(175aa) Constructing Ethanol Utilization Pathway (EUP) in Escherichia coli

Hong Liang^1,2, Xiaoqiang Ma¹, Yurou Liu¹, Wenbo Ning³, Anthony J. Sinskey^1,4, Gregory Stephanopoulos^1,5, Kang Zhou^1,3

1. Disruptive & Sustainable Technologies for Agricultural Precision, Singapore-MIT Alliance for Research and Technology, Singapore
2. Department of Chemistry, National University of Singapore, Singapore
3. Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore
4. Department of Biology, Massachusetts Institute of Technology
5. Department of Chemical Engineering, Massachusetts Institute of Technology

Acetyl-CoA is a key compound in central metabolism of living cells. Starting from acetyl-CoA, many important natural products can be produced by using engineered microbes, such as flavonoids, vitamins, lipids and biopolymers. The shortest biosynthetic pathway of acetyl-CoA is from acetic acid, which merely requires one enzymatic step, but it consumes at least one ATP. The pH also increases during the assimilation of acetate acid, which complicates fermentation process especially at shake flask scale. Ethanol is a neutral molecule, which will not change pH when it is consumed and may reduce the possibility of contamination as most bacteria cannot utilize ethanol. It is well known that ethanol can be naturally converted into acetyl-CoA in mammalian and yeast cell by a three-step pathway which requires ATP for assimilating the intermediate of acetic acid. We constructed a two-step, ATP-neutral ethanol utilization pathway (EUP) in E. coli to improve production of acetyl-CoA-derived compounds.

We first reconstructed an EUP in E. coli by using acetaldehyde dehydrogenase (ada) from Dickeya zeae and alcohol dehydrogenase (adh2) from Saccharomyces cerevisiae. The EUP could convert ethanol into acetyl-CoA without ATP consumption, and required one cofactor, NAD⁺. We then optimized expression of these two genes, and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) by using a constitutive promoter (pgyrA). NAD⁺-dependent EUP was further enhanced by overexpression of a NADH-dependent oxidase (nox) as it helped recycle NAD⁺ from NADH. The engineered E. coli strain with EUP and nox was able to consume ~ 8 g/L of ethanol in 90 hours. We further investigated the enzymatic kinetics of EUP in vitro and the competing pathway of EUP in vivo to reveal the limiting step of EUP. Last, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered strain with EUP and PHB biosynthetic pathway produced 1.4 g/L of PHB by using 10 g/L of ethanol with 1 g/L of Complete Supplement Mixture (CSM) in 90 hours, suggesting the feasibility of using ethanol as sole carbon source to produce acetyl-CoA-derived compound. The EUP developed in this study can potentially be a platform to produce value-added acetyl-CoA-derived compounds.