Engineered Bacterial Biofilm Formation Enhancing Phenol Removal And Cell Tolerance

A microbial biofilm consisting of an aggregation of microbes embedded in extracellular polymeric substances matrix,can provide microbial resistances to toxic chemicals and harsh environments.Phenol is a toxic environmental pollutant and a typical lignin-derived phenolic inhibitor.In this study,we genetically engineered Escherichia coli cells by expression of diguanylate cyclases(DGCs)to promote proteinaceous and aliphatic biofloc substance formation.Compared to the planktonic E.coli cells,the biofloc-forming cells improved phenol degradation rate by up to 2.2 folds,due to their substantially improved tolerance(up to 141%)to phenol and slightly enhanced biological activity(20%)of phenol hydroxylase(Phe H).The engineered bioflocs also improved E.coli tolerance to other toxic compounds such as furfural,5-hydroxymethylfurfural,and guaiacol.Additionally,the strategy of engineered biofloc formation was applicable to Pseudomonas putida and enhanced its tolerance to phenol.CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated)is a defense system of bacteria and archaea against foreign nucleic acid invasion.d Cas9(dead Cas9)and dd Cpf1(DNase-dead Cpf1)effector proteins that have lost DNA cleaving activity but left binding activity,can be applied for transcriptional regulation.Gp2 protein binds to RNA polymerase and further reduces the affinity of RNA polymerase to DNA,resulting in reduction of the relative transcription.The omega subunit mutant(I13N)of RNA polymerase(Ome R)is reported to enhance transcription by recruiting RNA polymerase.In this study,to dynamically regulate DGC expression for promoting phenol biodegradation,we constructed Cas-Gp2 and Cas-Ome R complexes by fusion of d Cas(d Cas9 or dd Cpf1)with Gp2 and Ome R,respectively.The complexes were tested for transcription regulation,but the results showed the repression or activation has little changes compared to controls.This study highlights a strategy to form engineered bioflocs for improved cell tolerance and removal of toxic compounds,enabling its universality of use in bioproduction and bioremediation.