Metabolic engineering of Clostridium autoethanogenum for selective alcohol production

Fungmin Liew,Anne M Henstra,Michael Kӧpke,Klaus Winzer,Sean D Simpson,Nigel P Minton

doi:10.1016/j.ymben.2017.01.007

Fungmin Liew, Anne M Henstra + Show 4 more

Open Access

https://doi.org/10.1016/j.ymben.2017.01.007

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Abstract

Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols.

Highlights

The deleterious environmental impact caused by the continuing extraction and exploitation of fossil fuels for energy, coupled with their inherent finite nature, are the principle drivers for the development of sustainable alternatives
We developed an allelic exchange method for C. autoethanogenum based on the use of a pseudo-suicide vector reliant on the pCD6 replicon (Heap et al, 2009) and a plasmidencoded counter selection marker composed of an orotate phosphoribosyltransferase gene of C. acetobutylicum
To create a double mutant, the aor1 gene was first inactivated using ClosTron mutagenesis in the ΔpyrE strain, before in-frame deletion (IFD) of aor2 was undertaken by allelic exchange using the pyrEbased KO vector and counter selection using FOA

Summary

Introduction

The deleterious environmental impact caused by the continuing extraction and exploitation of fossil fuels for energy, coupled with their inherent finite nature, are the principle drivers for the development of sustainable alternatives. In this regard, gas fermentation has emerged as a promising technology that converts industrial waste gases or syngas containing CO, CO2 and H2 into fuels without impacting on food production. Gas fermentation has emerged as a promising technology that converts industrial waste gases or syngas containing CO, CO2 and H2 into fuels without impacting on food production It is reliant on bacterial process organisms that are able to utilise single carbon gases as a source of carbon typified by a group of strictly anaerobic bacteria known as acetogens. One such acetogen is Clostridium autoethanogenum (Abrini et al, 1994). It is able to grow on CO as a sole source of carbon and energy and synthesize ethanol, 2,3-butanediol and lactate (Köpke et al, 2011)

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