Comprehensive molecular characterization of Methylobacterium extorquens AM1 adapted for 1-butanol tolerance.

Song Yang,Mary E Lidstrom,Wen-Jing Chen,Bo Hu,David A C Beck,Jing Yang,Qian-Wen Wang,Yi-Ming Yang

doi:10.1186/s13068-016-0497-y

Abstract

BackgroundThe toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels production. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, has been engineered to generate 1-butanol from cheap carbon feedstocks through a synthetic metabolic pathway. However, M. extorquens AM1 is vulnerable to solvent stress, which impedes further development for 1-butanol production. Only a few studies have reported the general stress response of M. extorquens AM1 to solvent stress. Therefore, it is highly desirable to obtain a strain with ameliorated 1-butanol tolerance and elucidate the molecular mechanism of 1-butnaol tolerance in M. extorquens AM1 for future strain improvement.ResultsIn this work, adaptive laboratory evolution was used as a tool to isolate mutants with 1-butanol tolerance up to 0.5 %. The evolved strains, BHBT3 and BHBT5, demonstrated increased growth rates and higher survival rates with the existence of 1-butanol. Whole genome sequencing revealed a SNP mutation at kefB in BHBT5, which was confirmed to be responsible for increasing 1-butanol tolerance through an allelic exchange experiment. Global metabolomic analysis further discovered that the pools of multiple key metabolites, including fatty acids, amino acids, and disaccharides, were increased in BHBT5 in response to 1-butanol stress. Additionally, the carotenoid synthesis pathway was significantly down-regulated in BHBT5.ConclusionsWe successfully screened mutants resistant to 1-butanol and provided insights into the molecular mechanism of 1-butanol tolerance in M. extorquens AM1. This research will be useful for uncovering the mechanism of cellular response of M. extorquens AM1 to solvent stress, and will provide the genetic blueprint for the rational design of a strain of M. extorquens AM1 with increased 1-butanol tolerance in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0497-y) contains supplementary material, which is available to authorized users.

Highlights

The toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels produc‐ tion
This research will be useful for uncovering the mechanism of cellular response of M. extorquens AM1 to solvent stress, and will provide the genetic blueprint for the rational design of a strain of M. extorquens AM1 with increased 1-butanol tolerance in the future
At 0.3 % 1-butanol, a portion of the cultures were able to grow to similar OD600 of that at 0.15 % 1-butanol, suggesting that critical genetic mutations related to 1-butanol tolerance may occur in these cultures

Summary

Introduction

The toxicity of alcohols is one of the major roadblocks of biological fermentation for biofuels produc‐ tion. M. extorquens AM1 is vulnerable to solvent stress, which impedes further development for 1-butanol production. A few studies have reported the general stress response of M. extorquens AM1 to solvent stress. C1 and C2 metabolism and development of a generation genetic tool set enable the direction of carbon flux in M. extorquens AM1 from methanol assimilation to the synthesis of higher value added products such as 1-butanol, a second generation biofuel [5,6,7]. The vulnerability of M. extorquens AM1 to solvent stress impedes its further development as a biofuel-producing platform. Improvement of solvent tolerance through genetic manipulation requires substantial knowledge on molecular mechanisms of cell response to solvent stress, which could be complicated as revealed in other microorganisms [8,9,10]. It is difficult to improve 1-butanol tolerance of M. extorquens AM1 through manipulating a single gene or a few genes as a cluster

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Conclusion