Molecular understanding ofEubacterium limosumchemostat methanol metabolism

Abstract

Methanol is a promising renewable energy carrier that can be used as a favourable substrate for biotechnology, due to its high energy efficiency conversion and ease of integration within existing infrastructure. Some acetogenic bacteria have the native ability to utilise methanol, along with other C1substrates such as CO2and formate, to produce valuable chemicals. Continuous cultures favour economically viable bioprocesses, however, the performance of acetogens has not been investigated at the molecular level when grown on methanol. Here we present steady-state chemostat quantification of the metabolism ofEubacterium limosum, finding maximum methanol uptake rates up to 640±22 mmol/gDCW/d, with significant fluxes to butyrate. To better understand metabolism of acetogens under methanol growth conditions, we sampled chemostats for proteomics and metabolomics. Changes in protein expression and intracellular metabolomics highlighted key aspects of methanol metabolism, and highlighted bottleneck conditions preventing formation of the more valuable product, butanol. Interestingly, a small amount of formate in methylotrophic metabolism triggered a cellular state known in other acetogens to correlate with solventogenesis. Unfortunately, this was prevented by post-translation effects including an oxidised NAD pool. There remains uncertainty around ferredoxin balance at the methylene-tetrahydrofolate reductase (MTHFR) and at the Rnf level.

Characterisation of acetogen formatotrophic potential usingE. limosum

“…Intriguingly, despite formate being the less reduced substrate, it produced more hydrogen at 21±7.8 mmol/gDCW/d compared to 1.1±0.56 mmol/gDCW/d for methanol/CO 2 ( Figure 1A ). We noted in Wood et al . (2022) that formate seems to trigger hydrogen production, which is further reinforced with this data.…”

“…Characteristics of Eubacterium limosum in autotrophic chemostats, with conditions as summarised in Table 1. Methylotrophic data is provided for comparison from (Wood et al 2022). (A) specific uptake and production rates.…”

“…Given the lack of success in identifying the missing product, we performed proteomics and metabolomics analyses to gain an understanding of formatotrophic E. limosum metabolism, and compared it to methanol metabolism (Wood et al 2022). We identified 934 proteins that met the criteria for differential analysis between the formate and methanol/CO 2 growth conditions ( i.e .…”

“…Reactions are shown with substrates, products and redox mediators, however without stoichiometric balances along pathways. Enzymes and associated protein accession numbers (elim_cxxxx) are shown for each reaction, along with log 2 fold changes comparing methylotrophic (Wood et al 2022) and formatotrophic growth. Intracellular metabolites concentrations (μmol/gDCW) are shown for select metabolites as the average ± standard deviation of biological replicate triplicates.…”

“…Methylotrophic data is provided for comparison from(Wood et al 2022). (A) specific uptake and production rates.…”