Lepidopteran mevalonate pathway optimization in Escherichia coli efficiently produces isoprenol analogs for next-generation biofuels

Bo Pang; Jia Li; Christopher B Eiben; Ethan Oksen; Carolina Barcelos; Rong Chen; Elias Englund; Eric Sundstrom; Jay D Keasling

doi:10.1016/j.ymben.2021.10.007

Lepidopteran mevalonate pathway optimization in Escherichia coli efficiently produces isoprenol analogs for next-generation biofuels

Metab Eng. 2021 Nov:68:210-219. doi: 10.1016/j.ymben.2021.10.007. Epub 2021 Oct 18.

Authors

Bo Pang¹, Jia Li², Christopher B Eiben³, Ethan Oksen⁴, Carolina Barcelos⁴, Rong Chen⁵, Elias Englund³, Eric Sundstrom⁴, Jay D Keasling⁶

Affiliations

¹ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, United States; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, 94720, United States.
² Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, United States; State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, School of Life Sciences, Hubei University, Wuhan, Hubei, 430062, PR China; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States.
³ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, United States; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States.
⁴ Advanced Biofuels & Bioproducts Process Development Unit, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States.
⁵ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, United States; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; School of Public Health, Hangzhou Normal University, Hangzhou, Zhejiang, 311121, PR China.
⁶ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, 94608, United States; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, United States; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, 94720, United States; Novo Nordisk Foundation Center for Biosustainability, Technical University Denmark, DK 2970 Horsholm, Denmark; Center for Synthetic Biochemistry, Shenzhen Institutes for Advanced Technologies, Shenzhen, Guangdong, 518055, PR China. Electronic address: [email protected].

PMID: 34673235
DOI: 10.1016/j.ymben.2021.10.007

Abstract

Terpenes constitute the largest class of natural products with over 55,000 compounds with versatile applications including drugs and biofuels. Introducing structural modifications to terpenes through metabolic engineering is an efficient and sustainable way to improve their properties. Here, we report the optimization of the lepidopteran mevalonate (LMVA) pathway towards the efficient production of isopentenyl pyrophosphate (IPP) analogs as terpene precursors. First, we linked the LMVA pathway to NudB, a promiscuous phosphatase, resulting in the production of the six-carbon analog of 3-methyl-3-buten-1-ol (isoprenol), 3-ethyl-3-buten-1-ol (C6-isoprenol). Using C6-isoprenol as the final product, we then engineered the LMVA pathway by redirecting its upstream portion from a thiolase-dependent pathway to a beta-oxidation pathway. The beta-oxidation LMVA pathway transforms valeric acid, a platform chemical that can be produced from biomass, into C6-isoprenol at a titer of 110.3 mg/L, improved from 5.5 mg/L by the thiolase LMVA pathway, which used propionic acid as a feedstock. Knockout of the E. coli endogenous thiolase genes further improved the C6-isoprenol titer to 390 mg/L, implying efficient production of homo isopentenyl pyrophosphate (HIPP). The beta-oxidation LMVA-NudB pathway also converts butanoic acid and hexanoic acid into isoprenol and isoprenol's seven-carbon analog, 3-propyl-3-buten-1-ol (C7-isoprenol), respectively, suggesting the beta-oxidation LMVA pathway produces IPP and C7-IPP from the corresponding fatty acids. Fuel property tests revealed the longer chain isoprenol analogs have lower water solubilities, similar or higher energy densities, and comparable research octane number (RON) boosting effects to isopentenols. This work not only optimizes the LMVA pathway, setting the basis for homoterpene biosynthesis to expand terpene chemical space, but provides an efficient pathway to produce isoprenol analogs as next-generation biofuels from sustainable feedstocks.

Keywords: Beta oxidation; Biofuel; Homo isopentenyl pyrophosphate; Lepidopteran mevalonate pathway; Thiolase.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Biofuels
Escherichia coli / genetics
Escherichia coli Proteins* / genetics
Metabolic Engineering
Mevalonic Acid*
Pyrophosphatases

Substances

Biofuels
Escherichia coli Proteins
NudB protein, E coli
Pyrophosphatases
Mevalonic Acid