Molecular Mechanism Underlying The Self-Flocculation Of Zymomonas Mobilis ZM401

The self-flocculation of microbial cells is a unique phenotype with many advantages from the viewpoint of bioprocess engineering: Cells can be separated from culture through sedimentation instead of centrifugation for regular cells with intensive capital investment on centrifuges,thus this procedure saves energy;repeated batch fermentation can be performed to save non-productive time associated with the cleaning of the fermentation system and regular inoculation,since cells can separate through sedimentation after previous batch fermentation,and be reused as inoculum to start next batch operation quickly;when continuous fermentation is applied,self-flocculating cells can be self-immobilized within fermenters for high density to improve productivities of the fermenters.Zymomonas mobilis is an excellent ethanol-producer,which presents two significantly different morphologies: unicellular cells observed in the model strain ZM4 and others,and the self-flocculating phenotype presented by ZM401,a mutant that was developed from ZM4 through chemical mutation treated with nitroso-guanidine?NTG?,but molecular mechanism underlying its self-flocculation still remains unknown,which hinders the development of strategies for process optimization and control.Based on the analysis of possible substances for the self-flocculation of ZM401,we developed a strategy for de-flocculating the bacterial flocs using different hydrolytic enzymes that can hydrolyze starch,protein and cellulose specifically,through which cellulose was identified to be responsible for the unique morphology.Furthermore,we performed comparative genome analysis for ZM401 by comparing its genome with that of ZM4 to screen mutations potentially related to its cellulose biosynthesis,and detected a nucleotide deletion in a putative gene upstream of its cellulose synthase operon,leading to a frame-shift mutation,which disrupted not only the transcription terminator of the putative gene,but also the start codon of the bcsA for them to be fused together for a new gene bcsA₄01.Although such a mutation does not affect the major function of BcsA in cellulose biosynthesis,two more transmembrane helixes were predicted for BcsA₄01,which might improve its efficiency for cellulose synthesis and developed as fibrils for entanglement to ZM401.The key role of bcsA₄01 in cellulose synthesis for the self-flocculation of ZM401 was validated by its knockout and complementation.Since Z.mobilis possesses both type I and IV restriction and modification?R-M?systems,which substantially compromise the efficiency of its genetic modifications,we developed mutant strains with both type I and IV R-M systems deactivated,laying a technical foundation for genetic manipulations on the species through knockout and/or overexpression of genes to be studied.Cyclic diguanylate?c-di-GMP?is an important second messenger in regulating cellulose synthesis in bacteria.We confirmed the role of c-di-GMP in the self-flocculation of ZM401 through studies on the functions of genes related to its anabolism and catabolism.It was demonstrated that an increase of intracellular c-di-GMP level benefits the self-flocculation of Z.mobilis,on the other hand,decreasing the content of intracellular c-di-GMP prevents the bacterial cells from self-flocculating.RT-qPCR analysis for the expression of genes encoding the Bcs complex indicated that c-di-GMP may involve in the transcriptional regulation of this operon,and complementation of the gene with mutation on the c-di-GMP binding site could not endow ZM401?bcsA with the self-flocculating phonotype.Therefore,we speculate that cdi-GMP could regulate cellulose biosynthesis in Z.mobilis at post-translation leve for its selfflocculation,and underlying mechanism needs to be explored.Based on the aforementioned progress,we successfully engineered the non-flocculating ZM4 with the self-flocculating phenotype through the overexpression of bcsABC₄01 together with the deactivation of its native c-di-GMP degradating pathway,which would be a platform for engineering other bacterial species with the self-flocculating phenotype to explore its advantages for industrial applications.