Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida

Oligonucleotides, as well as plasmids and strains constructed and used in this work, are described in Supplementary Data 2–4 as well as Supplementary Table 2. Plasmids and strains are available in the public domain of the JBEI Registry (https://public-registry.jbei.org). Source data are provided in the Source Data file. The generated mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD063733 (DBTL0) [https://www.ebi.ac.uk/pride/archive/projects/PXD063733], PXD063737 (DBTL1) [https://www.ebi.ac.uk/pride/archive/projects/PXD063737], PXD063738 (DBTL2) [https://www.ebi.ac.uk/pride/archive/projects/PXD063738], PXD063740 (DBTL3) [https://www.ebi.ac.uk/pride/archive/projects/PXD063740], PXD063743 (DBTL4) [https://www.ebi.ac.uk/pride/archive/projects/PXD063743], PXD063744 (DBTL5) [https://www.ebi.ac.uk/pride/archive/projects/PXD063744], and PXD063746 (DBTL6) [https://www.ebi.ac.uk/pride/archive/projects/PXD063746]⁶⁵. DIA-NN is freely available for download from https://github.com/vdemichev/DiaNN. All proteomics and GC-FID data were also deposited into a publicly available DRYAD database (https://doi.org/10.5061/dryad.gtht76hzh). Source data are provided with this paper.

ART is freely available for non-commercial use by academic institutions and for a small licensing fee for commercial use; the license can be accessed at https://github.com/JBEI/ART. All code required to reproduce this study, including ART, automation workflows, and figure generation, is provided as Jupyter Notebooks and can be accessed at https://github.com/JBEI/Isoprenol_CRISPRi or https://doi.org/10.5281/zenodo.17178684⁶⁶.

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Author notes

1. These authors contributed equally: David N. Carruthers, Patrick C. Kinnunen.

2. These authors jointly supervised this work: Hector Garcia Martin, Taek Soon Lee.

Authors and Affiliations

1. Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

   David N. Carruthers, Patrick C. Kinnunen, Yan Chen, Jennifer W. Gin, Ian S. Yunus, Stephen Tan, Tijana Radivojevic, Christopher J. Petzold, Aindrila Mukhopadhyay, Hector Garcia Martin & Taek Soon Lee

2. Joint BioEnergy Institute, Emeryville, CA, USA

   David N. Carruthers, Patrick C. Kinnunen, Yuerong Li, Yan Chen, Jennifer W. Gin, Ian S. Yunus, William R. Galliard, Stephen Tan, Tijana Radivojevic, Paul D. Adams, Anup K. Singh, Jess Sustarich, Christopher J. Petzold, Aindrila Mukhopadhyay, Hector Garcia Martin & Taek Soon Lee

3. Lawrence Livermore National Laboratory, Livermore, CA, USA

   Yuerong Li & Anup K. Singh

4. Sandia National Laboratories, Livermore, CA, USA

   William R. Galliard & Jess Sustarich

5. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

   Paul D. Adams & Hector Garcia Martin

6. Department of Bioengineering, University of California Berkeley, Berkeley, CA, USA

   Paul D. Adams

Contributions

D.N.C., P.C.K., I.S.Y., H.G.M., and T.S.L. conceptualized the study. Y.C. and J.W.G. collected and curated proteomics data. P.C.K. wrote code to use ART. D.N.C., and S.T. developed the automation workflow. D.N.C. and Y.L. performed the biological experiments. W.R.G. and J.S. developed the HTME, and W.R.G. performed the electroporations. D.N.C., P.C.K., and T.R. performed data analysis. P.D.A., A.K.S., C.J.P., A.M., H.G.M., and T.S.L., secured funding and resources. All authors revised and approved the manuscript.

Corresponding authors

Correspondence to Hector Garcia Martin or Taek Soon Lee.

Competing interests

A.K.S. and J.S. are founders of XLSI Bio. H.G.M. and W.R.G. are scientific advisors to XLSI Bio. The remaining authors declare no competing interests.

Peer review information

Nature Communications thanks Paul Jensen and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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