Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals

Rebecca M. Lennen, Hyun Gyu Lim, Kristian Jensen, Elsayed T. Mohammed, Patrick V. Phaneuf, Myung Hyun Noh, Sailesh Malla, Rosa A. Börner, Ksenia Chekina, Emre Özdemir, Ida Bonde, Anna Koza, Jérôme Maury, Lasse E. Pedersen, Lars Y. Schöning, Nikolaus Sonnenschein, Bernhard O. Palsson, Alex T. Nielsen, Morten O.A. Sommer, Markus J. Herrgård*Adam M. Feist*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

45 Downloads (Pure)


Although strain tolerance to high product concentrations is a barrier to the economically viable biomanufacturing of industrial chemicals, chemical tolerance mechanisms are often unknown. To reveal tolerance mechanisms, an automated platform was utilized to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals (1,2-propanediol, 2,3-butanediol, glutarate, adipate, putrescine, hexamethylenediamine, butanol, isobutyrate, coumarate, octanoate, hexanoate), reaching tolerance at concentrations 60%–400% higher than initial toxic levels. Sequencing genomes of 223 isolates from 89 populations, reverse engineering, and cross-compound tolerance profiling were employed to uncover tolerance mechanisms. We show that: 1) cells are tolerized via frequent mutation of membrane transporters or cell wall-associated proteins (e.g., ProV, KgtP, SapB, NagA, NagC, MreB), transcription and translation machineries (e.g., RpoA, RpoB, RpoC, RpsA, RpsG, NusA, Rho), stress signaling proteins (e.g., RelA, SspA, SpoT, YobF), and for certain chemicals, regulators and enzymes in metabolism (e.g., MetJ, NadR, GudD, PurT); 2) osmotic stress plays a significant role in tolerance when chemical concentrations exceed a general threshold and mutated genes frequently overlap with those enabling chemical tolerance in membrane transporters and cell wall-associated proteins; 3) tolerization to a specific chemical generally improves tolerance to structurally similar compounds whereas a tradeoff can occur on dissimilar chemicals, and 4) using pre-tolerized starting isolates can hugely enhance the subsequent production of chemicals when a production pathway is inserted in many, but not all, evolved tolerized host strains, underpinning the need for evolving multiple parallel populations. Taken as a whole, this study provides a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 223 chemical tolerant isolates.

Original languageEnglish
JournalMetabolic Engineering
Pages (from-to)179-192
Number of pages14
Publication statusPublished - 2023


  • Adaptive laboratory evolution
  • Biochemical production
  • Chemical tolerance
  • Osmotolerance


Dive into the research topics of 'Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals'. Together they form a unique fingerprint.

Cite this