Transcription engineering for biosensor-based screening of metagenomic libraries

基于生物传感器的宏基因组文库筛选的转录工程

• 批准号: 8933964
• 负责人: Nicholas Richard Sandoval
• 金额: $ 5.31万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2016-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8933964
• 关键词: Address; Antibiotics; Area; Bacillus subtilis; Bacteria; Binding Sites; Biochemical; Biosensor; Butanols; Carcinogens; Cells; Chemicals; Collection; Communities; DNA-Directed RNA Polymerase; Disease; Distant; Drug Metabolic Detoxication; Engineering; Enzymes; Escherichia coli; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genome engineering; Genomic DNA; Genomics; Genotype; Green Fluorescent Proteins; Health; High-Throughput Nucleotide Sequencing; Housing; Human; Human body; Laboratories; Lactobacillus plantarum; Lead; Libraries; Life; Link; Measures; Metagenomics; Metals; Methods; Microbe; Microbiology; National Research Service Awards; Nature; Nutrient; Organism; Phenotype; Plasmids; Population; Production; Prokaryotic Cells; Reporter; Reporter Genes; Resistance; Resources; Role; Sigma Factor; Soil; Solutions; Sorting - Cell Movement; System; Technology; Testing; Time; Toxin; Transcription Coactivator; Vitamins; Work; base; extracellular; genetic element; gut microbiota; high throughput screening; improved; interest; metagenome; microbial; microbiome; novel; promoter; public health relevance; screening; small molecule; vector; Address; Antibiotics; Area; Bacillus subtilis; Bacteria; Binding Sites; Biochemical; Biosensor; Butanols; Carcinogens; Cells; Chemicals; Collection; Communities; DNA-Directed RNA Polymerase; Disease; Distant; Drug Metabolic Detoxication; Engineering; Enzymes; Escherichia coli; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genome engineering; Genomic DNA; Genomics; Genotype; Green Fluorescent Proteins; Health; High-Throughput Nucleotide Sequencing; Housing; Human; Human body; Laboratories; Lactobacillus plantarum; Lead; Libraries; Life; Link; Measures; Metagenomics; Metals; Methods; Microbe; Microbiology; National Research Service Awards; Nature; Nutrient; Organism; Phenotype; Plasmids; Population; Production; Prokaryotic Cells; Reporter; Reporter Genes; Resistance; Resources; Role; Sigma Factor; Soil; Solutions; Sorting - Cell Movement; System; Technology; Testing; Time; Toxin; Transcription Coactivator; Vitamins; Work; base; extracellular; genetic element; gut microbiota; high throughput screening; improved; interest; metagenome; microbial; microbiome; novel; promoter; public health relevance; screening; small molecule; vector

DESCRIPTION (provided by applicant): Effective screening of metagenomic libraries will enable the exploration of the great diversity of microbes in nature that are responsible for many interesting and valuable functions necessary for human life. For example, in the human gut, the bacterial community detoxifies harmful chemicals, synthesizes vitamins, and helps digest nutrients that the human body cannot digest by itself, among other vital functions. Unfortunately, the vast majority of bacteria cannot be cultured in a laboratory, so the genetic basis of the biocatalytic activity often goes undiscovered. The microbiology workhorse organism E. coli can house genetic collections of these bacterial communities' genomic DNA (known as metagenomic libraries). Screening of metagenomic libraries is inefficient due to two main limitations. First, E. coli is often unable to recognize heterologous promoters in the metagenomic library. Second, searching for biocatalytic functions often involves screening for the presence of small molecules, which can be time and resource intensive. The proposed solution is to develop a culture-independent method for high-throughput screening of metagenomic libraries. In the first aim, it is proposed that by expressing transcription machinery (sigma factors) from prokaryotes phylogenetically distant from the host, the host's RNA polymerase will better recognize heterologous promoters, improving transcription of the genetic elements in metagenomic libraries. To improve heterologous gene expression from metagenomic libraries, sigma factors will be expressed in host strains from a range of bacterial species (including Bacillus subtilis and Lactobacillus plantarum). Transcription of heterologous libraries will be quantified by fusing the green fluorescent protein gene to small library inserts and measuring fluorescence with flow cytometry. The expression level of heterologous sigma factors will be optimized by engineering the promoters and ribosomal binding sites (RBS) in order to obtain a good expression strain. Using this system, a metagenomic library will be constructed from soil bacteria and screened for genes imparting enhanced butanol tolerance. In the second aim, a transcriptional activator biosensor system will be incorporated in E. coli to sense a biochemical of interest. The biosensor can sense the intracellular biochemical concentration and induce transcription of the gfp gene. The biosensor will be tuned by promoter and RBS engineering for optimized fluorescence-activated cell sorting, a high-throughput method. Finally, in the third aim, the biosensor system will be incorporated into the heterologous sigma factor expressing strain and transformed with a large-insert, fosmid-based synthetic metagenomic library. A precursor chemical will be supplied to the culture; if the precursor is converted to the biochemical sensed by the biosensor, the cell will fluoresce and can be isolated. These isolates will be characterized to determine the genetic element responsible for the enzymatic activity. Once developed, this method has the potential to quickly and accurately identify enzymatic genes from difficult-to-culture organisms, thereby providing a new means by which to explore a larger part of the large metagenomic space.

描述（由申请人提供）：对宏基因组库的有效筛选将使自然界中的微生物多样性探索，这些微生物是负责人类生活所需的许多有趣且有价值的功能。例如，在人类的肠道中，细菌群落对有害化学物质排毒，合成维生素，并帮助消化营养物质，这些营养物质本身不能自身消化，以及其他重要功能。不幸的是，绝大多数细菌不能在实验室中培养，因此生物催化活性的遗传基础常常未被发现。微生物学horsemante有机体大肠杆菌可以容纳这些细菌群落基因组DNA（称为元基因组库）的遗传收集。由于两个主要局限性，元基因组库的筛选效率低下。首先，大肠杆菌通常无法识别宏基因组库中的异源启动子。其次，搜索生物催化功能通常涉及筛查小分子的存在，这可能是时间和资源密集型。所提出的解决方案是开发一种与文化无关的方法，用于对宏基因组文库进行高通量筛选。在第一个目的中，有人提出，通过表达质原核的转录机制（Sigma因子），从宿主那里远离宿主，宿主的RNA聚合酶可以更好地识别异源启动子，从而改善了宏观库中遗传元素的转录。为了改善宏基因组文库的异源基因表达，Sigma因子将在来自一系列细菌种类（包括枯草芽孢杆菌和植物杆菌）的宿主菌株中表达。异源文库的转录将通过将绿色荧光蛋白基因融合到小文库插入物并用流式细胞仪测量荧光来量化。异源sigma因子的表达水平将通过工程启动子和核糖体结合位点（RB）来优化，以获得良好的表达菌株。使用该系统，将通过土壤细菌构建宏基因组文库，并筛选出赋予增强丁醇耐受性的基因。在第二个目标中，转录激活因生物传感器系统将被纳入大肠杆菌，以感知感兴趣的生化。生物传感器可以感觉到细胞内生化浓度并诱导GFP基因的转录。生物传感器将通过启动子和RBS工程来调整，以优化荧光激活的细胞排序，这是一种高通量方法。最后，在第三个目标中，生物传感器系统将被合并到表达菌株的异源Sigma因子中，并用大型插入，基于fosmid的合成元基因组库进行转化。将向培养物提供前体化学物质；如果将前体转化为生物传感器感应的生化，则细胞将荧光并可以分离。这些分离株将被表征以确定负责酶活性的遗传元件。一旦开发，该方法就有可能从难以培养的生物体中快速准确地识别酶基因，从而提供了一种新的手段来探索大型大元基因组空间的大部分。