Molecular mechanism of carotenoid esterification in microalgae

类胡萝卜素主要在光合作用生物(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，但必须通过饮食补充。因此，了解类胡萝卜素的生物合成对于基础研究和应用研究都具有重要意义。类胡萝卜素有两类：胡萝卜素(碳氢类胡萝卜素)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为叶黄素酯。 到目前为止，几乎所有自由形式叶黄素生物合成的步骤都在微藻中被鉴定出来。然而，叶黄素酯化的分子机制还很不清楚。本研究的目的是以雨生红球藻为模型系统，确定叶黄素酯化的基因/酶。这种藻类能够以虾青素酯的形式积累高含量的虾青素，这是一种典型的叶黄素，已被用作研究虾青素生物合成的模型系统。 根据虾青素和虾青素酯的分子结构推测，催化虾青素酯化的酶可能是雨生青霉虾青素酰基转移酶(HpAATs)。HpAATs可能属于二酰甘油酰基转移酶(DGAT)家族，因为在体外实验中，DGAT特异性抑制剂抑制了虾青素酯的合成。在拟议的研究计划中，将确定HpAAT。它们的生化性质将通过体外实验进行研究，它们的生物学功能将通过体内实验进行鉴定。将从高等植物和其他微藻物种中分离和比较HpAATs的同源物，以探索在广泛的光合作用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，雨生红豆杉是工业化生产虾青素的主要品种。虾青素的酯化反应是雨生红藻中虾青素积累的主要调控步骤。因此，我进一步假设HpAAT酶对虾青素的生产是重要的。为了验证这一假设，将用一种新的定向进化系统产生高活性的HpAAT变体。然后，这些“超级”HpAAT将在雨生红藻中过度表达，以测试它们在增加虾青素积累方面的贡献。 总体而言，这项假设驱动的研究将促进2名博士生、1名硕士研究生和6名本科生的培养。这一开创性的研究结果将有助于理解光合作用生物中类胡萝卜素的酯化作用，并有助于开发新的生物技术来提高类胡萝卜素的高价值生产。

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.