Manipulation of Synechocystis sp. PCC 6803 as a platform for functional identification of genes involved in carotenoid metabolism

摘要

Carotenoids are widely distributed in nature. They function as light-harvesting/photoprotective pigments, furnish flowers and fruits with distinct colours, and benefit human health as essential phytonutrients (Rodriguez-Concepcion et al., 2018). All photosynthetic organisms synthesize lycopene and b-carotene, and higher plants share a more complicated set of carotenoids, including carotenes and their oxygenated derivatives. However, some carotenoids only exist in particular species, such as astaxanthin in the green alga Haematococcus pluvialis and lactucaxanthin in lettuce (Lactuca sativa) (Rodriguez-Concepcion et al., 2018). The elucidation of enzymes that catalyse their biosynthesis would enable the manipulation of their production by genetic engineering and synthetic biology strategies. It is usually challenging to determine the catalytic activities of these enzymes by in vitro assays using recombinant proteins in an aqueous system, because most of their substrates and products are lipid-soluble. Therefore, in vivo assays are also used for functional characterization. The pigment complementation system in Escherichia coli is able to synthesize a wide range of carotenoid substrates and has enabled the discovery of a large number of enzymes in this pathway (Cunningham and Gantt, 2007). However, the activities of some enzymes rely on additional cofactors and/or membrane structures. For example, the lycopene b-cyclase (LCYB) CruA requires a bound chlorophyll a molecule, which is absent in E. coli, for its activity (Xiong et al., 2017). Although functional complementation and/or overexpression in Arabidopsis are also widely used for characterizing new enzymes, the complicated and dynamic repertoire of endogenous carotenoids, together with the possible redundancy of enzymes, might mask the functions of the transgenes (Quinlan et al., 2007).