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Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli

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Abstract

Main conclusion

MpBHY codes for a carotene β-ring 3(,3′)-hydroxylase responsible for both zeaxanthin and lutein biosynthesis in liverwort. MpCYP97C functions as an ε-ring hydroxylase (zeinoxanthin 3′-hydroxylase) to produce lutein in liverwort.

Abstract

Xanthophylls are oxygenated or hydroxylated carotenes that are most abundant in the light-harvesting complexes of plants. The plant-type xanthophylls consist of α-xanthophyll (lutein) and β-xanthophylls (zeaxanthin, antheraxanthin, violaxanthin and neoxanthin). The α-xanthophyll and β-xanthophylls are derived from α-carotene and β-carotene by carotene hydroxylase activities, respectively. β-Ring 3,3′-hydroxylase that mediates the route of zeaxanthin from β-carotene via β-cryptoxanthin is present in higher plants and is encoded by the BHY (BCH) gene. On the other hand, CYP97A (or BHY) and CYP97C genes are responsible for β-ring 3-hydroxylation and ε-ring 3′-hydroxylation, respectively, in routes from α-carotene to lutein. To elucidate the evolution of the biosynthetic routes of such hydroxylated carotenoids from carotenes in land plants, we identified and functionally analyzed carotenoid hydroxylase genes of liverwort Marchantia polymorpha L. Three genes homologous to higher plants, BHY, CYP97A, and CYP97C, were isolated and named MpBHY, MpCYP97A, and MpCYP97C, respectively. MpBHY was found to code for β-ring hydroxylase, which is responsible for both routes starting from β-carotene and α-carotene. MpCYP97C functioned as an ε-ring hydroxylase not for α-carotene but for zeinoxanthin, while MpCYP97A showed no hydroxylation activity for β-carotene or α-carotene. These findings suggest the original functions of the hydroxylation enzymes of carotenes in land plants, which are thought to diversify in higher plants. In addition, we generated recombinant Escherichia coli cells, which produced rare and novel carotenoids such as α-echinenone and 4-ketozeinoxanthin, through pathway engineering using bacterial carotenogenic genes that include crtW, in addition to the liverwort MpLCYb, MpLCYe and MpBHY genes.

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Abbreviations

BCH:

β-Carotene 3,3′-hydroxylase

BHY:

β-Carotene 3,3′-hydroxylase

CD:

Circular dichroism

CYP:

Cytochrome P450

LCYb:

Lycopene β-cyclase

LCYe:

Lycopene ε-cyclase

NMR:

Nuclear magnetic resonance

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Acknowledgments

The authors thank Dr. Hiroshi Shimada, Hiroshima University, for the construction of plasmid pACHP-Beta. We also thank Dr. Takayuki Kohchi, Kyoto University; Dr. Katsuyuki T. Yamato, Kinki University and Dr. Kimitsune Ishizaki, Kobe University for their assistance in searching liverwort sequences.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Correspondence to Miho Takemura.

Additional information

Gene accession numbers

The nucleotide sequence reported in this paper has been submitted to DDBJ under accession numbers, AB981062 (MpBHY), AB981063 (MpCYP97A), AB981064 (MpCYP97B) and AB981065 (MpCYP97C).

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Takemura, M., Maoka, T. & Misawa, N. Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli . Planta 241, 699–710 (2015). https://doi.org/10.1007/s00425-014-2213-0

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Keywords

  • α-Xanthophyll
  • CYP97
  • Liverwort
  • Carotene hydroxylase
  • Marchantia polymorpha