A new metabolic trait in an acetogen: Mixed acid fermentation of fructose in a methylene‐tetrahydrofolate reductase mutant of Acetobacterium woodii

Abstract

To inactivate the Wood–Ljungdahl pathway in the acetogenic model bacterium Acetobacterium woodii, the genes metVF encoding two of the subunits of the methylene‐tetrahydrofolate reductase were deleted. As expected, the mutant did not grow on C1 compounds and also not on lactate, ethanol or butanediol. In contrast to a mutant in which the first enzyme of the pathway (hydrogen‐dependent CO2 reductase) had been genetically deleted, cells were able to grow on fructose, albeit with lower rates and yields than the wild‐type. Growth was restored by addition of an external electron sink, glycine betaine + CO2 or caffeate. Resting cells pre‐grown on fructose plus an external electron acceptor fermented fructose to two acetate and four hydrogen, that is, performed hydrogenogenesis. Cells pre‐grown under fermentative conditions on fructose alone redirected carbon and electrons to form lactate, formate, ethanol as well as hydrogen. Apparently, growth on fructose alone induced enzymes for mixed acid fermentation (MAF). Transcriptome analyses revealed enzymes potentially involved in MAF and a quantitative model for MAF from fructose in A. woodii is presented.

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

“…In a previous study we have found conversion of fructose to molecular hydrogen, formate, ethanol and lactate as end products in a Δ metVF mutant of A. woodii (Moon et al 2023a ). Here, we aimed to redirect fructose metabolism to lactate.…”

“…This enzyme complex is known to be responsible for lactate oxidation during growth of A. woodii on lactate (Weghoff et al 2015 ). Recently, it has been reported that the lctBCD genes were highly expressed in the Δ metVF mutant grown on fructose where lactate was formed as a side product (Moon et al 2023a ). Therefore, to verify that a possible lactate formation was indeed catalyzed by LctBCD we genetically deleted the LDH/ETF complex.…”

“…Since ethanol was only produced in very minor amounts, and since A. woodii has eleven different alcohol dehydrogenases, it was not attempted to genetically delete ethanol production. Hydrogen was produced in huge amounts (Moon et al 2023a ) and therefore we analyzed whether H 2 production would be abolished in the ∆ hdcr , and the Δ hydBA/hdcr double mutant. The growth phenotype of these mutants has been described before; in brief, they do not grow on fructose, H 2 + CO 2 , methanol, or formate (Moon et al 2023b ).…”

“…Recently, we discovered a novel metabolic trait in A. woodii , mixed acid fermentation of fructose (Moon et al 2023a ). A mutant in which the central enzyme of the WLP, the methylene-tetrahydrofolate reductase was genetically deleted, was able to grow on fructose.…”

“…A mutant in which the central enzyme of the WLP, the methylene-tetrahydrofolate reductase was genetically deleted, was able to grow on fructose. But acetate was not the only product; in addition molecular hydrogen, formate, ethanol and lactate were produced as end products (Moon et al 2023a ). This finding offered the possibility to engineer strains that convert fructose or even C1 compounds to reduced end products such as ethanol or lactate.…”