Lactate formation from fructose or C1 compounds in the acetogen Acetobacterium woodii by metabolic engineering.

Anaerobic, acetogenic bacteria are promising biocatalysts for a sustainable bioeconomy since they capture and convert carbon dioxide to acetic acid. Hydrogen is an intermediate in acetate formation from organic as well as C1 substrates. Here, we analyzed mutants of the model acetogen Acetobacterium woodii in which either one of the two hydrogenases or both together were genetically deleted. In resting cells of the double mutant, hydrogen formation from fructose was completely abolished and carbon was redirected largely to lactate. The lactate/fructose and lactate/acetate ratios were 1.24 and 2.76, respectively. We then tested for lactate formation from methyl groups (derived from glycine betaine) and carbon monoxide. Indeed, also under these conditions lactate and acetate were formed in equimolar amounts with a lactate/acetate ratio of 1.13. When the electron-bifurcating lactate dehydrogenase/ETF complex was genetically deleted, lactate formation was completely abolished. These experiments demonstrate the capability of A. woodii to produce lactate from fructose but also from promising C1 substrates, methyl groups and carbon monoxide. This adds an important milestone towards generation of a value chain leading from CO2 to value-added compounds. KEY POINTS: • Resting cells of the ΔhydBA/hdcr mutant of Acetobacterium woodii produced lactate from fructose or methyl groups + CO • Lactate formation from methyl groups + CO was completely abolished after deletion of lctBCD • Metabolic engineering of a homoacetogen to lactate formation gives a potential for industrial applications.

Reprogramming the metabolism of an acetogenic bacterium to homoformatogenesis.

Methyl groups are abundant in anoxic environments and their utilization as carbon and energy sources by microorganisms involves oxidation of the methyl groups to CO2, followed by transfer of the electrons to an acceptor. In acetogenic bacteria, the electron acceptor is CO2 that is reduced to enzyme bound carbon monoxide, the precursor of the carboxyl group in acetate. Here, we describe the generation of a mutant of the acetogen Acetobacterium woodii in which the last step in methyl group oxidation, formate oxidation to CO2 catalyzed by the HDCR enzyme, has been genetically deleted. The mutant grew on glycine betaine as methyl group donor, and in contrast to the wild type, formed formate alongside acetate, in a 1:2 ratio, demonstrating that methyl group oxidation stopped at the level of formate and reduced electron carriers were reoxidized by CO2 reduction to acetate. In the presence of the alternative electron acceptor caffeate, CO2 was no longer reduced to acetate, formate was the only product and all the carbon went to formate. Apparently, acetogenesis was not required to sustain formatogenic growth. This is the first demonstration of a genetic reprogramming of an acetogen into a formatogen that grows by homoformatogenesis from methyl groups. Formate production from methyl groups is not only of biotechnological interest but also for the mechanism of electron transfer in syntrophic interactions in anoxic environments.