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A Simple and Versatile Cell-Free Expression Method for Producing Secondary Metabolites.

Jaime Lorenzo N Dinglasan1, Namil Lee2,3,4, Nam Ngoc Pham5,6

  • 1US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.

ACS Synthetic Biology
|December 25, 2025
PubMed
Summary
This summary is machine-generated.

We developed a generalizable cell-free expression system using Streptomyces lysates for natural product discovery. This system enables high-yield protein production and the biosynthesis of complex molecules, including megasynthases.

Keywords:
Streptomycescell-free expressionnatural product synthesissecondary metabolitestype I polyketide synthase

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Area of Science:

  • Biochemistry
  • Synthetic Biology
  • Natural Product Discovery

Background:

  • Secondary metabolites are crucial natural products with significant industrial bioactivities.
  • Lysate-based cell-free expression (CFE) accelerates the discovery and engineering of these natural products.
  • Streptomyces extracts offer a potentially more compatible environment for CFE compared to E. coli, but current systems are underdeveloped and not easily scalable.

Purpose of the Study:

  • To develop a generalizable and scalable cell-free expression system using Streptomyces lysates.
  • To enable high-yield protein expression and natural product biosynthesis in a cell-free environment.
  • To demonstrate the capability of Streptomyces CFE for expressing and catalyzing complex enzymes like type I polyketide synthases (T1PKS).

Main Methods:

  • Developed a simple and generalizable set of reaction conditions for CFE using lysates from Streptomyces venezuelae and Streptomyces lividans.
  • Validated the system by producing the polyketide flaviolin and the cyclic dipeptide albonoursin.
  • Demonstrated the expression and catalytic activity of a large (∼250 kDa) type I polyketide synthase (T1PKS) in S. lividans lysate.

Main Results:

  • Achieved high-yield protein expression (180-230 μg/mL) in both S. venezuelae and S. lividans lysates.
  • Successfully produced flaviolin and albonoursin, demonstrating pathway-level biosynthesis.
  • The S. lividans lysate supported the expression and catalytic activity of a T1PKS, producing 2-methyl-3-pentanone without supplements, outperforming E. coli systems.
  • This is the first reported instance of coupled expression and catalysis of a megasynthase in a Streptomyces CFE system and a T1PKS in any bacterial CFE system.

Conclusions:

  • Established a robust and scalable Streptomyces-based CFE protocol that overcomes limitations of previous systems.
  • The developed system facilitates parallel evaluation of diverse lysate systems for natural product biosynthesis.
  • Provides a foundation for high-throughput engineering of T1PKS and other complex biosynthetic machinery in vitro.