Engineering Microbial Consortia Using Fosmid Pool Selection

Synthetic ecology is an emerging component of biological engineering in which co-cultured microorganisms are used to tackle specific metabolic processes through syntrophic or distributed metabolic interactions. While initial studies using engineered microbes to compliment amino acid auxotrophies or complete catabolic pathways have been conducted^1,2, less is known about the underlying principles shaping microbial community interactions in natural and engineered ecosystems. Such communities contain a vast reservoir of genetic diversity and metabolic problem solving power that in principle can be harnessed for synthetic biology applications. To this end we assembled synthetic microbial consortia with an emergent toluene resistance phenotype as a model system to assess roles of sele­­cted genes and pathways in emergent or distributed metabolic processes. We used a pooled selection approach on large insert metagenomic libraries hosted in E.coli. Large insert (fosmid) libraries contain unique environmental DNA fragments between 30-45kb inserted into the pCC1fos backbone and transformed into E.coli EPI300 host cells. By applying strong selective pressure to the pooled fosmid libraries we enriched for clones conferring fitness advantages while removing other clones possessing non-selective traits. Both DNA and RNA were extracted from enriched fosmid pools and sequenced on the Illumina HiSeq platform revealing an abundance of genes encoding hydrocarbon resistance and degradation processes spanning both Archaeal and Bacterial domains of life with reduced complexity. This approach enables discovery of multiple genes or gene cassettes driving distributed metabolic interactions in heterologous host cells. The discovered functions can be used in the design of new strains with enhanced resistance traits and promote the development of robust hydrocarbon-degrading consortia in E. coli hosts for industrial and bioremediation applications.

1. Mee, M. T., Collins, J. J., Church, G. M. & Wang, H. H. Syntrophic exchange in synthetic microbial communities. Proc. Natl. Acad. Sci. U. S. A. 111, E2149-56 (2014).

2. Wintermute, E. H. & Silver, P. a. Emergent cooperation in microbial metabolism. Mol. Syst. Biol. 6, 407 (2010).