Biosensor-based High-throughput Screening For Enhancing The Biosynthesis Of Adipic Acid

As a platform chemical,adipic acid has enormous application in various industries.To synthesize adipic acid via chemical catalysis has disadvantages such as low efficiency,severe pollution,etc.Therefore,the biosynthesis of adipic acid has drawn broad attention these years.The reverse adipate degradation pathway is an efficient metabolic pathway to synthesize adipic acid,but the metabolic burden induced by problems such as lengthy pathway and imbalance expression of the pathway genes could lead to genetic heterogeneity,thus limiting further increase of the titer of adipic acid.To overcome these challenges,four aspects of researches were conducted utilizing Escherichia coli K12 MG1655 as host strain:（1）The construction and optimization of a constitutive expression-based reverse adipate degradation pathway（RADP）.The reverse adipate degradation pathway was first expressed in Escherichia coli K12 MG1655.The soluble expression of the pathway enzymes was verified by SDS-PAGE analysis.Then,fermentation in shake-flask level utilizing modified MOPS medium was conducted and the results were analyzed by LC-MS,which revealed that 13.10mg·L^-1 adipic acid was synthesized.Regulation of the carbon flux was then conducted in order to further improve the titer of adipic acid.CRISPR-Cas9 system was employed to manipulate the genome of the host by which by-product formation-related genes ato B,pfl B,adh E,ldh A and pox B were deleted.Shake-flask fermentation of the resulting strain was then conducted and28.32 mg·L^-1 adipic acid was eventually obtained.（2）The construction and optimization of a transcription factor-based adipic acid biosensor.The adipic acid biosensor was constructed based on a transcriptional regulator Ben M.The host of the adipic acid biosensor was selected among Escherichia coli K12 MG1655,JM109 and BL21（DE3）through measuring the dynamic range of the biosensor in each strain,and Escherichia coli K12 MG1655 was eventually chosen as the host of the adipic acid biosensor.Starting from this strain,4 h was determined as the best detection time.Then,the culturing strategy of the adipic acid biosensor was optimized.The results revealed that the dynamic range reached 13.91-fold,which was 2.93-fold higher than that of the control group.The performance of the optimized adipic acid biosensor was eventually evaluated,which showed that the hill coefficient reached 0.9956 and the biosensor showed little response to adipic acid-like chemicals.（3）The construction and application of a high-throughput screening method based on adipic acid biosensor.The correlation between the fluorescence intensity and the titer of adipic acid was first evaluated to verify the feasibility of the application of adipic acid biosensor in a high-throughput screening method,the results showed that the correlation coefficient reached0.8968.Then,a high-throughput screening platform including transformation—well plate fermentation—shake flask fermentation was constructed.To verify the feasibility of the method,one round of screening was conducted.The results showed that the titer of adipic acid reached188.08 mg·L^-1,which was 14.36-fold higher than that of the control group.Next,to further improve the titer of adipic acid,the established high-throughput screening method was coupled with fermentation optimization,in which medium,carbon source,metal ion and precursor optimization was sequentially conducted.The results showed that the titer of adipic acid reached 531.88 mg·L^-1 under shake-flask level,which was 40.60-fold higher than that of the control group.（4）Optimization of expression profile of the pathway genes to enhance the titer of adipic acid.To start with,the RADP genes were first modularized.Then,the original promoter of each module was substituted by promoters with different intensities to regulate the expression level of the RADP genes in the host.The established adipic acid biosensor-based high-throughput screening method was utilized to screen each combination.The results showed that the best combination resulted in an adipic acid titer of 120.66 mg·L^-1 under shake-flask level,which achieved an 9.21-fold improvement comparing to the control group.In addition,it was found that the combination that overexpressed the module“paa J-paa H”showed relatively high adipic acid production,which indicated that these two enzymes might be vital in the reverse adipate degradation pathway.