Setting out to isolate uncultivated deep marine sediment microorganisms, we engineered and operated a methane-fed continuous-flow bioreactor system for more than 2,000 days to enrich such organisms from anaerobic marine methane-seep sediments 15 (Supplementary Note 1). We successfully enriched many phylogenetically diverse yetto-be cultured microorganisms, including Asgard archaea members (Loki-, Heimdall-and Odinarchaeota) 15. For further enrichment and isolation, samples of the bioreactor community were inoculated in glass tubes with simple substrates and basal medium. After approximately one year, we found faint cell turbidity in a culture containing casamino acids supplemented with four bacteria-suppressing antibiotics (Supplementary Note 2) that was incubated at 20 °C. Clone librarybased small subunit (SSU) rRNA gene analysis revealed a simple community that contained Halodesulfovibrio and a small population of Lokiarchaeota (Extended Data Table 1). In pursuit of this archaeon, which we designated strain MK-D1, we repeated subcultures when MK-D1 reached maximum cell densities as measured by quantitative PCR (qPCR). This approach gradually enriched the archaeon, which has an extremely slow growth rate and low cell yield (Fig. 1a). The culture consistently had a 30-60-day lag phase and required more

34The origin of eukaryotes remains enigmatic. Current data suggests that eukaryotes may 35 have risen from an archaeal lineage known as "Asgard archaea". Despite the eukaryote-36 like genomic features found in these archaea, the evolutionary transition from archaea to 37 eukaryotes remains unclear due to the lack of cultured representatives and corresponding 38 physiological insight. Here we report the decade-long isolation of a Lokiarchaeota-related 39Asgard archaeon from deep marine sediment. The archaeon, "Candidatus 40Prometheoarchaeum syntrophicum strain MK-D1", is an anaerobic, extremely slow-41 growing, small cocci (~550 nm), that degrades amino acids through syntrophy. Although 42 eukaryote-like intracellular complexities have been proposed for Asgard archaea, the 43 isolate has no visible organella-like structure. Ca. P. syntrophicum instead displays 44 morphological complexity -unique long, and often, branching protrusions. Based on 45 cultivation and genomics, we propose an "Entangle-Engulf-Enslave (E 3 ) model" for 46 eukaryogenesis through archaea-alphaproteobacteria symbiosis mediated by the physical 47 complexities and metabolic dependency of the hosting archaeon. 48 49 How did the first eukaryotic cell emerge? So far, among various competing evolutionary 50 models, the most widely accepted are the symbiogenetic models in which an archaeal 51 host cell and an alphaproteobacterial endosymbiont merged to become the first eukaryotic 52 cell 1-4 . Recent metagenomic discovery of Lokiarchaeota (and the Asgard archaea 53 superphylum) led to the theory that eukaryotes originated from an archaeon closely 54 related to Asgard archaea 5,6 . The Asgard archaea genomes encode a repertory of proteins 55 hitherto only found in Eukarya (eukaryotic signature proteins -ESPs), including those 56 involved in membrane trafficking, vesicle formation/transportation, ubiquitin and 57 cytoskeleton formation 6 . Subsequent metagenomic studies have suggested that Asgard 58 archaea have a wide variety of physiological properties, including hydrogen-dependent 59 anaerobic autotrophy 7 , peptide or short-chain hydrocarbon-dependent organotrophy 8-11 60 and rhodopsin-based phototrophy 12,13 . A recent study suggests that an ancient Asgard 61 archaea degraded organic substances and syntrophically handed off reducing equivalents 62 (e.g., hydrogen and electrons) to a bacterial partner, and further proposes a symbiogenetic 63 model for the origin of eukaryotes based on this interaction 14 . However, at present, no 64 single representative of the Asgard archaea has been cultivated and, thus, the physiology 65 and cell biology of this clade remains unclear. In an effort to close this knowledge gap, 66 3 we successfully isolated the first Asgard archaeon and here report the physiological 67 characteristics, potentially key insights into the evolution of eukaryotes. 68 69 Isolation of an Asgard archaeon 70Setting out to isolate uncultivated deep marine sediment microorganisms, we engineered 71 and operated a methane-fed continuous-fl...