Direct cell-to-cell exchange of matter in synthetic clostridium syntrophies enabling CO2 fixation and an expanded metabolic space.

In microbial fermentations to produce metabolites, at least 33% of the sugar-substrate carbon is lost as CO2 during pyruvate decarboxylation to acetyl-CoA. Previous attempts to reduce this carbon loss focused on engineering a single organism. In nature, microorganisms live in complex communities where syntrophic interactions result in superior resource utilization. Microbial communities are ubiquitous in nature and have a wide range of applications, including production of biofuels and chemicals. Syntrophic and other microbial co-cultures/consortia carry out efficient bio-transformations that are the result of multiple complementary metabolic systems working together. It is now well appreciated that the capabilities of multi-microorganism systems cannot be predicted by the sum of their parts. Rather, synergistic interactions at different levels often result in better overall performance of these systems. Importantly, integration of diverse metabolic systems through syntrophic dependencies make co-culture systems robust to environmental fluctuations. Clostridium organisms are of major importance for developing new technologies to produce biofuels and chemicals. Three major types of Clostridium organisms have been the focus of studies for the sustainable production of fuels and chemicals. Solventogenic clostridia utilize a large variety of biomass-derived carbohydrates (hexoses, pentoses, disaccharides, and hemicellulose), and can produce C2-C4 chemicals. Acetogenic clostridia can fix inorganic H2, CO2, and CO to generate C2 acids and alcohols. Other specialized clostridia possess diverse biosynthetic capabilities for production of a wide variety of metabolites including C4 – C8 carboxylic acids and alcohols, which could serve as commodity chemicals, biofuels, or biofuel precursors. Here, we first examined a synthetic syntrophy consisting of the solventogen Clostridium acetobutylicum, which converts simple and complex carbohydrates into a variety of chemicals, and the acetogen C. ljungdahlii, which fixes CO2. This synthetic co-culture achieved carbon recoveries into C2-C4 alcohols almost to the limit of substrate-electron availability, with minimal H2 and CO2 release. The syntrophic co-culture produced robust metabolic outcomes over a broad range of starting population ratios of the two organisms. Significantly, the co-culture exhibited unique direct cell-to-cell interactions and material exchange among the two microbes, which enabled unforeseen rearrangements in the metabolism of the individual species that resulted in the production of non-native metabolites, namely isopropanol and 2,3-butanediol. Next, we expanded this co-culture system to include C. kluyveri, which can metabolite ethanol and acetate to produce C6 and C8 carboxylic acids. Both C. acetobutylicum and C. ljungdahlii produce ethanol and acetate, which makes C. kluyveri and ideal partner for a triple synthetic co-culture system capable to converting biomass-derived carbohydrates to C6 and C8 chemicals. Supported by the National Science Foundation through the US Army Research Office (ARO; Award No. W911NF-17-1-0343) and the US Department of Energy (DOE; Award No. DE-SC0019155)

DNA transfer between two different species mediated by heterologous cell fusion in Clostridium coculture

ABSTRACTProkaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns

Morphology-Induced Defects Enhance Lipid Transfer Rates

Molecular transfer between nanoparticles has been considered to have important implications regarding nanoparticle stability. Recently, the interparticle spontaneous lipid transfer rate constant for discoidal bicelles was found to be very different from spherical, unilamellar vesicles (ULVs). Here, we investigate the mechanism responsible for this discrepancy. Analysis of the data indicates that lipid transfer is entropically favorable, but enthalpically unfavorable with an activation energy that is independent of bicelle size and long- to short-chain lipid molar ratio. Moreover, molecular dynamics simulations reveal a lower lipid dissociation energy cost in the vicinity of interfaces (“defects”) induced by the segregation of the long- and short-chain lipids in bicelles; these defects are not present in ULVs. Taken together, these results suggest that the enhanced lipid transfer observed in bicelles arises from interfacial defects as a result of the hydrophobic mismatch between the long- and short-chain lipid species. Finally, the observed lipid transfer rate is found to be independent of nanoparticle stability

Effects of Nanoparticle Morphology and Acyl Chain Length on Spontaneous Lipid Transfer Rates

We report on studies of lipid transfer rates between different morphology nanoparticles and lipids with different length acyl chains. The lipid transfer rate of dimyristoylphosphatidylcholine (di-C₁₄, DMPC) in discoidal “bicelles” (0.156 h^–1) is 2 orders of magnitude greater than that of DMPC vesicles (ULVs) (1.1 × 10^–3 h^–1). For both bicellar and ULV morphologies, increasing the acyl chain length by two carbons [going from di-C₁₄ DMPC to di-C₁₆, dipalmitoylphosphatidylcholine (DPPC)] causes lipid transfer rates to decrease by more than 2 orders of magnitude. Results from small angle neutron scattering (SANS), differential scanning calorimetry (DSC), and fluorescence correlation spectroscopy (FCS) are in good agreement. The present studies highlight the importance of lipid dynamic processes taking place in different morphology biomimetic membranes