Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

Flaiz, Maximilian; Ludwig, Gideon; Bengelsdorf, Frank R.; Dürre, Peter

doi:10.21203/rs.3.rs-171102/v1

Cited by 4 publications

(3 citation statements)

References 66 publications

(88 reference statements)

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“…Historically, in a closely related strain to E. limosum , Butyribacterium methylotrophicum , butanol production was suspected via aldehyde dehydrogenase ( ald ) (Grethlein et al ., 1991 ). A recent investigation showed butanol production from methanol with recombinant E. limosum strains by introducing the bifunctional acetaldehyde/alcohol dehydrogenase from Clostridium acetobutylicum (Flaiz et al ., 2021 ). However, it is now well accepted that alcohols are predominately formed via the promiscuous aldehyde:ferredoxin oxidoreductase ( AOR ) enzyme in acetogens (Diender et al ., 2016 ; Richter et al ., 2016 ; Liew et al ., 2017 ; Valgepea et al ., 2017 ; Greene et al ., 2019 ).…”

Section: Resultsmentioning

confidence: 99%

“…The Eubacterium limosum genome shows a native butanol production pathway, a more reduced product than butyrate (Song and Cho, 2015 ); however, this has yet to be seen experimentally. It is noted, a recent investigation showed non‐native butanol and acetone production from methanol with recombinant Eubacterium limosum (Flaiz et al ., 2021 ). Unlike butyrate, butanol has a sizeable chemical market and holds promise for use as a drop‐in fuel (Wang et al ., 2014 ; Wood et al ., 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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High methanol‐to‐formate ratios induce butanol production in Eubacterium limosum

Wood

Marcellin

Plan

et al. 2021

Microbial Biotechnology

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Summary Unlike gaseous C 1 feedstocks for acetogenic bacteria, there has been less attention on liquid C 1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol‐to‐formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol‐C gDCW ‐1 day ‐1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM‐C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h ‐1 ) and cell density (maximum 1.2 ± 0.043 gDCW l ‐1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases – growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3‐butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum , particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High methanol‐to‐formate ratios induce butanol production in Eubacterium limosum

Wood

Marcellin

Plan

et al. 2021

Microbial Biotechnology

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“…Anecdotally, adaptive laboratory evolution could be used to address this negative phenotype, particularly given some E. limosum strains are known to not form sticky polymers that support biofilms (Flaiz et al 2021).…”

Section: Introductionmentioning

confidence: 99%

Characterisation of acetogen formatotrophic potential usingE. limosum

Wood

Gonzalez-Garcia

Daygon

et al. 2022

Preprint

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Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such asEubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in acetogens is poorly understood, and a systems-level characterization in continuous cultures is yet to be reported. Here we present the first steady-state dataset forE. limosumformatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280±56 mmol/gDCW/d), however, most carbon went to CO2(150±11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of pta appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of Pfl to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth.

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