Sulfur reduction coupling with anaerobic ammonium oxidation power the origin of life

The geochemical energy that drives the transition from non-life to life is as yet unknown. We found that sulfurous species reduction coupling with anaerobic ammonium oxidation (Sammox), could provide the primordial redox equivalents and energy for the reductive acetyl-CoA pathway combined with incomplete reductive tricarboxylic acid (rTCA) cycle and reductive amination. Fe-S mineral/metal alloy catalysis and thiols/thioesters as energy couplers could enhance the efficiency of Sammox-powered proto-anabolic networks. Genomic analysis of Sammox bacterium Ralstonia thioammoniphilus GX3-BWBA implied that this ancient metabolism in modern microbes should contain two stages according to ammonium transformation, -oxidation of ammonium to nitrite and denitrification. The incomplete rTCA cycle and reductive acetyl-CoA pathway were all identified in R. thioammoniphilus metabolic networks, which were responsible for chemolithotrophic metabolism. Sammox drove the coupling of the biochemical transformation of C, H, O, N, S, and/or Fe simultaneously in Hadean alkaline hydrothermal systems, thereby permitting the emergence of life. The results bridged the gap in the transition from geochemistry to biochemistry.


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The transition from non-life to life occurred in the context of geochemical energy 3 9 derived from element-coupled transformation 1-3 . The chemistry of life is based on 4 0 redox reactions, that is, successive transfers of electrons and protons from the six 4 1 major elements, i.e., C, H, O, N, S, and P 1,4 . The H 2 /CO 2 redox couple, which can 4 2 occur in submarine hydrothermal vents, has been proposed to drive the reductive 4 3 acetyl-CoA pathway, an ancient metabolic route 3,5-7 . Nevertheless, the primordial 4 4 energy source of the H 2 /CO 2 redox couple suffers from the difficulty that the anabolic biochemical pathway whose origins are proposed to trace back to 5 1 geochemistry 9,11,12 .

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Native iron/metals were recently shown to promote the reductive acetyl-CoA 5 3 pathway and rTCA cycle and strongly support the feasibility of these two primordial 5 4 synthetic pathways 13,14 , although they were generally considered to be rare near the 5 5 Earth's surface 13 and cannot support the long elemental transition from geochemistry pathways are therefore more logical 3,15-18 . However, the initial driving force for the 5 9 rise of proto-anabolic networks is still unclear. Moreover, the roles of N and S 6 0 . CC-BY-NC-ND 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 5, 2018. Duve's thioester world and the boom of S biochemistry. As a result, the S isotope nitrite also showed a slight increase during the ammonium oxidation process (Fig. 5d).

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The concentrations of sulfurous species and ammonium showed no significant Notably, quite a few iron oxides could be abiotically reduced by sulfide 2 0 7 generated from Sammox in an iron-rich environment, then an illusion of ferric iron including copper-containing nitrite reductase (nirK), nitric oxide reductase (norB), and nitrous oxide reductase (nosZ) (Fig. 6a). This result confirmed that nitrite was the pathway which is reduction of nitrite to dinitrogen (Eq. 5).
We did not find any other anaerobic ammonium oxidation-related functional 2 2 9 genes in the genome of R. thioammoniphilus (Fig. 6a). Thus, the ammonium oxidation 2 3 0 in Sammox was different from that in complete ammonium oxidation (Comammox) 2 3 1 and anaerobic ammonium oxidation (Anammox) 26 .

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Our main concern is how ammonium was oxidized by thiosulfate/sulfate into Sammox, we predicted that ferredoxin oxidoreductase (e.g., nitrite/sulfite reductase 2 3 6 . CC-BY-NC-ND 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 5, 2018. coupling with ammonium oxidation to nitrite (Eqs. 3-6, Extended Data Schematic 1).

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Here, thiosulfate should be first transformed into sulfite, which could be reduced by Sammox reaction were used to drive nitrite reduction to dinitrogen. As a highly 2 4 4 exergonic reaction, energy released from nitrite reduction drove CO 2 fixation. to the incorporation of P 19 .

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As we have inferred that the transition of geochemistry to biochemistry of S and 2 5 8 . CC-BY-NC-ND 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 5, 2018. . https://doi.org/10.1101/461707 doi: bioRxiv preprint N should start at the same starting point. Sulfite and nitrite reductases belong to the 2 5 9 only class of enzymes that share a common architecture as well as a requirement for a 2 6 0 siroheme cofactor 42 . The phylogenetic tree indicated that the putative multifunctional 2 6 1 sulfite/nitrite reductase of archaea and eukaryota gathered into one and three clusters, and nitrite 42 . This phenomenon suggested that S and N biochemistry may have a 2 6 8 common evolutionary origin derived from Sammox.

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Similar to the sulfite/nitrite reductase, some biomolecules as remnants of and archaea (Extended Data proposed to have an assimilatory role using dithiol dihydrolipoate as the sulfur in iron-sulfur proteins, such as ferredoxin 43,44 . This implies that rhodanese may be the 2 8 0 . CC-BY-NC-ND 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 5, 2018. . https://doi.org/10.1101/461707 doi: bioRxiv preprint primary mechanism for the formation of the iron-sulfur center of primordial enzymes 2 8 1 to catalyze proto-anabolic networks in LUCA, thus indicating its ancient nature.

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Rhodanese can also catalyze a sulfur dissimilatory metabolic reaction which is the first stage of Sammox.
Scheme 1 2 8 7 The structural similarity between rhodanese and Cdc25 phosphatases indicated phosphatases may originate from the rhodanese family because P incorporation 2 9 0 occurred later than S incorporation in metabolic networks. This evidence implied the 2 9 1 relationship between S and P metabolic evolution.

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Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession 5 2 5 QMKS00000000. The version described in this paper is version QMKS01000000.

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Protein sequences will be downloaded from NCBI, and get sequences in the  The best hit abstracted using Blast alignment tool for function annotation. Seven Easyfig 54 .

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General procedure for prebiotic Sammox-driven CO 2 fixation 5 5 1 Each 50 ml basal solution was transferred into 60 ml serum bottles and sealed and cooled as room temperature after washed by He gas (purity=99.999%). Additional trace elemental mixture solutions were filter sterilized or autoclaved individually. was added into serum bottle. The mixed solution was termed as Sammox reaction 5 5 7 system.

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The above mentioned stock solutions or aliquots were aseptically added to the organic products.

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This study was performed using a series of experiments: (I) 3.0 mM thiosulfate + 5 6 6 . CC-BY-NC-ND 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 5, 2018. General procedure for thiol/thioester promoted Sammox-driven CO 2 fixation.

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A solution of methanethiol was added to Sammox reaction system. The reaction to room temperature before derivatization and gas chromatography-mass 5 7 6 spectrometry analysis.

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This study was performed using a series of experiments: (I) 3.0 mM thiosulfate +  We design this experimental set to verify if Sammox-driven reductive general procedure is the same as above. Pyruvate (1.0 mM, final concentration) was 5 8 7 added into Sammox reaction system as substrate. Serum bottles were heated at 100 o C 5 8 8 . CC-BY-NC-ND 4.0 International license not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was this version posted November 5, 2018. . https://doi.org/10.1101/461707 doi: bioRxiv preprint