Project Details
Projekt
Synthetic coculture of Clostridium carboxidivorans and engineered Clostridium kluyveri for medium chain alcohol production from CO rich syngas (SynCoClos)
Subject Area
Biological Process Engineering
Term
since 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 427995829
Synthetic co-cultures of the autotrophic C. carboxidivorans and the chain-elongation C. kluyveri have been established successfully in controlled stirred-tank reactors with continuous CO/CO2-gassing in the first funding period of SynCoClos. The measurement of individual cell concentrations by flow cytometry after fluorescence in situ hybridization (FISH-FC) has been proven as a valuable tool for studying the clostridial co-culture processes for the autotrophic production of medium chain organic acids and alcohols. CO sensitivity of C. kluyveri seems to be the limiting factor in a co-culture with CO/CO2-converting C. carboxidivorans. Growth of C. kluyveri is strongly reduced at elevated CO concentrations but the cells remain viable and metabolically active. At low CO concentrations, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced and a constant decay of C. carboxidivorans was observed in the co-cultivation process.To improve this situation, attention will be turned to increasing the CO tolerance of C. kluyveri. To this end, automated Adaptive Laboratory Evolution (ALE) in fully controlled stirred-tank bioreactors will be used to select for C. kluyveri strains that are not inhibited by increased CO partial pressure. Genome sequencing of the evolved C. kluyveri strains will be applied to enable the identification of mutations in the genome. Selected mutations will be tested by reverse engineering. In parallel, rational construction of C. kluyveri strains expressing a combined aldehyde and alcohol dehydrogenase (AdhE) will enhance formation of reduced end products and should also result in an increased ability to select for clones with increased CO resistance. Selected genetically CO-adapted C. kluyveri strains will be combined with C. carboxydivorans in a synthetic co-culture to improve medium chain alcohol production from CO/CO2. Community flux balance analysis will be applied to identify beneficial ratios of C. carboxidivorans and CO-resistant C. kluyveri as well as limiting metabolic activities in the co-cultivation process to ensure maximum hexanol formation from CO/CO2 in the synthetic co-culture. The synergistic and interactive combination of process engineering and metabolic engineering strategies of SynCoClos will enable the quantitative characterization, scientific understanding and use of this clostridial co-cultivation process for the scalable production of medium chain alcohols from CO/CO2.Metabolic analysis and engineering work to improve Clostridium kluyveri will be performed by the group of Wolfgang Liebl/Armin Ehrenreich, Microbiology at TUM, Freising. Reaction engineering studies on co-cultivation of Clostridium carboxidivorans and evolved as well as engineered Clostridium kluyveri and modeling of co-cultivation processes will be performed in close cooperation by the group of Dirk Weuster-Botz, Biochemical Engineering at TUM, Garching.
DFG Programme
Priority Programmes
Co-Investigator
Dr. Armin Ehrenreich
