A lycopene ß-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and ß-carotene at different ratios.

Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene ß-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and ß-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-ß-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays.

A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize potential carotenoid substrates, followed by analysis of the pigment changes in the carotenogenic bacteria via high-performance liquid chromatography (HPLC). Two crucial steps in this method are (i) the quantitative extraction of the carotenoids out of E. coli and (ii) the reproducible and complete separation of the pigments by HPLC. Here, we present a protocol for the extraction and analysis of carotenoids with a broad range of polarities from carotenogenic E. coli. The solvent mixture used for extraction keeps both the lipophilic carotenes and the more polar xanthophylls in solution and is compatible with the eluent gradient of the subsequent HPLC analysis. The C30-column used is particularly suitable for the separation of various cis-isomers of carotenoids, but also for separation of stereoisomers such as α- and ß-carotene or lutein and zeaxanthin.