Total in vitro biosynthesis of the nonribosomal macrolactone peptide valinomycin

Lei Zhuang, Shuhui Huang, Wan Qiu Liu, Ashty S. Karim, Michael C. Jewett, Jian Li*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

45 Scopus citations


Natural products are important because of their significant pharmaceutical properties such as antiviral, antimicrobial, and anticancer activity. Recent breakthroughs in DNA sequencing reveal that a great number of cryptic natural product biosynthetic gene clusters are encoded in microbial genomes, for example, those of Streptomyces species. However, it is still challenging to access compounds from these clusters because many source organisms are uncultivable or the genes are silent during laboratory cultivation. To address this challenge, we develop an efficient cell-free platform for the rapid, in vitro total biosynthesis of the nonribosomal peptide valinomycin as a model. We achieve this goal in two ways. First, we used a cell-free protein synthesis (CFPS) system to express the entire valinomycin biosynthetic gene cluster (>19 kb) in a single-pot reaction, giving rise to approximately 37 μg/L of valinomycin after optimization. Second, we coupled CFPS with cell-free metabolic engineering system by mixing two enzyme-enriched cell lysates to perform a two-stage biosynthesis. This strategy improved valinomycin production ~5000-fold to nearly 30 mg/L. We expect that cell-free biosynthetic systems will provide a new avenue to express, discover, and characterize natural product gene clusters of interest in vitro.

Original languageEnglish (US)
Pages (from-to)37-44
Number of pages8
JournalMetabolic Engineering
StatePublished - Jul 2020


  • Cell-free systems
  • In vitro biosynthesis
  • Natural product
  • Nonribosomal peptide
  • Synthetic biology
  • Valinomycin

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Bioengineering
  • Biotechnology


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