Molecular Mechanisms of Marine Polysaccharide Depolymerization and Metabolism.

海洋植物生物量[即微藻和大型藻类(海藻)]估计约为陆地植物生物量的1/200，但海洋中光合作用固定碳的质量归一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。海藻的细胞壁和贮藏聚合物是海藻生物量的主体，它含有一组复杂的多糖，在结构上与陆地植物的不同。这种通过光合作用固定的碳主要通过微生物的新陈代谢作用回到全球碳循环中，但对这一过程背后的微生物学和生物化学的研究仍然很少。该研究计划的总体目标是解决微生物如何解聚海藻多糖，最终目的是为代谢海藻多糖所需的途径提供分子水平的细节。具体目标是：1.分离未鉴定的海藻多糖降解菌，对其基因组进行测序，然后利用已有的基因组对这些基因组进行利用，以描绘潜在的海藻多糖代谢途径。分析所选细菌对海藻生物量或特定多糖的特异性和/或全基因组表达。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。我们的初步方法将是从当地水域中分离出好氧海藻降解海洋细菌，并利用现代DNA测序技术(如Lillin HiSeq)对其基因组进行测序。除了现有的基因组外，这些新的基因组将提供丰富的资源，以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)，以提供处理海藻多糖所需的酶系统的广泛看法(目标1)。然后将利用基因组信息分析基因表达的方法(qPCR、RNAseq或微阵列分析)，以响应作为生长底物的特定海藻生物量，例如完整的海藻或纯化的多糖(目标2)。最后，我们主要关注的最大和最密集的目标是使用分子生物学、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射线结晶学)的组合来充分了解海藻多糖解聚和成分利用的分子细节(目标3)。这项研究计划将提供对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的碳是如何在我们的海洋中循环的，并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，在一个温室气体水平和气候变化不断上升的世界里，人们的目光越来越多地转向“绿色”科学、可再生资源、碳中性足迹和替代燃料来源。海藻是一种可再生资源，其细胞壁和储藏多糖是生产生物燃料的极具吸引力的碳水化合物来源，与陆地原料相比具有许多极具吸引力的优势。最终，拟议的研究计划将产生一个知识平台，可用于设计策略，将海藻细胞壁多糖酶转化为可发酵的糖，可转化为生物燃料。

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.