Molecular mechanism of carotenoid esterification in microalgae

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Carotenoids are mainly synthesized in photosynthetic organisms (higher plants, algae, and cyanobacteria) and have important biological functions. Carotenoids are also important for humans and animals but must be supplied through the diet. Therefore, understanding of carotenoid biosynthesis is important for both primary and applied research. There are 2 classes of carotenoids: carotenes (hydrocarbon carotenoids) and xanthophylls (oxygen derivatives of carotenes such as astaxanthin). In higher plants and microalgae, many xanthophylls are further esterified by fatty acids to xanthophyll esters. *** To date, almost all steps in free-form xanthophyll biosynthesis have been identified in microalgae. However, the molecular mechanism of xanthophyll esterification is largely unknown. The aim of the proposed research is to identify the genes/enzymes responsible for xanthophyll esterification with microalga Haematococcus pluvialis as a model system. This algal species can accumulate high content of astaxanthin, a typical xanthophyll, in the form of astaxanthin ester and has been used as a model system to study astaxanthin biosynthesis.*** Based on the molecular structures of astaxanthin and astaxanthin esters, I hypothesize that the enzymes catalyzing astaxanthin esterification are H. pluvialis astaxanthin acyltransferases (HpAATs). HpAATs may belong to the diacylglycerol acyltransferase (DGAT) family because astaxanthin esterification are inhibited by DGAT specific inhibitors in in vitro assay. In the proposed research program, HpAATs will be identified. Their biochemical properties will be studied by in vitro assay and their biological functions in H. pluvialis will be identified by in vivo assay. The orthologs of HpAATs from higher plants and other microalgal species will be isolated and comparatively characterized to explore the molecular mechanism of xanthophyll esterification in a broad range of photosynthetic organisms. *** Astaxanthin is a high-value carotenoid and H. pluvialis is the major species being used in industrial astaxanthin production. Astaxanthin esterification was recently revealed to be the principle regulatory step of astaxanthin accumulation in H. pluvialis. Therefore, I further hypothesize that HpAAT enzymes are important for astaxanthin production. To test this hypothesis, high activity HpAAT variants will be generated with a novel Directed Evolution system. These “super” HpAATs will then be over-expressed in H. pluvialis to test their contribution in increasing astaxanthin accumulation. *** Collectively, this hypothesis driven research will promote the training of 2 PhD students, 1 MSc student and 6 undergraduate researchers. The results of this seminal research will contribute to the understanding of carotenoid esterification in photosynthetic organisms and to the development of new biotechnology for improving high-value carotenoid production.**