Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

Elsayed T Mohamed; Allison Z Werner; Davinia Salvachúa; Christine A Singer; Kiki Szostkiewicz; Manuel Rafael Jiménez-Díaz; Thomas Eng; Mohammad S Radi; Blake A Simmons; Aindrila Mukhopadhyay; Markus J Herrgård; Steven W Singer; Gregg T Beckham; Adam M Feist

doi:10.1016/j.mec.2020.e00143

Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

Metab Eng Commun. 2020 Aug 29:11:e00143. doi: 10.1016/j.mec.2020.e00143. eCollection 2020 Dec.

Authors

Elsayed T Mohamed¹, Allison Z Werner^{2

3}, Davinia Salvachúa², Christine A Singer², Kiki Szostkiewicz², Manuel Rafael Jiménez-Díaz^{4

5}, Thomas Eng^{4

5}, Mohammad S Radi¹, Blake A Simmons^{4

5}, Aindrila Mukhopadhyay^{4

5}, Markus J Herrgård¹, Steven W Singer^{4

5}, Gregg T Beckham^{2

3}, Adam M Feist^{1

4

6}

Affiliations

¹ Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
² Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA.
³ Center for Bioenergy Innovation, Oak Ridge, TN, USA.
⁴ Joint BioEnergy Institute, Emeryville, CA, USA.
⁵ Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁶ Department of Bioengineering, University of California, San Diego, CA, USA.

Abstract

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) individually and in combination (pCA + FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 h decrease in lag phase in 20 g/L pCA and a 2.4-fold increase in growth rate in 30 g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of pCA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in pCA + FA TALE-derived strains but did not affect growth in pCA + FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

Keywords: Adaptive laboratory evolution; FA, ferulic acid; Hydroxycinnamic acids; Microbial lignin conversion; Pseudomonas putida KT2440; TALE, tolerance adaptive laboratory evolution; pCA, p-coumaric acid.