase), Glbch (G. lutea ␤-carotene hydroxylase, a plant-type ␤-ring nonheme di-iron monooxygenase introducing hydroxy groups at C-3), and ParacrtW (Paracoccus ␤-carotene ketolase). Each gene was driven by a different endosperm-specific promoter (respectively, the low molecular weight wheat glutenin, barley hordein, rice prolamin, rice glutelin-1, and maize ␥-zein promoters; Tables S2 and S3). A population of regenerated plants was screened by genomic PCR revealing many different combina- tions of transgenes, including 9 lines (13%) containing all 5 carot- enoid input genes. Multiple independent transgenic lines contain- ing and expressing the same transgene complement were identified (Table S4). All of the transgenic plants showed normal morphology and development, reflecting the restriction of transgene expression to the seed endosperm. Visual inspection of the endosperm tissue revealed 7 distinct phenotypes based on endosperm color (Ph-1 to Ph-7; Fig. 1A). Analysis of steady-state mRNA levels revealed which transgenes were expressed in individual transgenic plants (Fig. 1B). We found a precise correlation between the phenotypes and expressed transgenes. Phenotype 1 (Ph-1), expressing Zmpsy1 Fig. 1. Phenotypes and genotypes of 7 combinatorial transformants. (A) En- alone, appeared similar in color to WT yellow maize, whereas Ph-2, dosperm colors of 7 different transgenic maize phenotypes. Ph-1 expressing expressing PacrtI alone, was very pale yellow in color. The combi- Zmpsy1 only accumulates and has a bright yellow color. Ph-2 express- nation of Zmpsy1 and PacrtI in Ph-3 generated an orange–red ing only PacrtI has a phenotype similar to WT M37W with a slight increase phenotype, whereas the combination of Gllycb in addition to in total carotenoids. Ph-3 (Zmpsy1 and PacrtI) accumulates a significant Zmpsy1 and PacrtI in Ph-4 produced a distinct orange–yellow color. amount of lycopene and has an orange–red color. Ph-4 expresses Gllycb in addition to Zmpsy1 and PacrtI and accumulates ␤-carotene, hence the Ph-5, Ph-6, and Ph-7 were more complex phenotypes resulting orange color. Ph-5, Ph-6, and Ph-7 express Zmpsy1ϩPacrtIϩGlbch, from the expression of a bacterial ketolase gene, ParacrtW,in Zmpsy1ϩPacrtIϩGllycb, and Zmpsy1ϩPacrtIϩGllycbϩGlbch, respectively, in addition to Zmpsy1ϩPacrtIϩGlbch, Zmpsy1ϩPacrtIϩGllycb,or addition to ParacrtW, thus showing a range of colors from orange to red Zmpsy1ϩPacrtIϩGllycbϩGlbch, respectively. These lines also depending on the accumulation of ketocarotenoids. (B) Northern blot analysis showed distinguishable orange to red phenotypes (Fig. 1A). (30 ␮g of total RNA per lane) to monitor transgene expression in WT M37W and the 7 transgenic phenotypes (lanes 1–7). Staining of rRNA with ethidium Reconstruction of the Carotenoid Pathway in White Maize Leads to the bromide was used as a loading control. Accumulation of Extraordinary Levels of Metabolic Intermediates and End Products. HPLC analysis showed that the different color Simultaneous expression of Zmpsy1, PacrtI, and Gllycb in Ph-4 phenotypes reflected the accumulation of different metabolites, ␮ confirming a direct correspondence between genotype and carot- dramatically reduced the levels of lycopene (11.50 g/g DW, ␮ SCIENCES enoid accumulation. The metabolic profiles of each transgene 7.73%) but increased the levels of zeaxanthin (34.53 g/g DW, 23.21%) compared with Ph-3 (Fig. 2). Phytoene, which is not

complement and resulting phenotype were qualitatively consistent APPLIED BIOLOGICAL in the multiple transgenic events tested, despite variation in the present in WT M37W endosperm, accumulated in Ph-1, Ph-3, and absolute levels of particular compounds (Fig. 2 and Table S1). Ph-1 Ph-4 (which express Zmpsy1) but not in Ph-2 (which does not (Zmpsy1 alone) showed a 53-fold increase of total carotenoids over express Zmpsy1)(Table S1). This finding indicates that the con- white maize [58.21 vs. 1.10 ␮g/g dry weight (DW)], whereas Ph-2 version of phytoene to lycopene is a subsequent limiting step for (PacrtI alone) and Ph-3 (Zmpsy1ϩPacrtI) showed 2.5- and 142-fold carotenoid biosynthesis, revealing where phytoene synthesis is increases, respectively (2.69 and 156.14 ␮g/g DW) (Table S1). The enhanced. Phenotypes and carotenoid content remained stable for total carotenoid content in Ph-4 (Zmpsy1ϩPacrtIϩGllycb) reached at least 2 generations (homozygous T2 plants). Multiple indepen- 148.78 ␮g/g DW. These data suggest that PSY1 is the key enzyme dent transgenic plants expressing the same transgene complement limiting carotenoid accumulation in the endosperm of white maize. exhibited identical qualitative metabolite profiles (Tables S1 and S4). The predominant carotenoids accumulating in Ph-1 endosperm were zeaxanthin (18.25 ␮g/g DW, 31.35% of total carotenoids), Competition Between Bacterial Ketolase and ␤-Carotene Hydroxylase (14.95 ␮g/g DW, 25.68%) and ␤-carotene (7.10 ␮g/g DW, Extends the Carotenoid Pathway and Allows the Synthesis of 12.20%), whereas those accumulating in Ph-3 were ␤-carotene Complex Mixtures of Ketocarotenoids. Ph-4 endosperm (57.35 ␮g/g DW, 36.73%) and lycopene (26.69 ␮g/g DW, 17.09%) (Zmpsy1ϩPacrtIϩGllycb) accumulated not only ␤-carotene but (Fig. 2). These findings suggest that both lycopene cyclases may be also such as lutein and zeaxanthin. The pathway can rate-limiting enzymes in the synthesis of cyclic in Ph-3. be extended even further to ketocarotenoids such as astaxanthin by

Zhu et al. PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 ͉ 18233 Downloaded by guest on October 1, 2021 Fig. 2. HPLC analysis of carotenoids in the endosperm of white maize (M37W), yellow maize (A632), and the 7 transgenic lines (T2 mature seeds). Extracts from Ph-1 and Ph-4 were separated on a Nucleosil C18 column; all other extracts were separated on a Hypersil C18 column.

expressing ParacrtW. We generated 3 unique phenotypes in which and ␤-carotene (34.81 ␮g/g DW; 23.72%), but less zeaxanthin the ParacrtW transgene was expressed in combination with Zmpsy1 (13.71 ␮g/g DW; 9.34%), most likely because of the absence of and PacrtI, differing in the additional expression of Glbch (Ph-5), GlBCH (Fig. S1). Ph-7 accumulated the 3 mono-ketocarotenoids Gllycb (Ph-6), or both Glbch and Gllycb (Ph-7, which expressed all echinenone, 3-hydroxy-echinenone, and adonixanthin, whereas 5 carotenogenic input transgenes). Ph-5 produced only adonixanthin. The missing echineneone and In terms of ketocarotenoid synthesis, HPLC analysis (Fig. 2) 3-hydroxy-echineneone in Ph-5 may be caused by a shortage of revealed the presence of adonixanthin (4-ketozeaxanthin) in Ph-5, ␤-carotene (8.72 ␮g/g DW), reflecting the absence of lycopene adonixanthin, echinenone (4-keto-␤-carotene), and 3-hydroxy- ␤-cyclase (Fig. S1). In contrast, Ph-6 produced not only the 3 echinenone in Ph-7, and these 3 carotenoids plus astaxanthin mono-ketocarotenoids, but also the important di-ketocarotenoid (3,3Ј-dihydroxy-4,4Ј-diketo-␤-carotene) in Ph-6. The predominant astaxanthin. The shared difference, when Ph-6 is compared with carotenoids accumulating in Ph-5 were zeaxanthin (27.47 ␮g/g DW; Ph-5 and Ph-7, is the lack of Glbch, which is therefore likely to 28.37% of total carotenoids) and lutein (18.11 ␮g/g DW; 18.71%) explain their distinct metabolic profiles. in addition to ketocarotenoids (10.62 ␮g/g DW; 10.96%) (Table S1). Ph-5 is ‘‘based on’’ Ph-3, having the Ph-3 genotype with Discussion additional genes Glbch and ParacrtW (Fig. S1). Ph-5 accumulated Gene transfer to plants provides 1 way to study and modify less ␤-carotene than Ph-3 (8.72 vs. 57.35 ␮g/g DW), probably metabolic pathways precisely, and multigene engineering allows reflecting the conversion of ␤-carotene into ␤-cryptoxanthin and entire pathways to be reconstructed free of endogenous regulation. zeaxanthin by GlBCH (Fig. S1). Similarly, Ph-7 is based on Ph-4, This in turn requires strategies to introduce multiple transgenes into having the Ph-4 genotype with additional genes Glbch and ParacrtW plants and ensure their coordinated expression over many gener- (Fig. S1), and it produced mainly ␤-carotene (25.78 ␮g/g DW; ations (12). Expressing multiple transgenes stably is one of the most 25.24%) and zeaxanthin (16.78 ␮g/g DW; 16.43%) in addition to significant hurdles currently limiting progress in plant molecular ketocarotenoids (17.98 ␮g/g DW; 17.61%) (Table S1). biology (14, 19), because the chances of failure for at least 1 of the Ph-6 (Zmpsy1ϩPacrtIϩGllycbϩParacrtW) accumulated sig- transgenes increases with the number of genes introduced, requir- nificant amounts of ketocarotenoids (35.85 ␮g/g DW; 24.29%) ing the generation of very large populations to ensure complete

18234 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809737105 Zhu et al. Downloaded by guest on October 1, 2021 pathway reconstruction. Alternative approaches such as individual rate-limiting step for carotenoid biosynthesis in these pheno- transformation followed by crossing to ‘‘stack’’ transgenes are types. The impact of endogenous enzyme activities on the unworkable for large numbers of transgenes because of the time product of a transgenic extended metabolic pathway has been taken to stack all transgenes in 1 line and the risk of segregation of discussed in the case of golden rice (32). However, contrary to unlinked genes in later generations. our Ph-3 maize line, rice grains expressing the same 2 genes We have devised a strategy to simplify the process of metabolic (Zmpsy1 and PacrtI) did not accumulate phytoene in endosperm analysis and engineering in plants based on combinatorial nuclear tissue (32). transformation to create metabolically diverse transgenic libraries. Significant amounts of lycopene accumulated in maize lines Ph-3 To demonstrate the principle we generated a carotenoid metabolic and Ph-4, in contrast to results obtained in rice expressing similar library in white maize variety M37W, which normally lacks caro- complements of genes (30, 31), similarly to transgenic canola seeds tenoids in the endosperm because of the absence of the enzyme expressing bacterial crtB (the P. ananatis (formerly Erwinia ure- PSY1 (Fig. S1). We constructed individual expression vectors for 5 dovora) phytoene synthase gene) and crtI,orcrtB, crtI and crtY/ carotenogenic genes each under the control of a different en- Bnlycb (29), and unlike transgenic potato tubers expressing bacte- dosperm-specific promoter to avoid potential gene silencing caused rial crtB and crtI,orcrtB, crtI and crtY (33), where lycopene could by promoter homology (20) and to ensure the coordinated, re- not be detected. In the case of rice, lack of lycopene was caused by stricted expression of all transgenes, a feat that has not been its complete conversion to downstream products (32). These results achieved in previous reports of multigene transfer using marker demonstrate that lycopene ␤-cyclase can be a rate-limiting step in genes to our knowledge (21). Direct DNA transfer with separate the conversion of lycopene to cyclic carotenes in transgenic white vectors usually results in transgene integration at a random single maize endosperm, in contrast to rice. The introduced transgenes locus, in the form of a multigene array (22, 23) containing any had no effect on the expression of endogenous carotenogenic genes, number of transgenes from 1 to n, with the distribution within the including psy1 and psy2 (phytoene synthase 1 and 2), pds (phytoene transgenic population tending to describe a normal curve as would desaturase), zds (␨-carotene desaturase) crtiso (carotenoid isomer- be expected from random sampling (24). Input transgenes once ase), lyce (lycopene ␧-cyclase), lycb (lycopene ␤-cyclase), and bch1 integrated remain linked and do not segregate in subsequent and bch2 (␤-carotene hydroxylase), as measured by mRNA blot and generation as is the norm for direct DNA transfer of multiple semiquantitative RT-PCR analysis (data not shown). transgenes irrespective of whether these are on 1 cointegrate vector Although the natural end point of the ␤,␤ branch of the carot- or independent plasmids (cotransformation) (19–24). Plants car- enoid pathway in maize is zeaxanthin, in vitro (34, 35) and in vivo rying 1–5 transgenes in a roughly normal distribution could be (36) studies in certain bacteria have shown that the pathway may identified from colored seed phenotypes arising from the accumu- continue to astaxanthin as a final product (Fig. S1). The formation lation of specific carotenoids, and these could be correlated with of all 7 theoretically possible ketolated intermediates and products mRNA and metabolite profiles to identify and quantify the specific in maize endosperm expressing ParacrtW and combinations of carotenoid molecules present in the endosperm. The genotypes and Zmpsy1, PacrtI, Gllycb, and Glbch is shown in Fig. 3. Astaxanthin phenotypes were inherited stably from generation to generation is formed from ␤-carotene by the addition of keto groups at the 4 once homozygosity had been achieved. Multiple independent trans- and 4Ј positions and hydroxyl groups at the 3 and 3Ј positions of the genic lines containing and expressing the same transgene comple- ␤- rings. These reactions are catalyzed by ␤-carotene keto- ment exhibited similar qualitative carotenoid profiles, indicating lase and ␤-carotene hydroxylase, respectively (Fig. 3). In the first that the combination of expressed transgenes was responsible for step, each enzyme can carry out its reaction independently, but the overall metabolic profile, but the amounts varied from line to further events depend critically on which reaction occurs first (34). line showing that microvariation in the expression levels of indi- Paracoccus ␤-carotene ketolase has a strong preference for caro- vidual transgenes influenced the levels of each specific compound tenoids with at least 1 nonhydroxylated ␤-ionone ring, e.g., ␤- (see Table S1). carotene, ␤-cryptoxanthin, echinenone, and 3-hydroxy-echinenone. The comparative analysis of these plants provides unequivocal In contrast, 3-hydroxylated ␤-ionone rings like zeaxanthin, 3Ј- confirmation that PSY1 is the key enzyme-limiting carotenoid hydroxy-echinenone, and adonixanthin are poor substrates for this biosynthesis in maize endosperm (17, 25). The overexpression of enzyme (35). We generated 3 transgenic maize phenotypes pro- phytoene synthase has a potent effect on carotenoid levels in ducing ketocarotenoids, all of which produced the mono- storage organs generally, e.g., resulting in a 1.9-fold increase in ketocarotenoid adonixanthin (4-ketozeaxanthin). Ph-5 accumu- tomato fruits (26), a 50-fold increase in canola seeds (27), and an lated adonixanthin alone (100% of total ketocarotenoids), whereas 8-fold increase in potato tubers (28). In our equivalent maize plants in Ph-6 and Ph-7, adonixanthin represented 62.72% and 69.41% of (Ph-1, overexpressing Zmpsy1), the total endosperm carotenoid total ketocarotenoids, respectively (Table S1). ParacrtW has also levels were elevated 53-fold, but the expression of 2 sequential been expressed simultaneously with a ␤-carotene hydroxylase gene enzymes (ZmPSY1 and PaCRTI in Ph-3) resulted in a further in tomato and tobacco plants (37), resulting in the accumulation of SCIENCES

tripling of the carotenoid levels. In addition, the expression of 1 major ketocarotenoid (echinenone) in tomato leaves and fruits, APPLIED BIOLOGICAL ZmPSY1 and PaCRTI in Ph-3 led to an accumulation of lycopene, and several ketocarotenoids including echinenone, 3-hydroxy- the product of the enhanced phytoene synthase and desaturase echinenone, 3Ј-hydroxy-echinenone, , adonixanthin, branch of the pathway. The addition of lycopene ␤-cyclase in Ph-4 and astaxanthin in tobacco leaves and nectary tissues. Constitutive alleviated this partial pathway limitation by converting lycopene expression of ParacrtW in Lotus japonicus caused the accumulation preferentially to ␤-carotene and further derivatives (Fig. 2). of echinenone, canthaxanthin, adonixanthin, and astaxanthin in The ratio of ␤-to␧-ring derivatives was 1.42 in Ph-1 (Zmpsy1), 3.28 flower petals (38). in Ph-3 (Zmpsy1ϩPacrtI), and 3.11 in Ph-4 (Zmpsy1ϩPacrtIϩGllycb). The product of the ParacrtW transgene uses the same substrate As observed in transgenic canola (29) and rice (30, 31), the ␤,␤ as ␤-carotene hydroxylase, an unsubstituted ␤-ionone ring. The branch of the pathway appears to be favored, perhaps implying hydroxylase and ketolase thus compete at 4 stages for different the existence of a rate-limiting step in the ␤,␧ branch. Phytoene substrates in the extended carotenoid pathway: for ␤-carotene, for was not detected in WT M37W endosperm, but it accumulated the unsubstituted sides of ␤-cryptoxanthin, and for echinenone and in all of the transgenic varieties with the exception of Ph-2 (the 3-hydroxyechinenone (Fig. 3). Thanks to the restricted ketolation of only 1 lacking Zmpsy1). This finding suggests that the conversion a 3-HO-␤-ionone ring (35), nonketolated zeaxanthin and the mo- of phytoene to lycopene (catalyzed by endogenous desaturases nohydroxy-diketo carotenoid adonixanthin represent by-products and isomerases in Ph-1, and by PaCRTI in addition to endog- of the pathway to astaxanthin. Therefore, we propose that the enous desaturases and isomerase in the other phenotypes) is a astaxanthin in maize endosperm is derived mostly from adonirubin

Zhu et al. PNAS ͉ November 25, 2008 ͉ vol. 105 ͉ no. 47 ͉ 18235 Downloaded by guest on October 1, 2021 Fig. 3. Significant reaction steps involved in the biosynthetic pathway of individual ketocarotenoids from ␤-carotene in trans- genic maize transformed with ParacrtW. The dotted ovals indicate the different substrates competitively modified by CRTW and BCH, leading to the formation of monoketocaro- tenoids and diketocarotenoids.

(3-hydroxycanthaxanthin) via 3-hydroxyechinenone (the product of the use of crtW gene from Brevundimonas sp. SD212, which appears echinenone hydroxylation) or canthaxanthin (the product of echi- to be more efficient in ketolating 3-hydroxy-␤-ionone end groups nenone ketolation) (Fig. 3). The ketolase has to overcome the (39). Alternatively, it might be possible to use the astaxanthin hydroxylase twice, first by the ketolation of ␤-carotene, and then by synthase (ASY) gene from Xanthophyllomyces dendrohous, a mul- the ketolation of either echinenone or 3-hydroxy echinenone; tifunctional enzyme catalyzing all steps from ␤-carotene to astax- otherwise astaxanthin cannot be formed. Therefore, the accumu- anthin formation by the oxygenation of carbons 3 and 4 (40). The ␤ lation of astaxanthin is determined by the balance of the ketolase reaction involves the 4-ketolation of -carotene followed by 3-hy- relative to the hydroxylase. Only plants expressing ParacrtW pro- droxylation without the accumulation of 3-hydroxy intermediates. duce a surplus of ketolase, ensuring the formation of astaxanthin. As well as gaining insight into the mechanisms underlying Otherwise, the hydroxylase activity is too high and adonixanthin is carotenoid biosynthesis in plants, particularly bottlenecks in the endogenous pathway and the competition between ␤-carotene the only final keto-hydroxy product of the pathway (together with hydroxylase and ␤-carotene ketolase for substrates in the extended nonketolated zeaxanthin). High hydroxylase activity appears to be pathway leading to astaxanthin, the combinatorial library approach the case in Ph-5 and Ph-7, where total concentrations of ketolated provides a convenient strategy for the rapid identification of carotenoids are much lower than in Ph-6, and the pathway stops metabolic variants with desirable modified properties. For example, without the second ketolation at the level of adonixanthin (Fig. 3). we identified plants with extraordinarily high levels of unsubstituted Ph-6 had the highest ketocarotenoid levels and was the only line to carotenoids (carotenes) and plants with complex mixtures of hy- synthesize astaxanthin, probably reflecting the relatively low hy- droxycarotenoids and ketocarotenoids (xanthophylls). This ap- droxylase levels (only endogenous hydroxylase but no GlBCH proach is much simpler than traditional methods for the modifica- activity, because of the absence of the transgene) and high ketolase tion of the carotenoid pathway because it relies on probability and levels (high ParaCRTW activity) (Fig. 1B). ParacrtW mRNA levels random sampling to generate a library of metabolic variants and a were substantially lower in Ph-5 and Ph-7, both of which also rapid visual selection process to identify lines of interest. Although expressed Glbch and thus had greater hydroxylase levels, driving the metabolic library in this case relied on the availability of a flux away from the di-ketolated carotenoids. To summarize, we did suitable null background (white maize) and the convenience of not expect 100% conversion of ␤-carotene to astaxanthin, in color-based visual screening for the rapid evaluation of metaboli- agreement with previous studies expressing the same ketolase gene cally-diverse transgenic plants, there is no reason the same ap- (37, 38). In Ph-5, Ph-6, and Ph-7, all expressing ParacrtW,we proach should not be used to dissect and modify other pathways as expected and obtained high ketolase activity, resulting in ketocaro- long as suitable screening methods can be found. The approach is tenoids levels representing 11–24% of total carotenoids (Table S1). analogous to standard mutagenesis screens although the ‘‘mutants’’ The accumulation of astaxanthin in Ph-6 (and no additional hy- are generated not by random mutagenesis to create loss-of-function droxylase expressed in the form of GlBCH) compared with Ph-7 phenotypes, but by random multiplex transgene insertion to create (expressing Glbch), supports the proposed competition between partially reconstructed pathways. Combinatorial transformation the ketolase and hydroxylase activities in our transgenic lines (Fig. therefore provides an efficient approach for the dissection of complex metabolic pathways (including those where competition 3) in agreement with the previous in vitro (34, 35) and in vivo (36) between enzymes at multiple stages leads to intricate combinations studies. of different metabolites) while simultaneously allowing the same For many plants transformed with a ketolase gene such as pathways to be modified in a multitude of ways to facilitate ParacrtW the conversion of adonixanthin to astaxanthin appears to metabolic engineering approaches. be an important limiting step for astaxanthin biosynthesis. Our results demonstrate that the avoidance of adonixanthin accumula- Methods tion was crucial for astaxanthin production in transgenic maize Maize Combinatorial Transformation. Maize plants (Zea mays L., cv. M37W) were endosperm. A strategy that may help to overcome this problem is grown in the greenhouse or growth room with a 10-h photoperiod (28/20 °C

18236 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809737105 Zhu et al. Downloaded by guest on October 1, 2021 day/night temperature) and 60–90% relative humidity for 50 days, followed by 4-yl)phenyl phosphate (CSPD) (Roche) was detected on Kodak BioMax light film a 16-h photoperiod (21/18 °C day/night temperature) thereafter. Thirteen-day- (Sigma–Aldrich). old immature zygotic embryos were bombarded with 10 mg of gold particles coated with the carotenogenic constructs and the selectable marker bar (41) at a HPLC Analysis. Total carotenoids were extracted from freeze-dried endosperm in molar ratio of 3:1 as reported (42) and returned to osmoticum medium for 12 h 20 mL of 50/50 (vol/vol) tetrahydrofuran and methanol at 60 °C for 15–20 min, and before selection. Bombarded callus was selected on phosphinothricin- total carotenoids were quantified by measuring absorbance at 465 nm. For HPLC supplemented medium as described (43, 44), and transgenic plantlets were separation, the solvent was evaporated under a stream of N2 gas at 37 °C and regenerated and hardened off in soil. Seventy independent events were selected redissolved in 50 ␮l of acetone, and 20 ␮L of aliquot was injected immediately. for further analysis and either pollinated with nontransformed white maize Three different HPLC systems were used at flow rates of 1 mL/min. For ketocaro- (M37W) pollen or self-pollinated to produce T1 seeds. tenoid samples, a Hypersil HyPurity Elite C18 5␮ column was used with a mobile phase of acetonitrile/methanol/2-propanol (85:10:5 vol/vol/vol) and a column ␮ DNA Analysis of Transgenic Plants. Transgenic maize lines were characterized by temperature of 32 °C (40). All other samples were separated on a Nucleosil C18 3 PCR using 3 primer sets for each transgene, amplifying segments of the 5Ј end, column with the same mobile phase at 25 °C. In each case zeaxanthin and lutein middle, and 3Ј end of the transgene (Table S4). The corresponding plasmids were were separated in parallel runs on a C18 Vydac 218TP54 column with methanol used as positive controls. PCRs were carried out under standard conditions with as the mobile phase (46). Samples were monitored with a Kontron DAD 440 photodiode array detector with on-line registration of the spectra. All carote- 100 ng of genomic DNA and 0.5 units of GoTaq DNA polymerase in a 20-␮l noids were identified by cochromatography with authentic reference com- reaction volume. The reactants were denatured at 95° C for 3 min, followed by 30 pounds and comparison of their spectra. Those standards were also used for cycles of 94 °C for 45 s, 60 °C for 45 s, 72 °C for 90 s, and a final extension at 72 °C quantitation in combination with the extinction coefficients (47). for 10 min.

ACKNOWLEDGMENTS. We thank Dr. N. Misawa (Marine Biotechnology Insti- Northern Blot Analysis. Total RNA was isolated from near-mature endosperm tute of Japan, Kanaishi, Japan) for the P. ananatis crtI gene; Dr. P. D. Fraser tissue by using a RNeasy Plant Min Kit (Qiagen), and 30-␮g aliquots were frac- (University of London, London) for the Paracoccus crtW gene; Dr. D. Ludovid tionated on a denaturing 1.2% (wt/vol) agarose gel containing formaldehyde (Consejo Superior de Investigaciones Cientificas/Institut de Recercha Tecno- and blotted by using standard methods (45). The membrane was probed with logia Agroalimentaries, Barcelona) for the rice prolamin and maize ␥-zein digoxigenin-labeled partial cDNAs at 50 °C overnight using DIG Easy Hyb (Roche promoters; the Council for Scientific and Industrial Research (Pretoria, South Diagnostics). The partial cDNAs were converted into probes by using the DIG Africa) for the WT M37W maize; Dr. P. Beyer (University of Freiburg, Freiburg, Germany), and Prof. J. Hirschberg (The Hebrew University of Jerusalem, probe synthesis kit (Roche Diagnostics) and primer set 2 (Table S5). After washing Jerusalem) for critical comments on the manuscript. This work was supported and immunological detection with anti-DIG-AP (Fab-Fragments; Roche Diagnos- by Ministry of Education and Science Grant BFU2007-61413 and the Ramon y tics) according to the manufacturer’s instructions, chemiluminescence by diso- Cajal program of the Ministry of Education and Science. S.N. is the recipient of dium 3-(4-methoxyspiro {1,2-dioxetane-3,2Ј-(5Ј-chloro) tricyclo[3.3.1.13,7]decan}- Ministry of Education and Science Ph.D Fellowship BES-2005-9161.

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