Photosynthesis Research

, Volume 92, Issue 2, pp 245–259

A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: Adonis aestivalis as a case study

Research Article

DOI: 10.1007/s11120-007-9210-0

Cite this article as:
Cunningham, F.X. & Gantt, E. Photosynth Res (2007) 92: 245. doi:10.1007/s11120-007-9210-0

Abstract

Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, β-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.

Keywords

Adonis aestivalis Carotenoids Chloroplast Color complementation Escherichia coli Isoprenoids Phytoene Lycopene 

Abbreviations

BKT

Carotenoid β-ring 4-oxygenase or ketolase

CHYb

Carotenoid β-ring 3-hydroxylase of the nonheme diiron oxygenase type

CHYbP450

Carotenoid β-ring 3-hydroxylase of the cytochrome P450 type

CHYeP450

Carotenoid ε-ring 3-hydroxylase of the cytochrome P450 type

CrtIso

Carotene isomerase

DMAPP

Dimethylallyl diphosphate

DXP

1-deoxy-d-xylulose 5-phosphate

DXR

1-deoxy-d-xylulose 5-phosphate reductoisomerase

DXS

1-deoxy-d-xylulose 5-phosphate synthase

GAPD

Glyceraldehyde-3-phosphate dehydrogenase

GGPP

Geranylgeranyl diphosphate

GGPS

Geranylgeranyl diphosphate synthase

IPI

Isopentenyl diphosphate isomerase

IPP

Isopentenyl diphosphate

LCYb

Lycopene β-ring cyclase

LCYe

Lycopene ε-ring cyclase

LytB

1-hydroxy-2-methyl-butenyl 4-diphosphate reductase (IspH)

MECPS

2C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase (IspF)

MEP

Methylerythritol 4-phosphate

NSY

Neoxanthin synthase

PDS

Phytoene desaturase

PEP

Phosphoenolpyruvate

PPS

Phosphoenolpyruvate synthase

PSY

Phytoene synthase

VDE

Violaxanthin de-epoxidase

ZDS

ζ-carotene desaturase

ZEP

Zeaxanthin epoxidase

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  1. 1.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA

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