Enhancing Butanol Tolerance Of Escherichia Coli Based On Molecular Chaperones And Its Mechanism

Escherichia coli is one of the most important platform microorganisms for industrial biotechnology,due to its fast growing,clear genetic background and a set of readily available molecular tools for genetic manupulation etc.However,the application of E.coli in the biosynthesis of valuable products has often been hindered by its poor organic-solvent tolerance.Enhancing the organic solvent tolerance of E.coli is important for the green and efficient biosynthesis.In this study,molecular charperone engineering was adopted to enhance the butanol tolerance of E.coli and its molecular mechanism was also investigated.Firstly,under butanol pressure,the transcriptional levels of key response regulating factors in five stress response systems were determined using RT-PCR.The result showed that Cpx(Conjugative plasmid expressiong)and Bae(Bacterial adaptative response)participated in the response to butanol stress.Key components(Spy and NlpE)of Cpx and Bae systems were overexpressed and knocked out.Organic-solvent tolerance test revealed that periplasmic chaperone Spy was beneficial for the butanol tolerance of E.coli,resulting 13.4% improvement in OD600(1.475)at 0.8%(v/v)butanol in comparison with the control strain.Inspired by the positive role of Spy in the butanol tolerace of E.coli,further study was carrieded out in deeper mining of more chaperones of E.coli.Thirty molecular chaperones was cloned and overexpressed in E.coli and their function in the butanol tolerance was also investigated.Under 0.8% butanol,ClpB,YcdY and SecB could enhance the butanol tolerance with improvement in OD600 of 25.1%,18.2% and 37.9%,respectively.SecB was identified to be the most effiecint charperone in butanol tolerance.Randaom mutagenesis was therefore perfomed on SecB.After high-throughput screening,a variant SecBT10 A was identified with highest butanol tolerance.Compared with SecB,the OD600 of Sec BT10 A was improved for 27.1%,44.8% and 37.0% under 0.8%,1.0% and 1.2% butanol stress.To get insight into the molecular mechanism of SecB and SecBT10 A in the butanol tolerance of E.coli,saturation mutagenesis on T10 was firstly conducted.The results revealed that substitution for hydrophobic amino acids at T10 could improve the butanol tolerance.Based on analysis of butanol concentration and glucose consumption rate,SecB,SecBT10 A and control strains showed that butanol content in medium was almost the same,and all three strains displayed similar glucose compution rate.Microbial adhension to hydrocarbons(MATH)test was also conducted to characterize the surface properties of three strains,and higher hydrophobicity was found for strain SecBT10 A.Tolerance test against different organic solvents indicated that SecBT10 A has better tolerance toward short chain alcohols than long chain alcohols and cyclic hydrocarbons.Isothermal titration calorimetry analysis showed that the equilibrium dissociation constant(KD)and stoichiometric number(N)of SecBT10 A and SecB strains in the binding of preMBP were 5.15 ?M and 61.70 ?M,3.08 and 4.99,respectively.The binding affinity of SecBT10 A toward substrate preMBP was 12.0 folds of SecB,which is beneficial for the efficient transportation of organic solvent related proteins(such as MalE)and enhanced organic-solvent tolance.In summary,the organic solvent tolerance of E.coli was improved through charperone engineering and the organic-solvent tolerance related mechanism was also investigated.This study provides guidance for the engineering of microbial organic-solvent tolerance and insight into the function of molecular chaperones in the organic solvent tolerance mechanisms.