Metabolic Engineering And Fermentation Process Optimization Of L-Leucine Producing Strain

L-leucine,a branched chain amino acid,is one of the eight essential amino acids that cannot be synthesized in mammals.Its applications range from animal feed additives,to ingredients in cosmetics,and specialty nutrients in pharmaceutical or medical preparations.Microbial fermentation is the main method for industrial production of L-leucine.The breeding of strains and the control of fermentation process are the core sections in the production of L-leucine.With the deep understanding of L-leucine synthesis pathway and feedback mechanism,rational metabolic engineering techniques have been widely used in the development of high-performance L-leucine producing strains.However,there are few reports about the application of L-leucine engineering strains to industrial production,and most of them remain in the experimental stage.In fact,there are a lot of problems to be solved from experiment to production.Therefore,in this study,a strain of industrial L-leucine production Corynebacterium glutamicum CP was taken as the chassis strain,and the metabolic engineering of C.glutamicum CP toward L-leucine was carried out,and then the effects of various fermentation processes on the production of L-leucine were studied.Finally,the pilot scale-up of L-leucine production and the separation and purification of L-leucine were studied.The main research contents and results are as follows:（1）Metabolic engineering was employed on the producing strain,which improved the substrate utilization ratio and reduced the production of by-products.Pyruvate and acetyl-Co A are the main precursors for the synthesis of L-leucine.The mutant phosphoketolase（PKT）from B.adolescentis,whose transcriptional expression was optimized,was introduced into the engineered strain,and the availability to produce acetyl-Co A from glucose was increased.And genes of the competitive metabolic pathway were weakened and deleted.The production of L-leucine was increased,and the byproduct L-alanine was decreased.The strain C.glutamicum CP04(Δltb R::P_tuf-fxpkΔP_{glt A}::P_{dap A})was obtained and L-leucine titer was 25.2 g/L by flask fermentation.（2）The effects of various fermentation processes on the production of L-leucine by C.glutamicum CP04 were studied.When the concentration of biotin in the medium was 50μg/L and the inoculation size was 20%under fed-batch conditions,the L-leucine titer was 53.0 g/L and the by-product L-alanine titer was 6.4 g/L.In addition,the ultrasound-assisted fermentation process was employed to produce L-leucine,and the optimal combination of ultrasonic parameters was obtained as follows:ultrasonic power density of 94 W/L,ultrasonic frequency of 25 k Hz,ultrasonic interval of 31 min and ultrasonic duration of 37 s.The titer of L-leucine was 64.1 g/L,which was 20.9%higher than that by fed-batch fermentation.Under the ultrasound treatment,there was no significant difference in cell morphology,but the membrane permeability and enzyme activity were significantly improved.In order to solve the problems of fed-batch fermentation,process intensifications,such as cell-recycling fermentation,chemostat culture,were adopted to cultivate C.glutamicum CP04 to produce L-leucine.When the cell-recycling repeated batch fermentation was employed,the L-leucine titer was 26.5 g/L,and the L-alanine titers were 0.87 g/L.The yield and productivity were increased by 16.2%and 48.3%,respectively.L-leucine titer reached 21.3 g/L at the steady state,and the titer of L-alanine was 0.5 g/L by the cell-recycling continuous fermentation.The yield and productivity were increased by 18.3%and 60.8%,respectively.When the dilution rate of the chemostat culture was 0.04 h^-1,the concentration of ammonium acetate was 1.2 g/L,the titers of L-leucine and L-alanine were 24.8 g/L and0.8 g/L,respectively,and the yield and productivity were increased by 10.2%and 35.0%,respectively.After comprehensive consideration,cell-recycling repeated batch fermentation were selected for the scaling up study.（3）Under the optimized conditions of scale down fermentation process,the pilot scale fermentation process was studied at the level of 5 m³ fermenter.Compared with the small scale,the production of L-leucine decreased and the byproduct of L-alanine was more in the pilot scale.The mixing efficiency was improved by replacing the straight-blade agitator with the oblique-blade agitator,and the foam was reduced.The biomass,the titer of L-leucine and the yield increased 4.5%,12.0%and 8.2%,respectively,and the titer of the by-product L-alanine was 6.3 g/L.The cell-recycling repeated batch fermentation was employed to produce L-leucine in 5 m³ fermenter.The results showed that the average titers of L-leucine and L-alanine was 24.4 g/L and 0.9 g/L,respectively.The productivity and yield were increased by45.4%and 5.6%,respectively.（4）The separation and purification processes of ion exchange chromatography and electrodialysis were compared.The ultrafiltration membrane was employed to filtrate fermentation broth without bacterial pellet.The average membrane flux was 24.5 L/m²/h.The decolorization rate of the filtrate was 58.6%,and the recovery was 87.4%.The recovery ratio of L-leucine was 91.2%by ion exchange chromatography.By optimizing the electrodialysis process,the concentration,recovery ratio and purity of L-leucine were 16.2g/L,95.6%and 96.3%,respectively.The minimum processing time of decolorization was determined to be 20 min,and the optimal treatment temperature is 55℃.The activated carbon required for ion exchange chromatography purification was 0.5 g/L,while electrodialysis purification needed only 0.3 g/L activated carbon.The recovery ratio and purity of L-leucine purified by ion exchange chromatography were 76.8%and 95.6%,respectively,while the recovery ratio and purity of L-leucine purified by electrodialysis were87.6%and 98.7%,respectively.