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.
Most bacteria found in nature cannot be cultured in a laboratory but still are able to perform many interesting and valuable biocatalytic functions, such as novel antibiotic synthesis in soil bacteria and carcinogen detoxification by bacteria found in the human gut. Discovery of the underlying genetics behind these functionalities requires screening metagenomic libraries, but is hampered by poor gene expression and inefficient screening. Therefore, this project aims to develop enabling technologies for efficient screening of genes from the metagenome to discover beneficial functionalities.
|Sandoval, Nicholas R; Papoutsakis, Eleftherios T (2016) Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes. Curr Opin Microbiol 33:56-66|
|Gaida, Stefan M; Sandoval, Nicholas R; Nicolaou, Sergios A et al. (2015) Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries. Nat Commun 6:7045|