Roles Of The Pleiotropic Regulator AbrB In Two-phase Clostridium Acetobutylicum Fermentation

Clostridium acetobutylicum is a gram-positive,spore-forming and strictly anaerobic bacteria,and also an important industrial strain that life cycle includes acidogenesis,transition state and solventogenesis.These three physiological processes connect each other closely and together determine the strain fermentation phenotype.The successful transition from acidogenesis to solventogenesis is extremely significant for the final solvent synthesis in C.acetobutylicum.AbrB is a pleiotropic regulator that regulates many cellular processes and three putative homologues of abrB were identified in C.acetobutylicum.The previous study have observed that AbrB1 may act as a regulator at the transition between acidogenic and solventogenic growth in C.acetobutylicum.However,the questions include whether the other two AbrBs also play a role in C.acetobutylicum and how these three AbrBs coordinate their functions are not clear.In this study,we first constructed lacZ gene reporter system to analyze whether any of the abrB homologues in C.acetobutylicum were transcriptionally active.Then,Targetron technique was used to disrupt abrB1 and abrB3 genes in C.acetobutylicum.At the same time,we used homologous recombination to knock out abrB2.The abrB1-inactivated mutant produced significantly higher level of acetate and butyrate but less level of solvents than the control strain,which demonstrated that transition from acidogenesis to solventogenesis suffered damage and re-assimilation of acids was disrupted.On the contrary,abrB3 disruption exhibited much better growth,sugar consumption,acid reassimilation and solvent production than the strain 824 WT.While AbrB2 might not be essential because of no change observed in abrB2-inactivated mutant.Comparative transcriptome analysis was then performed to explain the phenotypic effects from disrupting abrB at the molecular level.The result showed that disrupting abrB1 and abrB3 influenced transcription of most genes involved in acid and solvent formation as well as the two-phase transition.Notably,after disrupting abrB1,the transcriptional level of ctfA and ctfB being responsible for acid re-assimilation genes did not change,which might suggest that AbrB1 may control other regulatory factor to shift acidogenesis to solventogenesis indirectly.In addition,there was no change about transcriptional level of solvent related genes after abrB2 knock-out.In contrast,the transcriptional level of adhEI and adhEII genes was significantly increased,which might be a major factor for the improved solvent-forming capacity of the mutant 824AbrB3.Moreover,a homologous alignment of AbrB3 from C.acetobutylicum was generated to analyze the key amino acid residues of this regulator.A series of mutant were screened through site-direct and saturation mutagenesis of AbrB3.The result showed that a mutant at 23 site of arginine(R23)exhibited a best phenotype.When R23 mutanted to aspartate,the mutant showed the ratio of ethanol increased by 11.37%.It is a pity that the ratio of butanol was not improved because of the butanol toxicity.In summary,this study performed a detailed investigation to clarify the function of these three abrBs and phenotypic effects caused by knocking out abrBs in C.acetobutylicum.In addition,this work discerned the function distribution and relationship between them.This provides the foundation for further strain improvement through metabolic engineering.