Metabolic engineering of astaxanthin-rich maize and its use in the production of biofortified eggs

Abstract

Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing β-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.

Keywords: astaxanthin; bidirectional promoter; laying hens; maize; metabolic engineering; multigene expression.

Figure 1

Schematic representation of reconstructed astaxanthin biosynthesis pathways in maize seed. Green indicates the enhancement of early precursor biosynthesis due to the expression of ZmPSY1 and PaCRTI. Grey represents a competitive metabolic pathway that converts lycopene into lutein; we aimed to limit lycopene conversion to lutein by down‐regulating the expression of LCY‐e. Red shows our attempt to produce astaxanthin through the elongation of the β‐carotene biosynthesis pathway. The box with the black dotted outline contains the details of the strategy highlighted in red. Hydroxylases (CBFD/P450/CrtZ) and ketolases (HBFD/BKT/P450) were employed to convert the β‐rings of β‐carotene into 3‐hydroxy‐4‐keto‐β‐rings. GGPS, geranylgeranyl pyrophosphate synthase; LCY‐e, lycopene ɛ‐cyclase; LCY‐b, lycopene β‐cyclase; ABA, abscisic acid.

Figure 2

Development of astaxanthin‐rich maize. (a) Schematic diagram of the constructs pBDEN‐CP‐BZ and pBDEN‐CP‐BZ‐RNAi, functional genes and sense (lcy‐e) and anti‐sense (lcy‐e′) fragments of ZmLCY‐e directed by the seed‐specific bidirectional promoter P_R5SGPA. (b–g) Engineered astaxanthin‐rich maize seeds at different developmental stages; (b–d) seeds derived from pBDEN‐CP‐BZ; (e–g) seeds derived from pBDEN‐CP‐BZ‐RNAi; (b, e) at 20 days after pollination (DAP); (c, f) at 30 DAP; (d, g) mature dry seeds. (h–l) High‐performance liquid chromatography (HPLC) analysis of astaxanthin in dried astaxanthin‐rich maize seeds; (h) a mixture of all‐E, 9Z and 13Z astaxanthin standards; (i) the transgenic event LX68‐1, derived from pBDEN‐CP‐BZ; (j) the transgenic event LX71‐2, derived from pBDEN‐CP‐BZ‐RNAi; (k) the yellow endosperm inbred line Z58 used as the recurrent parent; (l) quantitative analysis of isomers and total astaxanthin. all‐E, all‐trans geometric isomer; 9Z, cis‐9 geometric isomer; 13Z, cis‐13 geometric isomer. All data were derived from three technical repetitions.

Figure 3

Evaluation of backcrossed astaxanthin‐rich maize. (a–d) BC3F1 seeds from event LX71‐2 crossed with inbred line Z58 (a), C7‐2 (b), NM28 (c) and XF28 (d), respectively. (e) Quantitative analysis of isomers and total astaxanthin in backcrossed astaxanthin‐rich maize seeds. All data were derived from three technical repetitions.

Figure 4

Analysis of F1 progenies of astaxanthin‐rich maize generated using new ketolase and hydroxylase gene pairs. (a) Schematic diagram of constructs. (b–g) Transgenic maize seeds derived from each type of constructs. (b, e) transgenic maize samples derived from ‘SR’ and ‘CH+SR’ showing no astaxanthin peak; (c) samples derived from ‘CH’ showing a unique unidentified peak (black arrow) and three astaxanthin peaks; (d, f) samples derived from ‘BZ’ and ‘BZ + SR’ showing the same peaks as astaxanthin isomer standards; (g) samples derived from ‘BZ + CH + SR’ containing all peaks that appear in maize derived from other constructs. The peak indicated with a green arrow was a common unknown peak in all samples. (h) Quantitative analysis of isomers and total astaxanthin. (i) A mixture of all‐E, 9Z and 13Z astaxanthin standards. nd, not detected. All data were derived from three technical repeats.

Figure 5

Functional evaluation of astaxanthin from astaxanthin‐rich maize based on a feeding trial with laying hens. (a) Screening of the optimal amount of astaxanthin to add via the addition of Haematococcus pluvialis algal powder containing 2.84% astaxanthin to laying hen feed. A0, no addition; A5–A150, feeds with final astaxanthin contents of 5, 10, 15, 30 and 150 mg/kg, respectively. (b) Profile of accumulated astaxanthin in egg yolks produced by laying hens provided feed containing astaxanthin (24.32 mg/kg) from day 0 to day 14. (c) Egg yolks from hens in different groups. C, provided feed containing no astaxanthin; A, provided feed containing astaxanthin from algal powder; T, provided feed containing astaxanthin from transgenic maize. (d) Quantitative analysis of isomers and total astaxanthin contents in egg yolks at weeks 4 and 8 in the three treatments.

Figure 6

Evaluation of storage stability of astaxanthin in transgenic maize. The event LX71‐2‐derived BC3F1 seeds were divided into four batches, which were stored in the dark at different temperature. HPLC was employed to analyse the content of astaxanthin after 7 months. Z58, C7‐2, NM28 and NF28 were four inbred maize lines; RT, room temperature. All data were derived from three technical repetitions.