Seeking active RubisCOs from the currently uncultured microbial majority colonizing deep-sea hydrothermal vent environments

Abstract

Almost all the inorganic carbon on Earth is converted into biomass via the Calvin-Benson-Bassham (CBB) cycle. Here, the central carboxylation reaction is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which can be found in numerous primary producers including plants, algae, cyanobacteria, and many autotrophic bacteria. Although RubisCO possesses a crucial role in global biomass production, it is not a perfect catalyst. Therefore, research interest persists on accessing the full potential of yet unexplored RubisCOs. We recently developed an activity-based screen suited to seek active recombinant RubisCOs from the environment-independent of the native host's culturability. Here, we applied this screen to twenty pre-selected genomic fosmid clones from six cultured proteobacteria to demonstrate that a broad range of phylogenetically distinct RubisCOs can be targeted. We then screened 12,500 metagenomic fosmid clones from six distinct hydrothermal vents and identified forty active RubisCOs. Additional sequence-based screening uncovered eight further RubisCOs, which could then also be detected by a modified version of the screen. Seven were active form III RubisCOs from yet uncultured Archaea. This indicates the potential of the activity-based screen to detect RubisCO enzymes even from organisms that would not be expected to be targeted.

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

“…To identify the form of each variant, a set of manually curated variants was mapped to a phylogenetic tree and rubisco-like proteins were removed as they lack carboxylation activity, leaving % 33,000 non-redundant variants (see Materials and Methods for a detailed description of the pipeline). While rubisco enzymes that do not share sequence homology to currently identified variants are not detectable by this pipeline, so far, no evolutionarily distinct rubiscos were found (Böhnke & Perner, 2019). We, therefore, denote the set of % 33,000 sequences as the sequence space of wild rubisco's large subunit.…”

Single-cell RNA-seq reveals distinct metabolic “microniches” and close host-symbiont interactions in deep-sea chemosynthetic tubeworm

“…5E ), which is further oxidized to sulfate, producing adenosine triphosphate ( 54 , 55 ). Simultaneously, the elevated expression of RuBisCO in this microniche, which catalyzes the rate-limiting step in the CBB cycle, suggests that these periphery symbionts also fix atmospheric carbon dioxide into a biologically useful carbon source ( 17 , 56 ).…”

Microbial metabolic potential of hydrothermal vent chimneys along the Submarine Ring of Fire

“…These different habitats introduce a need for microbes to adapt quickly to an ever-changing environment which is supported by the enrichment of transposase genes and therefore an increased potential for horizontal gene transfer (46). Pagoda Chimney has the most taxonomic diversity of rbcLS genes, with a higher presence of archaeal rbcLS genes, likely due to the physical structure of the chimney allowing for many temperatures and chemical gradients (47). It has been shown that RuBisCO can also be used for nucleotide salvage rather than carbon fixation in archaea, which could explain the high abundance of archaeal rbcLS genes present (48).…”