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High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

ABSTRACT: Unlike gaseous C₁ feedstocks for acetogenic bacteria, there has been less attention on liquid C₁ 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.

SUBMITTER: Wood JC

PROVIDER: S-EPMC9049608 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

Wood Jamin C JC Marcellin Esteban E Plan Manuel R MR Virdis Bernardino B

Microbial biotechnology 20211128 5

Unlike gaseous C<sub>1</sub> feedstocks for acetogenic bacteria, there has been less attention on liquid C<sub>1</sub> 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, showi ...[more]

PMID: 34841673

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Project description:Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of synthesis gas (syngas), for autotrophic growth in order to produce biomass and metabolites such as acetyl-CoA via the Wood-Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution (ALE) for growth improvement of Eubacterium limosum ATCC 8486 under high CO conditions. The strain evolved under syngas conditions with 44% CO over 150 generations, resulting in a significant increased optical density (600 nm) and growth rate by 2.14 and 1.44 folds, respectively. In addition, the evolved populations were capable of proliferating under CO concentrations as high as 80%. These results suggest that cell growth is enhanced as beneficial mutations are selected and accumulated, and the metabolism is altered to facilitate the enhanced phenotype. To identify the causal mutations related to growth improvement under high CO concentrations, we performed whole genome resequencing of each population at 50-generation intervals. Interestingly, we found key mutations in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding genes, acsA and cooC. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth rate and CO tolerance level of the single clone were comparable to those of the evolved populations and wild type strain under CO conditions. Furthermore, the evolved strain produced 1.34 folds target metabolite acetoin when compared to the parental strain while introducing the biosynthetic pathway coding genes to the strains. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486.

Dataset Information

High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

Publications

High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

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