Effects Of Oxidative Stress On The Physiological State Of Escherichia Coli And The Synthesis Of 5-Aminolevulinic Acid

5-Aminolevulinic acid(ALA)is an essential precursor for the synthesis of heme,chlorophyll and vitamin B12,as a high value-added compound,that is widely used in agriculture and medicine.In recent years,microbial fermentation has been carried out to synthesize ALA by using bacteria constructed via key enzyme mining and metabolic engineering of key pathways.However,few studies have focused on the toxicity of high concentration ALA on host bacteria and the response of bacteria to ALA.In this study,the effects of ALA on the physiological state of Escherichia coli,and cell tolerance response were comprehensively studied.The results as follows:Firstly,this study examined the effect of initial high concentration of ALA on the growth of E.coli.It was found that the initial growth rate of the cells decreased by 36%under the stress of 15 g/L ALA,the cell growth stopped completely upon the addition of 18 g/L ALA.Membrane integrity and cell morphology assays showed that 52.6%of the cell membranes were damaged to varying degrees and the morphology of the cells having more slender under 15 g/L ALA.The above results indicate that high concentration of ALA has high cytotoxicity against E.coli.Secondly,this study examined the production of intracellular reactive oxygen species(ROS)in E.coli under ALA stress and its role in ALA cytotoxicity.The results showed that the intracellular ROS of E.coli increased by 3.4-fold under 15 g/L ALA stress.After the addition of antioxidant glutathione,the intracellular ROS level decreased significantly,meanwhile the cell growth restored,indicating that ROS is the main cause of high concentration of ALA on E.coli cytotoxicity.Further,by adding catalase(CAT)and superoxide dismutase(SOD),it was found that intracellular ROS decreased by 44%and 62%,respectively,and cell growth was restored,indicating hydrogen peroxide(H2O2)and superoxide anion(O2·-)are the main ROS types produced by ALA.Third,this study examined the response of E.coli antioxidant system to ALA stress.The results showed that the enzyme activities of CAT and SOD in E.coli increased by 2.5-fold and 91%respectively under the condition of 15 g/L ALA.At the same time,the antioxidation defense system-deficient strains became more sensitive to ALA.Moreover,overexpressing CAT and SOD made the cells tolerate ALA at 30 g/L and 18 g/L,respectively.The results suggest that the E.coli antioxidant defense system plays an important role in ALA tolerance.Finally,the above strategy for enhancing the cellular antioxidant system was used for the construction of ALA producers.All strains overexpressing katG,katE,sodA,sodB and sodC genes,respectively,achieved higher ALA titer by 13%,65%,28%,38%and 36%,accordingly,in shake flask fermentation compared with the control strain.The results indicated that enhanced antioxidant system could effectively increase ALA production.In subsequent,fermentation of the best KatE and SodB overexpressing strains produced 9.6 g/L and 8.7 g/L ALA in a 5 L fermentor,which were 81%and 64%higher than the control strains,respectively.At the same time,the cell growth was also significantly improved(56%and 28%,respectively).Further co-expression of katE and sodB genes generated 11.5 g/L ALA in the 5 L fermentor,the highest yield by one-step ALA fermentation to our best knowledge,which was 1.2-fold higher than that of the original strain.In this study,it is clearly confirmed that the ROS produced by ALA seriously affects the physiology of E.coli,whereas the antioxidant defense system plays a key role in the ALA tolerance process in E.coli.The overexpression of antioxidant genes increased not only the ALA tolerance but also the ALA production capacity,which provides a novel strategy for designing and developing ALA hyperproducers.