A Web-Enabled Database for Rapid Metagenomic Biocatalyst Discovery and Validation

 描述（由申请人提供）：Radiant Genomics提出开发一种集成的酶发现服务，即酶变体引擎（EVE），该引擎建立在迄今为止报道的最大克隆宏基因组序列集合的基础上。其目标是结合联合收割机一个公开的可访问的搜索引擎，丰富的注释序列数据库，阵列样品库，和LIMS自动化平台，以提供新的酶变体的最终用户以更低的成本，在更短的时间，并从更大的生物多样性池比替代方案，如DNA合成。重要的是，这项服务克服了酶发现的一个主要瓶颈，传统上集中在容易培养的生物上，现在已知这些生物只占生物多样性的不到1%。达到或超过了第一阶段研究和开发的里程碑。特别是，我们成功地证明了高测序效率的测序工作流程，这将使我们能够测序和组装我们的克隆文库，预计该文库编码约600 M基因，其中>99%来自未培养和基本上未研究的生物体。我们接下来展示了组合条形码化策略，其产生具有>30个内切酶的平均长度的组装体，这是宏基因组邻接性的显著改善。这一特征使得能够发现功能相关的基因簇，例如编码复杂天然产物和营养固定的基因。该等服务已成功纳入网上搜索引擎及电子商务平台，网址为www.eve.bio.最后，我们开发并展示了自动化LIMS基因恢复系统的基础设施，该系统每周可以从我们的阵列库中恢复数千个感兴趣的基因。第一阶段研究的成功得到了测序成本、效率和云计算的普遍改善的补充。EVE服务已经获得了商业吸引力，我们相信进一步的发展将有利于基础研究，同时对广泛的生物制造过程产生积极影响。基于客户反馈，该提案的目的是1）使用邻接保留策略对文库进行持续测序2）计算基础设施的缩放3）高级酶选择器的开发和4）第三方数据库集成。该计划的总体成果将是一个集中的搜索引擎，允许最终用户快速选择和接收生物信息学分析中识别的基因。与现有服务相比，这些基因将以更低的成本、更短的时间和更大的遗传多样性库获得。总的来说，我们相信我们的平台将提高我们对宏基因组环境的序列结构与功能关系和注释的理解，有助于弥合来自未探索的遗传多样性池的计算机模拟和生物化学表征之间的差距。

 DESCRIPTION (provided by applicant): Radiant Genomics proposes to develop an integrated enzyme discovery service, the Enzyme Variant Engine (EVE), built upon the largest cloned metagenomic sequence collection reported to date. The goal is to combine a publicly­accessible search engine, richly­annotated sequence database, arrayed sample library, and LIMS automation platform to deliver novel enzyme variants to end­users for lower cost, in less time, and from a greater pool of biodiversity than alternative options, such as DNA synthesis. Importantly, this service overcomes a major bottleneck in enzyme discovery that has traditionally focused on easily­cultivated organisms which are now known to represent less than 1% of biodiversity. Phase I research and development milestones were met or exceeded. In particular, we successfully demonstrated a high­efficiency sequencing workflow that will allow us to sequence and assemble our clone library, which is predicted to encode ~600M genes, >99% of which are derived from uncultivated and essentially unstudied organisms. We next demonstrated a combinatorial barcoding strategy that yields assemblies with an average length of >30 kilobases, a dramatic improvement in metagenomic contiguity. This feature enables the discovery of clusters of functionally related genes, such as those that encode complex natural products and nutrient fixation. These services were successfully integrated into an online search engine and e­commerce platform available at www.eve.bio. Finally, we developed and demonstrated infrastructure for an automated LIMS gene recovery system that can recover thousands of genes of interest from our arrayed library per week. The success of Phase I research was complemented by general improvements in sequencing cost­efficiency and cloud­computing. The EVE service has gained commercial traction and we believe further development will benefit basic research while positively impacting a broad range of biomanufacturing processes. Based on customer feedback, the aims of this proposal are 1) continued sequencing of the library using contiguity­preserving strategies 2) scaling of computational infrastructure 3) development of advanced enzyme selectors and 4) third­party database integration. The overall outcome of this program will be a centralized search engine which allows end­users to rapidly select and receive genes identified in bioinformatic analyses. These genes will be accessible for lower cost, in less time, and from a greater pool of genetic diversity than existing services. Overall, we believe that our platform will improve our understanding of sequence­to­function relationships and annotation for metagenomic environments, helping to bridge the gap between in silico and biochemical characterization from unexplored pools of genetic diversity.