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Applied and Environmental Microbiology, May 2009, p. 2705-2711, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01888-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Mutagenesis of the Bacterial RNA Polymerase Alpha Subunit for Improvement of Complex Phenotypes{triangledown}

Daniel Klein-Marcuschamer, Christine Nicole S. Santos, Huimin Yu,{dagger} and Gregory Stephanopoulos*

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 13 August 2008/ Accepted 20 February 2009

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.

* Corresponding author. Mailing address: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-4583. Fax: (617) 253-3122. E-mail: gregstep{at}mit.edu

{triangledown} Published ahead of print on 27 February 2009.

{dagger} Present address: Department of Chemical Engineering, Tsinghua University, Beijing 100084, China.

Applied and Environmental Microbiology, May 2009, p. 2705-2711, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01888-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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