TY - JOUR

T1 - Systems engineering of Escherichia coli for n-butane production

AU - Liu, Yilan

AU - Khusnutdinova, Anna

AU - Chen, Jinjin

AU - Crisante, David

AU - Batyrova, Khorcheska

AU - Raj, Kaushik

AU - Feigis, Michelle

AU - Shirzadi, Erfan

AU - Wang, Xiaotong

AU - Dorakhan, Roham

AU - Wang, Xue

AU - Stogios, Peter J

AU - Yakunin, Alexander F

AU - Sargent, Edward H

AU - Mahadevan, Radhakrishnan

N1 - Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.

PY - 2022/11/1

Y1 - 2022/11/1

N2 - Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance. Next, we did ribosome binding site (RBS) calculation to identify potential metabolic bottlenecks, and then mitigated the bottleneck with RBS engineering and precursor propionyl-CoA addition. Furthermore, we employed a model-assisted strain design and a nonrepetitive extra-long sgRNA arrays (ELSAs) and quorum sensing assisted CRISPRi to facilitate a dynamic two-stage fermentation. Through systems engineering, n-butane production was increased by 168-fold from 0.04 to 6.74 mg/L. Finally, the maximum n-butane production from acetate was predicted using parsimonious flux balance analysis (pFBA), and we achieved n-butane production from acetate produced by electrocatalytic CO reduction. Our findings pave the way for selectively producing n-butane from renewable carbon source.

AB - Rising concerns about climate change and sustainable energy have attracted efforts towards developing environmentally friendly alternatives to fossil fuels. Biosynthesis of n-butane, a highly desirable petro-chemical, fuel additive and diluent in the oil industry, remains a challenge. In this work, we first engineered enzymes Tes, Car and AD in the termination module to improve the selectivity of n-butane biosynthesis, and ancestral reconstruction and a synthetic RBS significantly improved the AD abundance. Next, we did ribosome binding site (RBS) calculation to identify potential metabolic bottlenecks, and then mitigated the bottleneck with RBS engineering and precursor propionyl-CoA addition. Furthermore, we employed a model-assisted strain design and a nonrepetitive extra-long sgRNA arrays (ELSAs) and quorum sensing assisted CRISPRi to facilitate a dynamic two-stage fermentation. Through systems engineering, n-butane production was increased by 168-fold from 0.04 to 6.74 mg/L. Finally, the maximum n-butane production from acetate was predicted using parsimonious flux balance analysis (pFBA), and we achieved n-butane production from acetate produced by electrocatalytic CO reduction. Our findings pave the way for selectively producing n-butane from renewable carbon source.

KW - Escherichia coli/genetics

KW - Metabolic Engineering

KW - Butanes/metabolism

KW - Acetates/metabolism

U2 - 10.1016/j.ymben.2022.10.001

DO - 10.1016/j.ymben.2022.10.001

M3 - Article

C2 - 36244545

VL - 74

SP - 98

EP - 107

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

ER -