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

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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.

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Metagenomics: application of genomics to uncultured microorganisms.

Jo Handelsman (2004)

Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na(+)(Li(+))/H(+) antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

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A synthetic pathway for the fixation of carbon dioxide in vitro.

Thomas Schwander, Lennart von Borzyskowski, Simon Burgener … (2016)

Carbon dioxide (CO2) is an important carbon feedstock for a future green economy. This requires the development of efficient strategies for its conversion into multicarbon compounds. We describe a synthetic cycle for the continuous fixation of CO2 in vitro. The crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network of 17 enzymes that converts CO2 into organic molecules at a rate of 5 nanomoles of CO2 per minute per milligram of protein. The CETCH cycle was drafted by metabolic retrosynthesis, established with enzymes originating from nine different organisms of all three domains of life, and optimized in several rounds by enzyme engineering and metabolic proofreading. The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2 fixation pathways, thereby opening the way for in vitro and in vivo applications.

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Beyond the Calvin cycle: autotrophic carbon fixation in the ocean.

Michael Hügler, Stefan Sievert (2010)

Organisms capable of autotrophic metabolism assimilate inorganic carbon into organic carbon. They form an integral part of ecosystems by making an otherwise unavailable form of carbon available to other organisms, a central component of the global carbon cycle. For many years, the doctrine prevailed that the Calvin-Benson-Bassham (CBB) cycle is the only biochemical autotrophic CO2 fixation pathway of significance in the ocean. However, ecological, biochemical, and genomic studies carried out over the last decade have not only elucidated new pathways but also shown that autotrophic carbon fixation via pathways other than the CBB cycle can be significant. This has ramifications for our understanding of the carbon cycle and energy flow in the ocean. Here, we review the recent discoveries in the field of autotrophic carbon fixation, including the biochemistry and evolution of the different pathways, as well as their ecological relevance in various oceanic ecosystems.