FNR/ArcA-2,3-butanediol regulatory axis enhances glucose uptake and adhE expression in high alcohol-producing Klebsiella pneumoniae

ABSTRACT Previously, we found that Klebsiella pneumoniae is a high-alcohol producer, which contributes to the development of non-alcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying its high-alcohol production remain unclear. By comparing biological phenotypes of K. pneumoniae with those of Escherichia coli , we found that the former is better at metabolizing high concentrations of glucose, exhibits faster growth rates, and produces higher levels of alcohol, with the 2,3-butanediol (2,3-BD) pathway being the key determinant underlying these differences. More importantly, species possessing the 2,3-BD pathway, including Klebsiella aerogenes , Klebsiella oxytoca , Enterobacter cloacae , and Enterobacter hormaechei , have all demonstrated high-alcohol production capabilities similar to that of K. pneumoniae , suggesting a conserved mechanism for alcohol production among 2,3-BD-positive Enterobacteriaceae . Deletion of the 2,3-BD pathway reduced the expression of the phosphotransferase system (PTS) and the alcohol dehydrogenase gene adhE , which normally confer enhanced glucose metabolism and alcohol production. Pathway deletion also downregulated multiple virulence determinants, including capsular polysaccharides (CPS), lipopolysaccharides (LPS), and type 1 and 3 fimbriae, thereby attenuating virulence in a murine pneumonia model. In addition, the regulator FNR enhanced alcohol production by upregulating adhE and 2,3-BD pathway genes, whereas ArcA activated the 2,3-BD pathway in an ArcB-independent manner, revealing a non-canonical signaling mechanism that couples metabolic adaptation with virulence. Collectively, our findings identify the “FNR/ArcA–2,3-BD pathway axis” as a key molecular mechanism driving high alcohol production in K. pneumoniae , highlighting the 2,3-BD pathway as a key link between bacterial metabolism and virulence and a potential therapeutic target for controlling K. pneumoniae infections. IMPORTANCE Klebsiella pneumoniae has emerged as a novel cause of NAFLD due to its high alcohol-producing capacity. However, the molecular basis of this phenotype remains unclear. In this study, we demonstrate that the FNR/ArcA-2,3-BD pathway axis drives high levels of alcohol production by enhancing glucose-PTS system and adhE expression, and that this pathway is conserved among Enterobacteriaceae with strong alcohol-producing abilities. Moreover, loss of the 2,3-BD pathway reduces the expression of multiple virulence factors and attenuates pathogenicity in a murine pneumonia model. These findings identify the FNR/ArcA-2,3-BD axis as a potential target for mitigating excessive alcohol production and virulence in bacteria.