Global transcription machinery engineering

Abstract

The invention relates to global transcription machinery engineering to produce altered cells having improved phenotypes.

Description

FIELD OF THE INVENTION [0001] The invention relates to global transcription machinery engineering to produce altered cells having improved phenotypes. BACKGROUND OF THE INVENTION [0002] It is now generally accepted that many important cellular phenotypes, from disease states to metabolite overproduction, are affected by many genes. Yet, most cell and metabolic engineering approaches rely almost exclusively on the deletion or over-expression of single genes due to experimental limitations in vector construction and transformation efficiencies. These limitations preclude the simultaneous exploration of multiple gene modifications and confine gene modification searches to restricted sequential approaches where a single gene is modified at a time. [0003] U.S. Pat. No. 5,686,283 described the use of a sigma factor encoded by rpoS to activate the expression of other bacterial genes that are latent or expressed at low levels in bacterial cells. This patent did not, however, describe mutati...

AI Technical Summary

Benefits of technology

[0007] The invention utilizes global transcription machinery engineering to produce altered cells having improved phenotypes. In particular, the invention is demonstrated through the generation of mutated bacterial sigma factors with varying preferences for promoters on a genome-wide level. The cells resulting from introduction of the mutated sigma factors have rapid and marked improvements in phenotypes, such as tolerance of deleterious culture conditions or improved production of metabolites.

[0008] The introduction of mutant transcription machinery into a cell, combined with methods and concepts of directed evolution, allows one to explore a vastly expanded search space in a high throughput manner by evaluating multiple, simultaneous gene alterations in order to improve complex cellular phenotypes.

[0019] In some embodiments of the invention, a promoter binding region of the global transcription machinery is not disrupted or removed by the one or more truncations or detections. In other embodiments, the mutated global transcription machinery exhibits increased transcription of genes relative to the unmutated global transcription machinery, decreased transcription of genes relative to the unmutated global transcription machinery, increased repression of gene transcription relative to the unmutated global transcription machinery, and / or decreased repression of gene transcription relative to the unmutated global transcription machinery.

[0025] In some embodiments, the cell is contained in a multicellular organism. In such embodiments, preferred phenotypes include one or more growth characteristics, generation time, resistance to one or more pests or diseases, production of fruit or other parts of a plant, one or more developmental changes, one or more lifespan alterations, gain or loss of function and / or increased robustness.

Problems solved by technology

However, no evolution-inspired approaches have been directed towards the systematic modification of the global transcription machinery as a means of improving cellular phenotype.

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