Extracellular Metabolite Profiling in CO2-Fixing Bacterium Rhodobacter sphaeroides Under Autotrophic Conditions

<b>Background/Objectives:</b><i>Rhodobacter sphaeriids</i> is considered a promising biomanufacturing platform due to its capacity to convert CO₂ into value-added products. To enhance the yield of CO₂-derived products, understanding extracellular metabolite dynamics during autotrophic growth is essential. However, the extracellular metabolite profiles of <i>R. sphaeroides</i> under autotrophic conditions have not been reported. <b>Methods:</b> In this study, we performed a comprehensive analysis of extracellular metabolites produced under autotrophic conditions using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and liquid chromatography time-of-flight mass spectrometry (LC-TOFMS). <b>Results:</b> A total of 62 putative metabolites were detected, of which 23 were measured above the quantification limit. Metabolites involved in glycolysis and gluconeogenesis constituted the largest proportion of extracellular metabolites, with lactic acid exhibiting the highest accumulation levels. To investigate the transcriptional changes associated with metabolite accumulation, we analyzed gene expression and observed the downregulation of glycolytic genes, including <i>pgi</i>, <i>gapB</i>, and <i>lctB</i>, whereas <i>cfxA</i>, encoding fructose-1,6-bisphosphate aldolase, was upregulated under autotrophic conditions compared to heterotrophic conditions. <b>Conclusions:</b> These results suggest that the carbon assimilation metabolic flux in <i>R. sphaeroides</i> shifts toward the CBB cycle and lactic acid overflow metabolism under autotrophic conditions. Collectively, these findings provide new insights into metabolic regulation during autotrophic growth and offer a basis for reducing extracellular byproduct formation and improving CO₂-based biological production in <i>R. sphaeroides</i>.