Methods for altering acetic acid production and enhancing cell death in bacteria

Abstract

Methods of increasing cultured cell growth yields and / or protein production from bacterial cell cultures are provided. More particularly, mutant bacterial cells having an alteration in the expression or activity of the cidABC operon, a gene therein, or a homolog or a regulator thereof, and methods for reducing acetic acid / acetate production in cultures are provided, as are methods for increasing cultured cell growth yields and / or protein production employing such cells. Methods for enhancing bacterial cell death and methods for identifying agents that increase the susceptibility of bacteria to cell death are also provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This is a continuation of co-pending U.S. patent application Ser. No. 11 / 597,013, filed Nov. 17, 2006, which is the §371 U.S. National Stage of PCT / US2005 / 017604, filed May 20, 2005, which in turn claims the benefit of U.S. Provisional Application No. 60 / 573,431, filed May 21, 2004, and U.S. Provisional Application No. 60 / 642,039, filed Jan. 6, 2005. These applications are incorporated herein in their entirety.STATEMENT OF GOVERNMENT SUPPORT[0002]This invention was made in part with United States government support pursuant to NIH grant no. R01AI038901; NIH-NRRI grant no. P20RR15587; and Department of Defense grant no. DAAD 19-03-0191. The United States government has certain rights in the invention.FIELD[0003]A first aspect of this disclosure relates to methods of increasing cultured cell growth yields and / or protein production from bacterial cell cultures. More particularly, this document relates to methods for reducing acetic acid / acet...

AI Technical Summary

Benefits of technology

[0009]The cidC gene affects the production / secretion of acetic acid by bacterial cells. Inactivating this gene, or the pyruvate oxidase encoded thereby, either directly or indirectly, allows for growth of bacteria in the presence of glucose without the accumulation of deleteriously high levels of acetic acid in the culture medium. A result of this is enhanced survival and increased longevity in culture as compared to previously available strains. In addition, the mutant cells exhibit increased growth once stationary phase has been reached. Both the maintenance of higher cell numbers (biomass maintenance) and increased longevity contribute to the enhanced commercial potential of these mutant bacterial cells, and other bacterial cells comprising similar mutations in the cidC gene, the cidABC operon, or a homolog thereof.

[0010]The present disclosure also provides bacterial host cells, expression methods and systems for enhanced bacterial production and secretion of heterologous or homologous products, such as proteins. In one embodiment, a bacterial cell is genetically engineered to have a deletion or mutation in a cidABC gene or operon, or homolog thereof, such that the activity of the cidC pyruvate oxidase is reduced, thereby measurably reducing the production of acetic acid by that bacterial cell. In another embodiment, a bacterial cell is genetically engineered to comprise a mutation or deletion in another gene or operon, which influences the cidABC operon such that the activity of the cidC pyruvate oxidase is reduced, thereby measurably reducing the production of acetic acid by that bacterial cell.

[0012]It has also been determined that agents and conditions that increase expression of the products encoded by the cidABC operon increase the susceptibility of bacteria to cell death. Based on this property of the cidABC operon, compositions and methods useful for the identification of agents that enhance bacterial cell death are disclosed herein, as are methods for enhancing cell death. In an embodiment, an agent that induces expression of the cidABC operon is administered in combination with an agent with bactericidal (antibiotic) properties to increase the rate or extent of bacterial cell death.

Problems solved by technology

A major limiting factor that the industry faces is the growth yield that can be achieved using these recombinant organisms.

This secreted acetate is converted into acetic acid, which becomes toxic at high levels, inducing cell death.

Unfortunately, the removal of acetate from the medium is costly and can outweigh the financial benefits of achieving higher growth yields.

However, the maximum growth potential that is possible with higher levels of glucose is, ultimately, negatively impacted by the increased cell death induced by toxic acetate byproducts that are generated by growth in the presence of this carbon source.

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