Functional Characterization of Proanthocyanidin Pathway Enzymes from Tea and Their Application for Metabolic Engineering

Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blightresistant tea cultivar TRI2043 and functionally...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 2013-03, Vol.161 (3), p.1103-1116
Main Authors: Pang, Yongzhen, Abeysinghe, I. Sarath B., He, Ji, He, Xianzhi, Huhman, David, Mewan, K. Mudith, Sumner, Lloyd W., Yun, Jianfei, Dixon, Richard A.
Format: Article
Language:English
Subjects:
Amino acids
BIOCHEMISTRY AND METABOLISM
Biological and medical sciences
Biosynthesis
Biosynthetic Pathways - genetics
CDNA libraries
Chromatography, High Pressure Liquid
Enzymes
Flavonoids
Flavonoids - chemistry
Flavonoids - metabolism
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation, Enzymologic
Gene Expression Regulation, Plant
Gene Library
Genes, Plant - genetics
Kinetics
Libraries
Medicago truncatula - genetics
Metabolic Engineering
Nicotiana - metabolism
Open reading frames
Phylogeny
Plant Leaves - genetics
Plant physiology and development
Plant Proteins - genetics
Plant Proteins - metabolism
Plant Roots - genetics
Plants
Plants, Genetically Modified
Proanthocyanidins - biosynthesis
Recombinant Proteins - metabolism
Sequence Analysis, DNA
Tea
Tea - enzymology
Tea - genetics
Transgenic plants
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Summary:Tea (Camellia sinensis) is rich in specialized metabolites, especially polyphenolic proanthocyanidins (PAs) and their precursors. To better understand the PA pathway in tea, we generated a complementary DNA library from leaf tissue of the blister blightresistant tea cultivar TRI2043 and functionally characterized key enzymes responsible for the biosynthesis of PA precursors. Structural genes encoding enzymes involved in the general phenylpropanoid/flavonoid pathway and the PA-specific branch pathway were well represented in the library. Recombinant tea leucoanthocyanidin reductase (CsLAR) expressed in Escherichia coli was active with leucocyanidin as substrate to produce the 2R, 3S-trans-flavan-ol (+)-catechin in vitro. Two genes encoding anthocyanidin reductase, CsANR1 and CsANR2, were also expressed in E. coli, and the recombinant proteins exhibited similar kinetic properties. Both converted cyanidin to a mixture of (+)-epicatechin and (-)-catechin, although in different proportions, indicating that both enzymes possess epimerase activity. These epimers were unexpected based on the belief that tea PAs are made from (-)-epicatechin and (+)-catechin. Ectopic expression of CsANR2 or CsLAR led to the accumulation of low levels of PA precursors and their conjugates in Medicago truncatula hairy roots and anthocyanin-overproducing tobacco (Nicotiana tabacum), but levels of oligomeric PAs were very low. Surprisingly, the expression of CsLAR in tobacco overproducing anthocyanin led to the accumulation of higher levels of epicatechin and its glucoside than of catechin, again highlighting the potential importance of epimerization in flavan-3-ol biosynthesis. These data provide a resource for understanding tea PA biosynthesis and tools for the bioengineering of flavanols.
ISSN:0032-0889
1532-2548
1532-2548
DOI:10.1104/pp.112.212050