Mutations in adaptively evolved Escherichia coli LGE2 facilitated the cost-effective upgrading of undetoxified bio-oil to bioethanol fuel

Dongdong Chang,Zhisheng Yu,Cong Wang,Fabrice Ndayisenga

doi:10.1186/s40643-021-00459-2

Abstract

Levoglucosan is a promising sugar present in the lignocellulose pyrolysis bio-oil, which is a renewable and environment-friendly source for various value-added productions. Although many microbial catalysts have been engineered to produce biofuels and chemicals from levoglucosan, the demerits that these biocatalysts can only utilize pure levoglucosan while inhibited by the inhibitors co-existing with levoglucosan in the bio-oil have greatly limited the industrial-scale application of these biocatalysts in lignocellulose biorefinery. In this study, the previously engineered Escherichia coli LGE2 was evolved for enhanced inhibitor tolerance using long-term adaptive evolution under the stress of multiple inhibitors and finally, a stable mutant E. coli-H was obtained after ~ 374 generations’ evolution. In the bio-oil media with an extremely acidic pH of 3.1, E. coli-H with high inhibitor tolerance exhibited remarkable levoglucosan consumption and ethanol production abilities comparable to the control, while the growth of the non-evolved strain was completely blocked even when the pH was adjusted to 7.0. Finally, 8.4 g/L ethanol was achieved by E. coli-H in the undetoxified bio-oil media with ~ 2.0% (w/v) levoglucosan, reaching 82% of the theoretical yield. Whole-genome re-sequencing to monitor the acquisition of mutations identified 4 new mutations within the globally regulatory genes rssB, yqhA, and basR, and the − 10 box of the putative promoter of yqhD-dgkA operon. Especially, yqhA was the first time to be revealed as a gene responsible for inhibitor tolerance. The mutations were all responsible for improved fitness, while basR mutation greatly contributed to the fitness improvement of E. coli-H. This study, for the first time, generated an inhibitor-tolerant levoglucosan-utilizing strain that could produce cost-effective bioethanol from the toxic bio-oil without detoxification process, and provided important experimental evidence and valuable genetic/proteinic information for the development of other robust microbial platforms involved in lignocellulose biorefining processes.

Highlights

As concerns on the environmental and energy issues are increasing due to the massive usage of fossil fuels, the development of bio-based systems for the production of biofuels and chemicals from renewable resources that serve as a replacement for unsustainable fossil fuelsPyrolysis process that does not require any solvent or enzyme to depolymerize lignocellulose can and cheaply decompose lignocellulose toChang et al Bioresour
Adaptive evolution generated highly inhibitor‐tolerant strain An evolutionary engineering approach based on longterm adaptation in lignocellulose-derived inhibitors to select strains for enhanced inhibitor tolerance was envisaged in the current study
Our previously engineered strain E. coli LGE2 (Chang et al 2021) was chosen for the evolution experiment to screen for robust strains with genetic changes that are linked to the cellular tolerance to the representative lignocellulosic inhibitors-acetic acid, furfural, and phenol

Summary

Introduction

As concerns on the environmental and energy issues are increasing due to the massive usage of fossil fuels, the development of bio-based systems for the production of biofuels and chemicals from renewable resources that serve as a replacement for unsustainable fossil fuelsPyrolysis process that does not require any solvent or enzyme to depolymerize lignocellulose can and cheaply decompose lignocellulose toChang et al Bioresour. Levoglucosan can further be bioconverted to various biofuels and chemicals by different engineered microorganisms (Kim et al 2015; Layton et al 2011; Linger et al 2016; Xiong et al 2016) introduced with a heterologous gene lgk from Lipomyces starkeyi (Dai et al 2009). These investigated studies all focused on the bioconversion of pure levoglucosan that is purified from the bio-oil; and this purification step greatly increases the overall cost and process complexity which make the approach not economically feasible for its industrial implementation. Physical and/or chemical detoxification processes can remove the bio-toxic chemicals co-existing with levoglucosan and make the treated bio-oil fermentable (Chi et al 2013; Rover et al 2014), this detoxification step becomes a major challenge toward its commercialization since it makes the whole conversion process more costly and complex

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Results

Conclusion