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Emulsion-based evolution of Escherichia coli for higher growth yield on D-xylose identifies central role of cyclic AMP

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Abstract

D-xylose is an abundant sugar found in plant biomass and can be used as a renewable feedstock for the microbial production of diverse biofuels and bioproducts. However, D-xylose metabolism is slow in many industrial microorganisms, at least as compared to glucose metabolism. Not surprisingly, a number of approaches have been developed for improving D-xylose metabolism in diverse microorganisms. In this work, we applied a previously developed evolution strategy based on media-in-oil emulsions for improving the growth yield of Escherichia coli NCM3722 on D-xylose. After 30 rounds of evolutions, we isolated multiple mutants with increased growth yield on D-xylose. In addition, we also observed similar increases in the growth rate. Three mutants were selected for whole-genome sequencing. Two mutants had an amber stop mutation in adenylate cyclase, which truncates nearly 60% of the enzyme. However, the ability of this mutant to grow on xylose indicated that truncated enzyme, lacking the C-terminal regulatory domain, is still active. The other mutant had a point mutation in the cyclic AMP receptor protein (CRP), near the high affinity binding site for cyclic AMP. Both mutations, when introduced into wild type E. coli, were able to increase the growth yields at levels similar to the isolated mutants. In addition to D-xylose, these mutant strains and their genetic mimics also exhibited higher growth rates and yields on glucose, lactose, and L-arabinose. These results suggest that the improved growth rates and yields are due to changes in the production and sensing of intracellular cyclic AMP concentrations and also suggest native concentrations are suboptimal with respect to the growth rate and yield under the growth conditions tested. Collectively, these results may prove useful for engineering strains of E. coli for high-density fermentations or protein production.

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Availability of data and materials

All data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This work was partially supported by the Energy Biosciences Institute and by the DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018420). Any opinions, findings and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the U.S. Department of Energy.

Funding

US Department of Energy, DE-SC0018420, CV Rao.

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Author notes

  1. James S. Orr, Edwin Zen and Xiaoyi Wang have equally contributed to this work.

Authors and Affiliations

  1. DOE Center for Advanced Bioenergy and Bioproducts Innovation, Urbana, IL, USA

    James S. Orr, Edwin Zen, Xiaoyi Wang & Christopher V. Rao

  2. Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL, 61801, USA

    Xiaoyi Wang & Christopher V. Rao

  3. Core Facility Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China

    Xiaoyi Wang

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  1. James S. Orr
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Contributions

JO, EZ, and CR conceived the project. JO, EZ, and XW designed and performed the experiments. JO, EZ, JL, XW, and CR analyzed the data. JO, XW, and CR wrote the manuscript and designed the figures. All authors discussed the results and contributed to the final manuscript.

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Correspondence to Christopher V. Rao.

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Orr, J.S., Zen, E., Wang, X. et al. Emulsion-based evolution of Escherichia coli for higher growth yield on D-xylose identifies central role of cyclic AMP. Syst Microbiol and Biomanuf 3, 730–738 (2023). https://doi.org/10.1007/s43393-022-00136-1

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  • DOI: https://doi.org/10.1007/s43393-022-00136-1

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