title: Mutagenesis of the bacterial RNA polymerase alpha subunit for improvement of complex phenotypes.
abstract: Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, L-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Klein-Marcuschamer
authors.forename: Christine Nicole S
authors.initials: CN
authors.lastname: Santos
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000440:Butanols / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D016296:Mutagenesis; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D000440:Butanols; D029968:Escherichia coli Proteins; D014443:Tyrosine; D006820:Hyaluronic Acid; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit