Synthetic coculture of Clostridium carboxidivorans and engineered Clostridium kluyveri for medium chain alcohol production from CO rich syngas (SynCoClos)

在SynCoClos的第一个资助期，在可控搅拌槽反应器中成功建立了自养型C. carboxidivorans和链延长型C. kluyveri的合成共培养物，并连续CO/ co2气体排放。荧光原位杂交（FISH-FC）后流式细胞术测量单个细胞浓度已被证明是研究梭菌共培养过程中自养生产中链有机酸和醇的有价值的工具。C. kluyveri的CO敏感性似乎是与CO/ co2转换C. carboxidivorans共培养的限制因素。在CO浓度升高的情况下，C. kluyveri的生长受到强烈抑制，但细胞保持活力和代谢活性。低CO浓度下，克氏梭菌的生长得到促进；然而，在共培养过程中，C. carboxidivorans形成醇的能力降低，并且观察到C. carboxidivorans不断衰减。为了改善这种情况，将把注意力转向提高C. kluyveri的CO耐受性。为此，将在完全可控的搅拌槽生物反应器中使用自动适应实验室进化（ALE）来选择不受CO分压增加抑制的克卢威梭菌菌株。进化的C. kluyveri菌株的基因组测序将用于鉴定基因组中的突变。选定的突变将通过逆向工程进行测试。同时，合理构建表达醛醇联合脱氢酶（AdhE）的克卢韦氏C. kluyveri菌株将促进还原终产物的形成，也将提高对CO抗性增强的克隆的选择能力。选择基因共适应的克卢维梭菌菌株将与C. carboxydivorans在合成共培养中结合，以提高CO/CO2中链酒精的产量。群落通量平衡分析将用于确定C. carboxidivorans和C. kluyveri的有益比例，以及限制共培养过程中的代谢活动，以确保在合成共培养中CO/CO2最大限度地生成己醇。SynCoClos的工艺工程和代谢工程策略的协同和互动组合将使这种梭状菌共培养工艺的定量表征、科学理解和使用成为可能，从而可大规模地从CO/CO2中生产中链醇。改善克卢威梭菌的代谢分析和工程工作将由Wolfgang Liebl/Armin Ehrenreich， TUM， Freising微生物学小组进行。Garching TUM生物化学工程的Dirk west - botz小组将密切合作，共同培养梭菌和进化的以及改造的克卢韦梭菌的反应工程研究和共同培养过程的建模。

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.