Development of a high throughput cell-free metagenomic screening platform

Metagenomics is the benchmark for exploring the diverse metabolic potential of microorganisms directly from any environment. Although it has been fruitful, there are numerous limitations which hamper its ability to adequately tap into novel functional space, and many microbial genomes continue to remain inaccessible. This study aimed to develop an innovative ultra-high throughput solution to overcome many of the limitations associated with classic functional metagenomics. Here, uncloned metagenomic DNA from the environment was expressed and activity screened in emulsion droplets using Escherichia coli and Rhodococcus erythropolis based cell free protein synthesis, followed by selection using flow cytometry, cell sorting, whole genome amplification and sequencing to identify the expressed gene sequences. Emulsions expressing β-xylosidase activity using both R. erythropolis and E. coli based protein synthesis were selected, and two different β-xylosidase populations were identified, corresponding to the source of the cell free protein synthesis machinery (R. erythropolis or E. coli). Heterologous expression and enzyme assays of four ORFs from the R. erythropolis-based FACS sorted emulsions showed that these genes are expressed up to 100 fold higher in R. erythropolis under their native promoters compared with expression in E. coli. Furthermore, these ORFs could not be identified through classical functional screening of a pCCFOS library in E. coli. Elimination of clone library construction saves time and cost while improving efficiency and elimination of cloning bias and heterologous expression limitations. This is the first report of screening uncloned metagenomic DNA and the development of an actinomycete based cell free protein synthesis.