Fitness Selection By Genome Wide Regulator Engineering and Gene Translation Regulation

Regulation of gene transcription and translation is fundamentally important for microbes to maintain normal cellular processes and respond to environmental changes. Modification of gene transcription (Alper et al., 2006; Alper & Stephanopoulos, 2007) and translation (Haft et al., 2014) was employed to adapt to novel environments through few mutations with large effects. The most typical case study, global transcription machinery engineering (gTME), has successfully been demonstrated by engineering sigma factors to improve three distinct phenotypes (ethanol tolerance, metabolite overproduction, and multiple phenotypes)(Alper & Stephanopoulos, 2007). However, limited regulators or landscape were rationally designed for kinds of environmental changes. Herein, global regulators, regulators regulating over teen genes and significant genes involved in transcription and translation were selected for modification. The potential interesting sites, including DNA binding sites, active sites and dimerization sites, were collected and designed. Saturated mutagenesis was then performed by CRISPR EnAbled Trackable genome Engineering (CREATE) for the selected interesting sites (Andrew Garst, submitted.). The library was subject to multiple environmental stresses like short chain alcohol, low pH, high temperature, salts, furfural and antibiotics. From these selections, multiple novel beneficial mutations were identified with enhanced tolerance to these stresses. These mutations might enable better understanding of different tolerance mechanisms.

Reference:

Alper, H., Moxley, J., Nevoigt, E., Fink, G.R., Stephanopoulos, G. 2006. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science, 314(5805), 1565-8.

Alper, H., Stephanopoulos, G. 2007. Global transcription machinery engineering: a new approach for improving cellular phenotype. Metab Eng, 9(3), 258-67.

Andrew Garst, M.B., Gur Pines, Sean Lynch, Andrea Halweg-Edwards, Zhiwen Wang, Ryan T. Gill. submitted. Trackable genome scale engineering with single nucleotide resolution.

Haft, R.J., Keating, D.H., Schwaegler, T., Schwalbach, M.S., Vinokur, J., Tremaine, M., Peters, J.M., Kotlajich, M.V., Pohlmann, E.L., Ong, I.M., Grass, J.A., Kiley, P.J., Landick, R. 2014. Correcting direct effects of ethanol on translation and transcription machinery confers ethanol tolerance in bacteria. Proc Natl Acad Sci U S A, 111(25), E2576-85.