Complementary DNA cloning and functional analysis of lycopene β-cyclase in the brown alga Undaria pinnatifida
To date, there is no functional evidence of the enzymes catalyzing carotenogenesis in brown algae. In this study, we have investigated the activity of brown algal lycopene β-cyclase that converts lycopene to β-carotene. A homology search for a lycopene β-cyclase candidate was performed after de novo transcriptome analysis of Undaria pinnatifida. The complementary DNA of the candidate, encoding 625 residues, termed Undaria pinnatifida lycopene β-cyclase-like protein (UpLCYB), was cloned. UpLCYB has a longer N-terminal region consisting of 76 residues than the 11 lycopene β-cyclase candidates of other brown algae. The N-terminal region consisting of 89 residues of the UpLCYB was predicted to be the chloroplast transition signal peptide. To assay the activity of UpLCYB, lycopene-producing Escherichia coli was prepared by introducing three genes encoding geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase from Flavobacterium sp. strain UMI-01. Recombinant UpLCYB (residues 90–625) was then expressed in this strain. The cells showed a yellow color, in contrast with cells without the UpLCYB gene, which exhibited a red color. Analysis of the extracted carotenoids from UpLCYB-expressing cells indicated that β-carotene was the main carotenoid, but only lycopene was detected in cells that did not express UpLCYB. These results suggest that UpLCYB plays a catalytic role in the conversion of lycopene to β-carotene.
KeywordsCarotenoid β-carotene Flavobacterium Carotenogenesis De novo transcriptome analysis N-terminus Signal peptide
This work was supported by Japan Society for the Promotion of Science KAKENHI grant no. 16H04977. We would like to express our gratitude to Dr. Tomohiro Hirose (Open Facility Division, Global Facility Center, Creative Research Institution, Hokkaido University) for performing the APCI-MS on an Exactive Mass Spectrometer and providing insight and expertise that greatly assisted our research.
A. I. conceived the experiment and wrote the manuscript. A. I. and T. I. conducted the experiments. A. I. and T. O. prepared the brown algal cDNA.
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