Discovery and characterization of algal enzymes synthesizing the biogeochemical sulfur-cycling agent dimethylsulfoniopropionate

具有讽刺意味的是，最微小的海洋微藻对全球产生了巨大的影响。更具体地说，许多种类的微型和大型藻类在酶作用下合成了一种重要的渗透压调节化合物--二甲基磺基丙酸二甲酯(DMSP)，具有多种拟议功能。DMSP主要由海洋细菌分解成挥发性化合物二甲基硫化物(DMS)。DMS约占全球大气硫收支的一半，并影响全球气候。由于海藻是海洋DMSP的唯一来源，因此也是DMS的来源，令人惊讶的是，几乎没有人对DMSP的合成进行过研究--没有一个酶是已知的！催化这一主要生物地球化学通量的酶的分子特性和结构是什么？它们是否保存在系统发育多样性的藻类物种中？基因鉴定能预测藻类产生DMSP的能力吗？我将使用新的基因组方法来回答这些问题。本课题的研究重点是发现藻类中DMSP含量的生化规律。我的短期目标是使用细菌互补分析、比较基因组学和经典的蛋白质纯化方法相结合的方法来发现编码DMSP生物合成途径酶的基因。天然酶的纯化将使基因鉴定技术能够发现编码DMSP代谢酶的基因。纯化的天然或重组DMSP酶将根据其生化结构、调节和动力学行为进行表征。识别编码DMSP代谢酶的基因将进一步加深我们对DMSP产生的理解：1)通过搜索其基因组来对藻类基因组进行测序；2)在对未测序基因组的藻类进行免疫印迹分析时使用针对纯化酶的抗体。这些工具还将通过监测转录本或蛋白质表达水平的变化，促进不同条件下DMSP酶调控的研究。最后，设计藻类DMSP进入其他生物的代谢途径可能具有商业价值。

It is ironic that the tiniest oceanic microalgae have a huge global impact. More specificially, many taxa of micro- and macro- algae enzymatically synthesize dimethylsulfoniopropionate (DMSP), an important osmoregulatory compound, with multiple proposed functions. DMSP is broken down to the volatile compound dimethylsulfide (DMS), primarily by marine bacteria. DMS accounts for about half the global atmospheric sulfur budget, and influences global climate. As marine algae are the only source of oceanic DMSP and thus, the source of DMS, it is surprising that almost no research on DMSP synthesis has been done - not one enzyme is known! What are the molecular identities and structures of the enzymes catalyzing this major biogeochemical flux? Are they conserved in phylogenetically diverse algal species? Will gene identification predict an alga's capacity to make DMSP? I will use novel genome-enabled methods to answer these questions. My research program focuses on discovering the biochemical regulation of DMSP contents in algae. My short term goal is to disover the genes encoding the DMSP biosynthesis pathway enzymes using a combination of bacterial complementation assays, comparative genomics and classical protein purification methods. Purification of the native enzymes will enable gene identification technologies to discover the genes encoding DMSP metabolic enzymes. Purified native or recombinant DMSP enzymes will be characterized with respect to their biochemical structure, regulation, and kinetic behaviour. Identification of genes encoding DMSP metabolic enzymes will further our understanding of how widespread DMSP production is in 1) sequenced algal genomes by searching their genomes and 2) by using antibodies raised against purified enzymes in immunoblot analyses of those algae without sequenced genomes. These tools will also facilitate investigations of DMSP enzyme regulation under different conditions by monitoring transcript or protein expression level changes. Lastly, engineering of the algal DMSP metabolic pathway into other organisms may have commercial value.