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Genome shuffling of Lactobacillus for improved acid tolerance

Nature Biotechnology volume 20, pages 707–712 (2002)Cite this article

Abstract

Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.

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Figure 1: Comparison of genome-shuffled libraries.
Figure 2: Characterization of libraries in 96-well megatiter plates.
Figure 3: Screening in 10 ml culture tubes.
Figure 4: Fermentations in shake flasks at pH 3.9.
Figure 5: Characterization of an isolate in a 1 liter bioreactor.

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Acknowledgements

We thank Keith Powell and Tony Cox for their useful suggestions and discussions and Ken Zahn for proofreading the manuscript. Special thanks go to Amy Giver, Henry Garcia, and Rob Pak for technical support. Funding for this project was provided in part by the US National Institute of Standards and Technology Advanced Technology Program.

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  1. Codexis (a subsidiary of Maxygen), 515 Galveston Drive, Redwood City, 94063, CA

    Ranjan Patnaik, Susan Louie, Vesna Gavrilovic, Kim Perry, Willem P.C. Stemmer & Stephen del Cardayré

  2. Cargill Dow, LLC, 15305 Minnetonka Boulevard, Minnetonka, 55345, MN

    Chris M. Ryan

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Correspondence to Stephen del Cardayré.

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Each of the authors is or was an employee of Maxygen, Codexis, or Cargill Dow, LLC.

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Patnaik, R., Louie, S., Gavrilovic, V. et al. Genome shuffling of Lactobacillus for improved acid tolerance. Nat Biotechnol 20, 707–712 (2002). https://doi.org/10.1038/nbt0702-707

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