Molecular mechanism of carotenoid esterification in microalgae

类胡萝卜素主要在光合生物（高等植物、藻类和蓝藻）中合成，具有重要的生物学功能。类胡萝卜素对人类和动物也很重要，但必须通过饮食提供。因此，了解类胡萝卜素的生物合成对基础研究和应用研究都很重要。类胡萝卜素有两类：胡萝卜素（烃类胡萝卜素）和叶黄素（胡萝卜素的氧衍生物，如虾青素）。在高等植物和微藻中，许多叶黄素被脂肪酸进一步酯化成叶黄素酯。 到目前为止，几乎所有的自由形式的叶黄素生物合成步骤已被确定在微藻。然而，叶黄素酯化的分子机理在很大程度上是未知的。建议的研究的目的是确定的基因/酶负责叶黄素酯化与微藻雨生红球藻作为一个模型系统。该藻能以虾青素酯的形式积累大量的典型叶黄素--虾青素，并已被用作研究虾青素生物合成的模式系统。 根据虾青素及其酯的分子结构，推测催化虾青素酯化的酶是H。雨生藻虾青素酰基转移酶（HpAATs）。HpAAT可能属于二酰基甘油酰基转移酶（DGAT）家族，因为在体外测定中，虾青素酯化被DGAT特异性抑制剂抑制。在拟议的研究计划中，将确定HpAAT。通过体外实验研究其生化特性，并探讨其在H.将通过体内测定来鉴定雨生植物。从高等植物和其他微藻物种的HpAAT的直系同源物将被分离和比较表征，以探索在广泛的光合生物中叶黄素酯化的分子机制。 虾青素是一种高价值的类胡萝卜素，H。雨生藻是用于工业虾青素生产的主要物种。近年来研究表明，虾青素酯化是H.雨生植物因此，我进一步假设，HpAAT酶是重要的虾青素生产。为了检验这一假设，将用新的定向进化系统产生高活性HpAAT变体。这些“超级”HpAAT随后将在H. pluvialis来测试它们在增加虾青素积累中的贡献。 总的来说，这项假设驱动的研究将促进2名博士生、1名硕士生和6名本科研究人员的培训。这一开创性的研究结果将有助于了解光合生物中的类胡萝卜素酯化作用，并有助于开发新的生物技术来提高高价值类胡萝卜素的生产。

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.