Molecular Mechanisms of Marine Polysaccharide Depolymerization and Metabolism.

据估计，海洋植物生物量[即微藻和大藻（海藻）]约为陆地植物生物量的1/200，但海洋中光合作用固定碳的大规模标准化年周转率大约比陆地快两个数量级。因此，这种极具活力的环境对全球碳循环非常重要。海藻的细胞壁和储存聚合物是海藻生物量的主体，由一组结构不同于陆生植物的复杂多糖组成。这种光合作用的固定碳主要通过微生物的代谢作用返回到全球碳循环中，但这一过程背后的微生物学和生物化学研究仍然很少。**本研究计划的总体目标是研究微生物如何解聚海藻多糖，最终目的是提供代谢海藻多糖所需途径的分子水平细节。具体目标是：*1。分离未鉴定的海藻多糖降解细菌，对其基因组进行测序，然后利用这些基因组与现有基因组结合，描绘潜在的海藻多糖代谢途径。分析选定细菌对海藻生物量或特定多糖的特异性和/或全基因组基因表达。使用结构-功能方法揭示细菌对海藻多糖的酶生物转化背后的分子细节。**我们最初的方法将是从当地水域分离出可降解海洋细菌的好氧海藻，并利用现代DNA测序技术（例如Illumin HiSeq）对其基因组进行测序。除了现有的基因组外，这些新的基因组将为利用生物信息学方法（例如，自动基因组注释，用dbCAN注释碳水化合物活性酶）挖掘提供丰富的资源，从而对加工海藻多糖所需的酶系统有更广泛的了解（目标1）。基因组信息将被用于分析特定海藻生物量作为生长基质的基因表达（qPCR、RNASeq或微阵列分析），如完整的海藻或纯化的多糖（目标2）。最后，我们关注的最大和最密集的目标是结合分子生物学，重组蛋白表达，蛋白质生物化学，酶学，和结构生物学（主要是x射线晶体学），以充分了解海藻多糖解聚和成分利用的分子细节（目标3）。**该研究计划将提供微生物如何降解海藻多糖的基本原理的详细见解。这对于揭示光合作用固定碳如何在海洋中循环以及将生化信息纳入全球碳循环的生物地球化学模型至关重要。此外，在一个温室气体和气候变化水平不断上升的世界里，人们越来越多地将目光转向“绿色”科学、可再生资源、碳中和足迹和替代燃料来源。它通常被忽视，但海藻是一种可再生资源，其细胞壁和储存多糖代表了生产生物燃料的有吸引力的碳水化合物来源，与陆地原料相比具有许多极其吸引人的优势。最终，拟议的研究计划将产生一个知识平台，可用于设计将海藻细胞壁多糖酶转化为可转化为生物燃料的可发酵糖的策略。

Ocean plant biomass [i.e. microalgae and macroalgae (seaweed)] is estimated to be ~1/200 that of terrestrial plant biomass yet the mass-normalized annual turnover rate of photosynthetically fixed carbon in the oceans is roughly two-orders of magnitude faster than on land. This extremely dynamic environment is therefore very important to the global carbon cycle. The cell wall and storage polymers of seaweed, which are the bulk of seaweed biomass, comprise a group of complex polysaccharides that are structurally different from those of terrestrial plants. This photosynthetically fixed carbon is returned to the global carbon cycle primarily through the metabolic action of microbes but the microbiology and biochemistry underlying the process remains poorly studied. **The overarching objective of this research program is to address how microbes depolymerize seaweed polysaccharides with the ultimate aim of providing molecular level detail for the pathways required to metabolize seaweed polysaccharides. The specific objectives are to:*1. Isolate uncharacterized seaweed polysaccharide degrading bacteria, sequence their genomes, then exploit these genomes with existing genomes to delineate potential seaweed polysaccharide metabolism pathways.*2. Analyse the specific and/or genome-wide expression of genes in selected bacteria in response to seaweed biomass or specific polysaccharides.*3. Use a structure-function approach to uncover the molecular details behind the enzymatic bioconversion of seaweed polysaccharides by bacteria.**Our initial approach will be to isolate aerobic seaweed degrading marine bacteria from local waters and exploit modern DNA sequencing technologies (e.g. Illumin HiSeq) to sequence their genomes. In addition to existing genomic, these new genomes will provide a rich resource to mine using bioinformatics approaches (e.g. automated genome annotation, annotation of carbohydrate-active enzymes with dbCAN) to give a broad view of the enzyme systems required to process seaweed polysaccharides (Objective 1). The genomic information will then be exploited using methods to analyse gene expression (qPCR, RNASeq, or microarray analysis) in response to particular seaweed biomass as growth substrates, such as intact seaweed or purified polysaccharides (Objective 2). Finally, the largest and most intensive objective that is receiving the bulk of our focus is to use a combination of molecular biology, recombinant protein expression, protein biochemistry, enzymology, and structural biology (primarily X-ray crystallography) to fully understand the molecular details of seaweed polysaccharide depolymerisation and utilization of the components (Objective 3).**This research program will provide detailed insight into the fundamentals of how microbes degrade seaweed polysaccharides. This is critical to unravelling how photosynthetically fixed carbon is recycled in our oceans and incorporating biochemical information into biogeochemical models of the global carbon cycle. Additionally, in a world that is seeing rising levels of greenhouse gases and climate change, eyes are being increasingly turned to "green" science, renewable resources, carbon neutral footprints, and alternative fuel sources. It has been generally overlooked but seaweed is a renewable resource whose cell wall and storage polysaccharides represent an attractive carbohydrate source for the production of bio-fuels, with many extremely appealing advantages over terrestrial feedstocks. Ultimately, the proposed research program will generate a knowledge platform that can be leveraged to design strategies for the enzymatic conversion of seaweed cell-wall polysaccharides into fermentable sugars that can be converted into biofuels.