Model-driven approach for the production of butyrate from CO₂/H₂ by a novel co-culture of <i>C. autoethanogenum</i> and <i>C. beijerinckii</i>

One-carbon (C1) compounds are promising feedstocks for the sustainable production of commodity chemicals. CO(2) is a particularly advantageous C1-feedstock since it is an unwanted industrial off-gas that can be converted into valuable products while reducing its atmospheric levels. Acetogens are microorganisms that can grow on CO(2)/H(2) gas mixtures and syngas converting these substrates into ethanol and acetate. Co-cultivation of acetogens with other microbial species that can further process such products, can expand the variety of products to, for example, medium chain fatty acids (MCFA) and longer chain alcohols. Solventogens are microorganisms known to produce MCFA and alcohols via the acetone-butanol-ethanol (ABE) fermentation in which acetate is a key metabolite. Thus, co-cultivation of an acetogen and a solventogen in a consortium provides a potential platform to produce valuable chemicals from CO(2). In this study, metabolic modeling was implemented to design a new co-culture of an acetogen and a solventogen to produce butyrate from CO(2)/H(2) mixtures. The model-driven approach suggested the ability of the studied solventogenic species to grow on lactate/glycerol with acetate as co-substrate. This ability was confirmed experimentally by cultivation of Clostridium beijerinckii on these substrates in batch serum bottles and subsequently in pH-controlled bioreactors. Community modeling also suggested that a novel microbial consortium consisting of the acetogen Clostridium autoethanogenum, and the solventogen C. beijerinckii would be feasible and stable. On the basis of this prediction, a co-culture was experimentally established. C. autoethanogenum grew on CO(2)/H(2) producing acetate and traces of ethanol. Acetate was in turn, consumed by C. beijerinckii together with lactate, producing butyrate. These results show that community modeling of metabolism is a valuable tool to guide the design of microbial consortia for the tailored production of chemicals from renewable resources

Selective enrichment and identification of cross-linked peptides to study 3-D structures of protein complexes by mass spectrometry

Chemical cross-linking of protein complexes combined with mass spectrometry is a powerful approach to obtain 3-D structural information by revealing amino residues that are in close spatial proximity. To increase the efficiency of mass spectrometric analysis, we have demonstrated the selective enrichment of cross-linked peptides from the 350 kDa protein complex RNA polymerase (RNAP) from Bacillus subtilis. Bis(succinimidyl)-3-azidomethyl glutarate was used as a cross-linker along with an azide-reactive cyclooctyne-conjugated resin to capture target peptides. Subsequently released peptides were fractionated by strong cation exchange chromatography and subjected to LC-MS/MS. We mapped 10 different intersubunit and 24 intrasubunit cross-links by xComb database searching supplied with stringent criteria for confirmation of the proposed structure of candidate cross-linked peptides. The cross-links fit into a homology model of RNAP. Cross-links between β lobe 1 and the β′ downstream jaw, and cross-links involving the N-terminal and C-terminal parts of the α subunits suggest conformational flexibility. The analytical strategy presented here can be applied to map protein–protein interactions at the amino acid level in biological assemblies of similar complexity. Our approach enables the exploration of alternative peptide fragmentation techniques that may further facilitate cross-link analysis