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

合成生物地球化学硫循环剂二甲磺基丙酸酯的藻类酶的发现和表征

• 批准号: 418730-2012
• 负责人: Waller, Jeffrey
• 金额: $ 2.62万
• 依托单位: Mount Allison University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=686596
• 关键词: Discovery; characterization; algal; enzymes; synthesizing

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.

具有讽刺意味的是，最微小的海洋微藻却对全球产生巨大影响。 更具体地说，许多微藻和大型藻类通过酶法合成二甲基磺酰基丙酸酯（DMSP），这是一种重要的渗透调节化合物，具有多种功能。 DMSP 主要被海洋细菌分解为挥发性化合物二甲硫醚 (DMS)。 DMS 约占全球大气硫预算的一半，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对 DMSP 合成进行研究 - 没有一种酶是已知的！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？它们在系统发育多样化的藻类物种中保守吗？基因鉴定能否预测藻类制造 DMSP 的能力？我将使用新颖的基因组方法来回答这些问题。 我的研究项目重点是发现藻类中 DMSP 含量的生化调节。 我的短期目标是结合细菌互补分析、比较基因组学和经典的蛋白质纯化方法，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 酶将在其生化结构、调节和动力学行为方面进行表征。 鉴定编码 DMSP 代谢酶的基因将进一步了解 DMSP 产生在以下方面的广泛性：1）通过搜索藻类基因组进行测序的藻类基因组；2）通过使用针对纯化酶产生的抗体对那些没有测序基因组的藻类进行免疫印迹分析。这些工具还将通过监测转录物或蛋白质表达水平的变化，促进不同条件下 DMSP 酶调节的研究。 最后，将藻类 DMSP 代谢途径工程化到其他生物体中可能具有商业价值。