A cryptic lactate cycle in CO fermentation by Clostridium ljungdahlii

ABSTRACT The valorization of C1 gases presents a route toward the utilization of industrial waste gases as resources. Acetogens such as Clostridium ljungdahlii serve as promising microbial cell factories for light-independent carbon fixation and high-value compound production, yet their efficiency is often limited by energy constraints and poorly understood metabolic pathways. Here, we investigated lactate metabolism in C. ljungdahlii during carbon monoxide (CO) fermentation via growth and product profiling, 13 C-labeling, gene knockouts, and transcriptomics. Although lactate cannot serve as the sole source of carbon and energy for C. ljungdahlii ’s growth, we found that lactate can be co-fermented with CO into acetate. 13 C-tracing further revealed concurrent lactate production and oxidation during CO fermentation, with the unlabeled lactate fraction increasing from 1.0% (day 2) to 5.0% (day 6), indicating the existence of an active lactate cycle. Genetic and transcriptional analyses showed that lactate production and oxidation were catalyzed by two distinct lactate dehydrogenases: LdhA (CLJU_RS15865) and the electron-bifurcating Ldh/EtfAB_1 complex (CLJU_RS10590–CLJU_RS10600), respectively. Disruption of the lactate cycle, particularly in the Ldh/EtfAB_1 mutant, improved energy metabolism and triggered intracellular metabolic rearrangements. This mutant produced approximately 10-fold more lactate and shifted the acetate/ethanol ratio from 3.7:1 to 2.1:1, accompanied by upregulation of the AOR (aldehyde:ferredoxin oxidoreductase)-mediated ethanol production pathway. Subsequent ldhA overexpression further elevated lactate titer to ~2.5 mM, confirming the potential for lactate production from CO. The results imply that the cryptic lactate cycle dissipates energy but aids in intracellular redox balancing during CO fermentation. These findings provide new insights into the metabolic engineering of acetogens to produce target compounds, such as lactate and ethanol. IMPORTANCE Acetogens possess a natural carbon fixation pathway, the Wood-Ljungdahl pathway, enabling them to fix CO or CO2 to produce acetate. Among various acetogens, Clostridium ljungdahlii exhibits strong tolerance to CO and thus holds great application potential for the resource utilization of syngas. However, the physiological and metabolic mechanisms underlying its autotrophic fermentation remain incompletely understood, which imposes limitations on metabolic engineering efforts. In this study, we systematically investigated lactate metabolism in C. ljungdahlii and revealed the existence of a cryptic lactate cycle during CO fermentation. By characterizing this cycle’s operation and its impacts on energy metabolism and carbon distribution, we elucidated its potential physiological functions. These findings not only expand our understanding of the physiological and metabolic behaviors of acetogens during autotrophic fermentation but also provide theoretical guidance for the future metabolic engineering of lactate, ethanol, and other compounds.