Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Understanding the Role of Tolerance on Microbial Production of Isoprenol

Authors:

Aparajitha Srinivasan1,2* (asrinivasan@lbl.gov), Hyungyu Lim1,3, Russel Menchavez1,2, Ian Yunus1,2, Myung Hyun Noh3, Megan White1,2, Yan Chen1,2, Christopher J. Petzold1,2, Thomas Eng1,2, Blake A. Simmons1,2, Bernhard O. Palsson1,3, Taek Soon Lee1,2, Adam M. Feist1,3, Aindrila Mukhopadhyay1,2, Jay D. Keasling1,2,4

Institutions:

1Joint BioEnergy Institute; 2Lawrence Berkeley National Laboratory; 3University of California–San Diego; 4University of California–Berkeley

URLs:

Goals

Discovery, optimization and enhancement of tolerance mechanisms in bacterial hosts to biomass-related inhibitors and final products to generate robust, scalable production platforms.

Abstract

Isoprenol (3-methyl-3-buten-1-ol), a precursor for diverse commodity chemicals (Baral et al. 2021), can be converted into 1,4-dimethylcyclooctane (DMCO), which can further be used as a sustainable aviation fuel blendstock (Baral et al. 2021). Bioproduction of isoprenol has been reported from various engineered bacterial hosts such as Escherichia coli, Corynebacterium glutamicum, and Pseudomonas putida (Kang et al. 2016; Sasaki et al. 2019; Wang et al. 2022; Banerjee et al. 2024). A critical factor affecting scalability for high titer rate–yield production of isoprenol in these microbial hosts is its toxicity. Therefore, it is imperative to understand and improve the tolerance to isoprenol in these bacterial hosts that affect both growth and production.

In the current study, this team specifically focused on P. putida KT2440, an ideal production chassis due to its fast growth rate, capability to utilize various substrates including lignin aromatics, and high stress tolerance, which are critical factors in industrial bioprocesses (Nikel and de Lorenzo 2018). P. putida has endogenous isoprenol catabolism pathways and hence the native regulatory cascades driving the microbial physiology could be perturbed upon heterologous production of isoprenol. The group used a top-down approach harnessing the power of adaptive laboratory evolution (ALE) to generate isoprenol tolerant phenotypes and further characterized them (Lim et al. 2021). For this purpose, the team tolerized wild-type (WT) as well as mutant strains of P. putida lacking isoprenol catabolism in glucose minimal medium by gradually increasing the concentration of isoprenol.

The group successfully obtained evolved strains that could robustly grow in the presence of 8 grams per liter isoprenol compared to the basal strain that was unable to grow at this concentration. Furthermore, the team utilized whole-genome sequencing, gene expression profiling (RNA sequencing), and global proteomics to understand determinants of isoprenol tolerance in these novel evolved isolates compared to the parent strains. Researchers also study how these profiles change when they heterologously express isoprenol production pathways in these evolved isolates and its subsequent effect on production. Taken together, unraveling and understanding the effect of the evolution-driven isoprenol tolerization mechanism and its effect on production in this important bacterial chassis will help scientists in rationally engineering robust isoprenol production platforms.

References

Banerjee, D., et al. 2024. “Genome-Scale and Pathway Engineering for the Sustainable Aviation Fuel Precursor Isoprenol Production in Pseudomonas putida,” Metabolic Engineering 82, 157–70. DOI:10.1016/j.ymben.2024.02.004.

Baral, N. R., et al. 2021. “Production Cost and Carbon Footprint of Biomass-Derived Dimethylcyclooctane as a High Performance Jet Fuel Blendstock,” ACS Sustainable Chemistry & Engineering 9(35),11872–82. DOI:10.1021/acssuschemeng.1c03772.

Kang, A., et al. 2016. “Isopentenyl Diphosphate (IPP)-Bypass Mevalonate Pathways for Isopentenol Production,” Metabolic Engineering 34, 25–35. DOI:10.1016/j.ymben.2015.12.002.

Lim, H. G., et al. 2021. “Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution,” ACS Sustainable Chemistry & Engineering 9(34),11512–23. DOI:10.1021/acssuschemeng.1c03765.

Nikel, P. I., and V. de Lorenzo. 2018. Pseudomonas putida as a Functional Chassis for Industrial Biocatalysis: From Native Biochemistry to Trans-Metabolism. Metabolic Engineering 50,142–55. DOI:10.1016/j.ymben.2018.05.005.

Sasaki, Y., et al. 2019. “Engineering Corynebacterium glutamicum to Produce the Biogasoline Isopentenol from Plant biomass Hydrolysates,” Biotechnology for Biofuels 12(41). DOI:10.1186/s13068-019-1381-3.

Wang, X., et al. 2022. “Engineering Isoprenoids Production in Metabolically Versatile Microbial Host Pseudomonas putida,” Biotechnology for Biofuels and Bioproducts 15(1). DOI: 10.1186/s13068-022-02235-6.

Funding Information

This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, BER program, of the U.S. DOE under Contract No. DE-AC02-05CH11231.