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  • Review Article
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Design of microbial catalysts for two-stage processes

Nature Reviews Bioengineering (2024)Cite this article

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Abstract

Microbial catalysts must partition incoming substrate between the synthesis of biomass and the synthesis of a desired product. Although biomass synthesis generates more catalyst and therefore potentially higher volumetric productivities, the synthesis of product increases specific production rates and product yields. Two-stage bioprocesses can accommodate this tradeoff through temporal separation of the growth and production phases. The biocatalyst first grows to optimal density; it is then switched to a growth-arrested state during which the product is synthesized. However, a substantial reduction in metabolic activity is often observed during cellular growth arrest, even in the presence of sufficient substrate. An ultimate bioengineering goal, therefore, is to create growth-arrested states that retain high metabolic activity. Achieving this goal brings the metabolic engineer to the intersection of microbial physiology, synthetic biology and biochemistry. In this Review, we describe various aspects of the design of microbial catalysts for two-stage bioprocesses for metabolite production, including synthetic biology tools to arrest cell growth using external or internal cues, and metabolic engineering tools to minimize interference from the native metabolic network and enhance substrate uptake and conversion. We highlight recent systems biology studies of nutrient-limited heterotrophs and phototrophs and conclude that the reduction in substrate uptake by cells in growth arrest is the consequence of reduced energy demand as well as imbalances in regulatory metabolites that typically arise during nutrient limitation. On the basis of these studies, we propose strategies for increasing metabolic activity in growth-arrested cells.

Key points

  • Microbial catalysts must balance production with growth and other essential functions.

  • The theoretically optimal solution to maximizing production over a finite time interval is a two-stage process with a growth phase followed by a production phase.

  • Tools from synthetic biology enable efficient and dynamic switching to a metabolic mode with limited growth and enhanced production.

  • A major drawback of the growth-arrested state is a commonly observed decrease in metabolic activity and substrate uptake.

  • The non-growing state is generally poorly understood in quantitative biology.

  • Advances in high-throughput phenotyping and computation-based tools offer new avenues to understand metabolic activity decline and increase biocatalyst performances and stability over time.

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Fig. 1: Transitioning from a single-stage to a two-stage microbial production process.
Fig. 2: Overview of the main tools used for arresting cell growth in two-stage process design.
Fig. 3: Feedback between growth rate and cell metabolism.
Fig. 4: Main barriers to redirecting resources to production and future engineering strategies for two-stage production.

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Authors and Affiliations

  1. Department of Bioengineering and Imperial College Centre for Synthetic Biology, Imperial College London, London, UK

    Kiyan Shabestary

  2. Analysis and Redesign of Biological Networks, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany

    Steffen Klamt

  3. Bacterial Metabolomics, CMFI, University Tübingen, Tübingen, Germany

    Hannes Link

  4. Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada

    Radhakrishnan Mahadevan

  5. Peter Debye Institute for Soft Matter Physics, Leipzig University, Leipzig, Germany

    Ralf Steuer

  6. School of Engineering Sciences in Chemistry, Biotechnology and Health, Science for Life Laboratory, KTH – Royal Institute of Technology, Stockholm, Sweden

    Elton P. Hudson

Authors
  1. Kiyan Shabestary
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  2. Steffen Klamt
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  3. Radhakrishnan Mahadevan
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  4. Ralf Steuer
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  5. Elton P. Hudson
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Contributions

K.S. and E.P.H. prepared the outline with input from all authors. K.S., S.K. and E.P.H. drafted the manuscript with input from all authors. All authors contributed text, and edited and reviewed the final manuscript.

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Correspondence to Elton P. Hudson.

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Nature Reviews Bioengineering thanks Ralf Takors and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Shabestary, K., Klamt, S., Link, H. et al. Design of microbial catalysts for two-stage processes. Nat Rev Bioeng (2024). https://doi.org/10.1038/s44222-024-00225-x

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