Functional Approaches for Annotating Secretome-bound Small ORFs in Microbial Genomes

生物技术计划化学、生物工程、环境和运输系统司注释微生物基因组中分泌体结合的小ORF的功能方法罗伯特·凯利北卡罗来纳州立大学CBET-0730091到目前为止，几乎所有的微生物功能基因组学和蛋白质组学研究都集中在细胞质上。“分泌组”或细胞外蛋白质组在很大程度上被忽视了，尽管它在理解自然和工业生物加工环境的微生物生理学和生态学方面至关重要。特别是，分泌组结合的小蛋白和小肽通常不会被注意到。这些生物分子不仅很难识别、以成熟的形式分离和表征，而且它们可能只对特定的、有时是短暂的刺激做出反应。这里假设微生物基因组包含许多尚未被注释的小开放阅读框(ORF)，这些ORF编码在微生物生物学和生物处理中具有核心重要性的新的分泌组结合的生物分子，包括参与物种间和物种内信号传递和抗微生物功能的多肽/蛋白质。该项目的科学和智力价值与开发定量的、全基因组的方法有关，以战略性地探索微生物分泌体的动态，着眼于分泌组结合的小ORF(约100个氨基酸或更少)。虽然这里的重点将是在纯培养和混合培养中生长的超嗜热性的模型maritima，但开发的方法也适用于较不嗜热的微生物。这一方法将通过寻找通过初步努力获得的分泌体结合多肽的线索来验证，此外还将探索这里确定的新的可能性。这个项目的具体目标是：(1)证明转录反应信息可以用于战略性地聚焦微生物分泌组的蛋白质组学分析，以鉴定和分离新的分泌体结合小蛋白和多肽的成熟形式；(2)研究与毛滴虫中假定的分泌体结合肽相关的种群依赖的信号机制，并确定较不嗜热细菌中的同源多肽是否在群体感应中发挥作用；以及(3)评估在毛滴虫分泌体结合小ORF上的新线索，这个项目的更广泛的影响与生物分子工程研究生在定量系统生物技术的新方面的培训有关。通常情况下，每年有5-10名本科生参与这项研究，并将招募几名本科生加入该项目，让他们在研究中接触到职业前景。将开发和扩展包含极端微生物酶的生产和纯化的实验室，以用于NCSU生物技术计划教授的课程。通过该网站提供的精选数据库将提供关于毛滴虫和其他微生物的ORF小注释的最新进展情况。

Biotechnology ProgramDivision of Chemical, Bioengineering, Environmental, and Transport SystemsFunctional Approaches for Annotating Secretome-Bound Small ORFs in Microbial GenomesRobert KellyNorth Carolina State UniversityCBET-0730091To date, almost all microbial functional genomics and proteomics studies have focused on the cytosol. The "secretome", or extracellular proteome, has been largely ignored, despite its critical importance in understanding the microbial physiology and ecology of both natural and industrial bioprocessing environments. In particular, secretome-bound small proteins and peptides typically go unnoticed. These biomolecules are not only difficult to identify, isolate in their mature forms, and characterize, but they may only be produced in response to specific, and sometimes transient, stimuli. Here, it is hypothesized that microbial genomes contain many yet to be annotated small open reading frames (ORFs) that encode novel secretome-bound biomolecules of central importance in microbial biology and bioprocessing including peptides/proteins implicated in inter- and intra-species signaling and anti-microbial functions. The scientific and intellectual merit of this project relates to the development of quantitative, genome-wide approaches to strategically probe the dynamics of microbial secretomes with an eye towards secretome-bound small ORFs (about 100 amino acids or less). While the focus here will be on the model hyperthermophile Thermotoga maritima grown in pure and mixed cultures, the methodology developed will be applicable to less thermophilic microorganisms as well. This approach will be validated by pursuing leads on secretome-bound peptides obtained through preliminary efforts, in addition to exploring new possibilities that are identified here. The specific objectives of this project are to: (1) demonstrate that transcriptional response information can be used to strategically focus proteomics analysis of microbial secretomes to identify and isolate mature forms of novel secretome-bound small proteins and peptides, (2) investigate population-dependent signaling mechanisms related to a putative secretome-bound peptide in T. maritima and determine if orthologous peptides in less thermophilic bacteria play roles in quorum sensing, and (3) evaluate new leads on secretome-bound small ORFs in T. maritima, with a focus on thermally stable anti-microbial peptides.The broader impacts of this project relate to the training of biomolecular engineering graduate students in a new aspect of quantitative systems biotechnology. Typically 5-10 undergraduates participate in this research each year and several will be recruited to the project to expose them to career prospects in research. Labs that incorporate production and purification of extremophilic enzymes will be developed and expanded for use in courses taught in the NCSU Biotechnology Program. A curated database provided through the website will provide updates on progress with small ORF annotation in T. maritima and other microorganisms.