A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based...

Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h^-1 on formate showed high specific substrate uptake rates (47 mmol g^-1 h^-1). Co-utilization of formate with H₂, CO, CO₂ or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7-16.4 mmol g^-1 h^-1) and major differences in redox and energy metabolism. Growth on formate, H₂/CO₂, and formate + H₂/CO₂ resulted in low energy availability (0.20-0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H₂/CO/CO₂, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70-92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.