Metabolism Perturbation Of Co-factor In Clostridium Acetobutylicum

Clostridial ABE (Acetone-Butanol-Ethanol) fermentation has a history stretching back more than100years, ranking second in scale to yeast ethanol fermentation. However its development encountered many setbacks, the most important reason is that ABE fermentation is affected by the fluctuation of petroleum price. When oil price goes down, ABE fermentation would lose its competence with fossil based chemicals. So increasing native competence of ABE fermentation is the important way to get rid of the petroleum obsession and to realize sustainable development. Metabolic engineering is an effective way to improve strain physiological metabolic performance. Co-factor as an important metabolic model was selected in this work as engineered target to improve the metabolic performance of Clostridium acetobutylicum.The effect of metabolic perturbation of co-factor on ABE fermentation of Clostridium acetobutylicum was studied in this dissertation, which includes three parts:(1) Introducing energy perturbation, shortening fermentation period and increasing solvent productivity. The F1part of F0F1-ATPase could hydrolyze ATP into ADP, thus the native F1-ATPase of C. acetobutylicum was overexpressed, which led to reduced cell energy state. This stimulated the cell growth and glucose consumption and shortened the fermentation period by10h. The increased ATP turnover rate improved the solvent productivity, titer and yield by76%,15%and4.2%. Metabolic flux analysis indicated that solventogensis in C. acetobutylicum was brought forward. It means reduced cell energy state would be a signal to trigger the shift from acidogenesis to solventogenesis.(2) Introducing redox requivalent perturbation, switching the classical ABE fermentation into high valued IBE fermentation completely. There are three solvents in the ABE fermentation:butanol, acetone and ethanol. In this work, a NADPH dependent secondary alcohol dehydrogenase was expressed, which led to completely conversion of acetone to higher valued isopropanol. The engineered strain could produce about24g/1mixed alcohol including15g/1butanol,7.6g/1isopropanol and1.28g/1ethanol with yield of31.4%to glucose after48h batch fermentation. In other hand, this work suggested that the pathway from pyruvate to butyryl-CoA was more robust than the pathway from acyl-CoA to alcohol when introducing redox requivalent perturbation. It indicated that inreasing the robustness of aldehyde/alcohol dehydrogenases of the pathway from CoA to alcohol may be help for butanol production.(3) Elucidating the effect on alcohol production of inactivated perturbation of redox equivalent dependent aldehyde/alcohol dehydrogenases in C. acetobutylicum, paving the way for enhanced butanol metabolic flux and improved butanol selectivity. Butanol has highest price compared with the other products in ABE fermentation, improving its selectivity would be an important way to enhance the economy of ABE fermentation. There are several NADH dependent aldehyde/alcohol dehydrogenases in C. acetobutylicum (AdhEl, AdhE2, BdhA, BdhB, Cac3392, Cap0059), they have different substrate specificity and expression order. The distribution of reducing power among these dehydrogenases will affect the butanol ratio in total solvent. In this study, we disrupted these genes by ClosTron system based on group II intron insertion. Fermentation evaluation on butanol selectivity was carried on. The results showed that adhel and bdhB genes were important for alcohol production in C. acetobutylicum especially adhel gene which inactivated mutant amolst lost the ability for alcohol production. However, the alcohol production performance of adhe2gene mutant showed significant improvement. The butanol titer could get16.3g/1, increased by12.5%, ethanol titer decresed by7%, the butanol/ethanol ratio increased to8.15:1from6.65:1of wild type strain. The butanol production of bdhA gene mutant strain has no significant difference compared with wild type strain, but its ethanol titer increased42.3%, which led to lower butanol/ethanol ratio (5.6:1). As to gene cac3392, the butanol production of this mutant had no remark difference with that of wild type strain, but ethanol titer was decresed by32.6%, which led to higher butanol/ethanol ratio (10.2:1). The interesting finding was the mutant of gene cap0059which produced higher butanol and lower ethanol, the butanol/ethanol ratio got to10.6:1. This work paved the way for increasing butanol metabolic flux, selectivity and homo-butanol fermentation in C. acetobutylicum.Energy and redox equivalent metabolic perturbation was introduced by co-factor engineering in C. acetobutylicum in this work. Shortened fermentation period and higher solvent productivity was achieved by energy perturbation, ABE fermentation was completely converted to higher valued IBE fermentation by single redox requivalent perturbation, the elucidation of aldehyde/alcohol dehydrogenases paved the way for higher butanol selectivity. All these results would contribute the competence of clostridial ABE fermentation and put an alternative way for sustainable and stable development of ABE fermentation.