Single‐microbe RNA sequencing uncovers unexplored specialized metabolic functions of keystone species in the human gut

The human body is inhabited by trillions of microorganisms that play a crucial role in health and diseases. Our understanding of the species and functional composition of the human gut microbiome is rapidly expanding, but it is still mainly based on taxonomic profiles or gene abundance measurements. As such, little is known about the species-function heterogeneity and dynamic activities in human microecosystem niches. By applying a novel gut-specific single-microbe ribonucleic acid (RNA) sequencing and analytical framework on three healthy donors with distinct enterotypes, we created a comprehensive transcriptional landscape of the human gut microbiome and dissected functional specialization in 38,922 single microbes across 198 species. We investigated the functional redundancy and complementarity involved in short-chain fatty acids related central carbon metabolism and studied the heterogeneity and covariation of single-microbe metabolic capacity. Comparing the human gut microbiome at different times throughout the day, we were able to map diurnal dynamic activities of the gut microbiome and discovered its association with sub-population functional heterogeneous. Remarkably, using single-microbe RNA sequencing, we systematically dissected the metabolic function heterogeneity of <i>Megamonas funiformis</i>, a keystone species in Asian populations. Together with in vitro and in vivo experimental validations, we proved <i>M. funiformis</i> can effectively improve mineral absorption through exogenous phytic acid degradation, which could potentially serve as a probiotic that reduces malnutrition caused by deficiency of mineral elements. Our results indicated that species-function heterogeneity widely exists and plays important roles in the human gut microbiome, and through single-microbe RNA sequencing, we have been able to capture the transcriptional activity variances and identify keystone species with specialized metabolic functions of possible biological and clinical importance.