Study On D-Glucaric Acid Biosynthesis Based On Metabolic Engineering

D-Glucaric acid?GA?,as a promising platform chemical,has been widely used in the field of food,pharmaceutical and chemical industries.The biosynthesis pathway of value-added GA has attracted increasing attention owing to its high efficiency,low cost and environmently friendiness when compared to the chemical oxidation method.However,the biosynthesis of GA was less explored,which limited its industrial application to some extent.Therefore,this dissertation aims to study the biosynthesis pathway of GA based on metabolic engineering and provide the theoretical basis for its industrial application.The main results of this dissertation are as follows:Firstly,we constructed a one-step biosynthesis pathway of GA from D-glucuronic acid?GlcA?by uronate dehydrogenases?UDHs?,which were obtained from different Agrobacterium tumefaciens strains?Agrobacterium tumefaciens?At?,AtLBA4404,AtGV3101,AtEHA105?.All of these UDHs showed their optimal activity at the conditions of pH 8.0,30 ^oC.Moreover,the UDH from AtLBA4404 showed the best catalytic efficiency than other two UDHs(800 vs 600 and 530 s^-1 mM^-1).To further boost the catalytic performance of the UDH from AtLBA4404,site-directed mutagenesis based on semi-rational design was carried out here.The A39P/H99Y/H234K triple mutant obtained showed a 400-fold improvement in half-life at 59^oC,a 5^oC improvement in value and a2.5-fold improvement in specific activity at 30^oC compared to wild-type UDH.Under optimal conditions,the yield of GA by the strain(Escherichia coli?E.coli?/pET-udh^{A39P/H99Y/H234K}-nox)was approximately 98%afer 180 min at a substrate concentration of 100 mM D-glucuronic acid?GlcA?.This strategy was pretty efficient in improving the yield of GA.However,its application was limited due to the drawback of the high cost of the raw material.Hence,it is essential to explore other ways to solve this problem.Secondly,to address the above challenge,an engineered E.coli for producing GA from D-glucose was investigated.Herein,we obtained an engineering strain?GA17?with a good performance by redirecting metabolic flux into the GA biosynthetic pathways,blocking the conversion pathways of GlcA and GA into by-products,introducing an in situ NAD⁺regeneration system and fine-tuning the activity of the key enzyme,myo-inositol oxygenase?Miox?.Subsequently,the optimization works about the culture medium and process conditions?temperature,pH,substrate type and dissolved oxygen level?were performed to improve the performance of the strain GA17.The yield of GA under the optimal conditions was 46%?50 mM D-glucose?.This approach could absolutely decrease the cost for the production of GA by using the renewable and low-cost D-glucose as the raw material.However,the yield was lower than that of the one-step method,and this problem could thus promote us for exploring other efficient approaches to get high yield of GA by utilizing low-cost materials.Lastly,to further realize the low-cost industrial production of GA,we developed the cell-free biocatalysis systems which could efficiently convert sucrose to GA in a single vessel.The cell-free biocatalysis systems containing seven enzymes?sucrose phosphorylase,phosphoglucomutase,myo-inositol 1-phosphate synthase,myo-inositol monophosphatase,myo-inositol oxygenase,uronate dehydrogenase and NADH oxidase?do not need the supplementation of ATP or NAD⁺through the whole process.In addition,the reaction conditions?temperature,pH,NAD⁺,buffer,enzyme composition,including fed-batch addition of myo-inositol oxygenase?were optimized to improve the synthesis performance of this cell-free biocatalysis systems.A relatively high yield of GA?75%?was obtained by using the cost-effective sucrose under the optimal conditions,and 50 mM of sucrose could be converted to 34.8 mM of GA.To our best knowledge,it is also the first study on the synthesis of GA by employing the cell-free biocatalysis systems.In summary,the feasibility of three different strategies for the production of GA was investigated.Based on the results of the first two approaches,a novel and high-efficient biosynthetic pathway of GA by using low-cost materials was put forward.This study could not only provide new ideas for explorating suitable strategies for the producing of GA in an efficient way,but also promote the appliciton of the metabolic engineering technique in the field of value-added chemicals.