Automated Strain Construction for Biosynthetic Pathway Screening in Yeast

  • ACS Synth Biol. 2025 Oct 17;14(10):4143-4151. doi: 10.1021/acssynbio.5c00554.
Maria C T Astolfi  1  2  3 ,  Sam D Yoder  2  3 ,  Marina Delfa-Lalaguna  2  4 ,  Peter H Winegar  2  3  5 ,  Sara K F Holm  2  6 ,  Mengziang Lei  2 ,  Xixi Zhao  2  3  5 ,  Stephen E Tan  2  3 ,  Randy Louie  2  3 ,  Nathan J Hillson  2  3 ,  Graham A Hudson  2  3  5 ,  Jay D Keasling  1  2  3  5  6  7
Affiliations
  • 1. Department of Bioengineering, University of California, Berkeley, Berkeley, California 94720, United States.
  • 2. Joint BioEnergy Institute, Emeryville, California 94608, United States.
  • 3. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States.
  • 4. Faculty of Chemical Sciences, Complutense University of Madrid, Madrid 28040, Spain.
  • 5. California Institute for Quantitative Biosciences (QB3 Institute), University of California, Berkeley, Berkeley, California 94720, United States.
  • 6. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark (DTU), Lyngby 2800, Denmark.
  • 7. Department of Chemical and Biomolecular Engineering, University of California at Berkeley, Berkeley, California 94720, United States.
PMID: 41063332 DOI: 10.1021/acssynbio.5c00554
Abstract

Automation accelerates the Design-Build-Test-Learn (DBTL) cycle for synthetic biology; however, most strain construction pipelines lack robotic integration. Here, we present the workflow design and source code for a modular, integrated protocol that automates the Build step in Saccharomyces cerevisiae. We programmed the Hamilton Microlab VANTAGE to integrate off-deck hardware via its central robotic arm, enabling automated steps that increased throughput to 2,000 transformations per week. We developed a user interface with the Hamilton VENUS software to support on-demand parameter customization. As a proof of concept, we screened a gene library in an engineered yeast strain producing verazine, a key intermediate in the biosynthesis of steroidal Alkaloids. Our pipeline rapidly identified pathway bottlenecks and genes that enhanced verazine production by 2.0- to 5-fold. This technical note provides resources for synthetic biologists designing yeast workflows for biofoundries to screen libraries for pathway discovery/optimization, combinatorial biosynthesis, and protein engineering.

Keywords
automation; high-throughput screen; metabolic engineering; microbial biosynthesis; robotics integration; strain engineering.
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