Functional analysis of the marine cellulolytic system of Saccharophagus degradans

降解蔗糖2-40(Saccharphagus degradans 2-40，Sde2-40)是一种海洋细菌，能快速降解高等植物和藻类细胞壁中发现的10种不同的复杂多糖。最近获得的基因组序列揭示了每个CP降解系统的基本元素，这些系统通常在高等植物细胞壁中发现，最明显的是纤维素。该细菌能够作为唯一的碳源和能源生长在Avicel上，这表明它表达了真正的纤维分解系统。虽然人们对土壤、瘤胃细菌和纤维素分解真菌的纤维分解系统有很好的了解，但对纤维素在海洋环境中如何降解却知之甚少。Sde2-40纤维素分解系统似乎由一组具有独特结构的分泌酶和表面排列的酶组成。预测的纤维素酶和辅酶的结构特征已被用来组装具有不寻常元素的Sde2-40降解纤维素的模型。拟议研究的目的是阐明2-40在海洋环境中降解和加工纤维素的机制。表达蛋白质的生化活性将用于确认预测的基因功能，并使用先前设计的微阵列进行转录组分析，以确定相对表达水平。将确定功能性酶的空间分布和底物特异性。基因置换突变将用于评估选定的酶或组分的作用。这些实验将使我们能够测试特定酶的作用并通过特定的途径估计通量，以开发一个关于Sde2-40如何降解纤维素的综合模型。除了在研究过程中对本科生和研究生进行培训和获得知识，例如将纤维素生物质应用于生物加工以生产生物乙醇，我们还参与了两个推广计划，旨在引入和招募代表不足的少数族裔进入微生物学研究。我们还为微生物遗传学课程开发了一个实验室模块。

Saccharophagus degradans 2-40 (Sde2-40) is a marine bacterium with the ability to rapidly degrade 10 different complex polysaccharides (CPs) found in higher plant and algal cell walls. The recently obtained genome sequence has revealed essential elements of the degradative systems for each CP typically found in higher plant cell walls, most notably cellulose. The bacterium is able to grow on Avicel as a sole carbon and energy source, indicating it expresses a true cellulolytic system. Although cellulolytic systems of soil and rumen bacteria and cellulolytic fungi are well characterized, little is known about how cellulose is degraded in the marine environment. The Sde2-40 cellulolytic system appears to be composed of a provocative assemblage of secreted and surface-arrayed enzymes with unique architectures. The structural features of the predicted cellulases and accessory enzymes have been used to assemble a model of the degradation of cellulose by Sde2-40 that has unusual elements. The goal of the proposed studies is to elucidate the mechanism by which 2-40 degrades and processes the cellulose in the marine environment. Biochemical activity of expressed proteins will be used to confirm predicted gene functions and transcriptome analysis employing previously designed microarrays used to establish relative expression levels. Spatial distribution and substrate specificities of functional enzymes will be determined. Gene replacement mutagenesis will be used to evaluate the role of selected enzymes or components. These experiments will allow us to test the role of specific enzymes and estimate fluxes through particular pathways to develop an integrated model for how Sde2-40 degrades cellulose.In addition to the training of undergraduate and graduate students during the course of this research and the gain of knowledge, such as the applications to bioprocessing of cellulosic biomass for bioethanol production, we are involved in two outreach programs designed to introduce and recruit underrepresented minorities into microbiological research. A laboratory module has also been developed for our Microbial Genetics course.