Biosynthesis Of ?-aminoadipic Acid In Escherichia Coli

?-Aminoadipic acid,which is an unnatural amino acid and is widely used in medical and chemical fields.Today,however,it still depends on chemical synthesis to produce,there are problems of high cost,low yield and serious pollution.Therefore,it is of great significance to use economical,green and friendly biosynthesis method to produce it.Our research designed two biosynthesis pathways of ?-aminoadipic acid and the enzymes used in the two pathways were screened by measuring kinetics constant.Then the designed pathways were transferred into Escherichia coli for fermentation and achieved the biosynthesis of?-aminoadipic acid successfully.Finally,E.coli was modified by metabolic engineering to gradually increase the target product yield.The specific research was as follows:1,Both synthesis pathways included two steps to produce the target product.The first step used lysine as the substrate to generate?-aminoadipate-?-semialdehyde(AASA),and the second step used?-aminoadipate-?-semialdehyde dehydrogenase(AASADH)catalyzing AASA to produce ?-aminoadipic acid.Firstly,in the first step of the saccharopine pathway,the in vitro activity of lysine ketoglutarate reductase(LKR)and saccharopine dehydrogenase(SDH)derived both from Saccharomyces cerevisiae(Sc)and Streptomyces avermitilis(Sa)were determined.It was found that only ScLKR and ScSDH had activity.At the same time,in the first step of the transaminase pathway,the in vitro activity of lysine aminotransferase(LAT)derived from Rhodococcus erythropolis(Re)and Flavobacterium lutescens(F1)were detected.We found that the FlLAT had higher activity.The conversion rate could reach 17.47 ± 0.33 ?M/min.Then,it was found that both AASADH derived from Re and Pseudomonas putida(Pp)were active and ReAASADH was superior,with a conversion rate of 0.037±0.0013?M/min.2,Because the transaminase pathway was shorter and the enzyme activity was higher,it was chosen and FlLAT and ReAASADH were used.The transaminase pathway was designed in E.coli and lysine was added to the medium for fermentation.The target product was detected,which indicated that the pathway was constructed successfully.Then,the lysine catabolism genes of E.coli were knocked out to realize the synthesis of?-aminoadipic acid from glucose.Through module optimization of exogenous genes,the optimal yield could reach 96.34 mg/L.Then the nitrogen source in the fermentation medium was optimized.3,The combinations of upstream metabolic genes were screened and the expression level was optimized.As a result the yield increased to 152.90 mg/L.Then,introducing the ddh gene derived from Corynebacterium glutamicum to replace the four genes of dapD,dapC,dapE and dapF in E.coli shortened the metabolic pathway and increased the yield to 184.26 mg/L.During the product detection process,a certain amount of lysine was accumulated in the fermentation broth.After knocking out the gene lysE encoding lysine efflux,the yield reached 300.39 mg/L.Using further modified strain(replacement promoter,etc.)for fermentation,the yield reached 1006.30 mg/L.Finally,the ddh gene was integrated into the genome of the transformed strain of fermentation,and the final yield could reach 1328.23 mg/L.