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Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...
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Upstream processing represents a critical phase in biomanufacturing, wherein biological systems such as microorganisms, mammalian cells, or insect cells are cultivated to produce therapeutic proteins, vaccines, enzymes, or other biologically derived products. This phase encompasses all steps from the selection and genetic manipulation of the production organism to the cultivation of cells in bioreactors under tightly controlled environmental conditions.Host Selection and Genetic OptimizationThe...
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Cell-free metabolic engineering: biomanufacturing beyond the cell.

Quentin M Dudley1, Ashty S Karim, Michael C Jewett

  • 1Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA; Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA; Institute for Bionanotechnology in Medicine, Northwestern University, Chicago, IL, USA.

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Summary

Cell-free metabolic engineering (CFME) offers a powerful alternative to traditional methods for producing chemicals. This in vitro approach overcomes limitations like low productivity and toxicity, enabling efficient biosynthesis of valuable products.

Keywords:
BiocatalysisBiotransformationCell-free metabolic engineeringMetabolic pathway debuggingSynthetic biology

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Area of Science:

  • Biotechnology
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • Industrial biotechnology faces challenges in sustainable chemical production, including low yields and product toxicity.
  • Current microbial metabolic engineering methods are limited in the scope of producible biochemicals.

Purpose of the Study:

  • To introduce cell-free metabolic engineering (CFME) as a novel approach to overcome limitations in biochemical production.
  • To highlight the advantages of CFME over traditional in vivo systems for metabolic engineering.

Main Methods:

  • Utilizing in vitro ensembles of catalytic proteins (purified enzymes or crude cell lysates).
  • Developing engineering foundations for cell-free systems to enable complex pathway activation.

Main Results:

  • Achieved activation of long enzymatic pathways (>8 enzymes) with near theoretical conversion yields.
  • Demonstrated productivities exceeding 100 mg L⁻¹ h⁻¹ and reaction scales over 100 L.
  • Advanced protein purification, spatial organization, and enzyme stability for cell-free applications.

Conclusions:

  • CFME provides unprecedented control and design freedom for optimizing biosynthetic pathways.
  • Facilitates rapid design-build-test iterations without organism re-engineering.
  • Enables molecular transformations where in vivo methods are limited by yield, productivity, or toxicity.