Construction Of E.coli Synthetic Trans 2-decenoic Acid Engineering Bacteria And Study On The Conditions Of Whole Cell Catalysis

As a type of fatty acid,?,?-unsaturated fatty acids can be further processed to produce various types of fine chemical products with good quality,which are widely used in food,medicine,spices and other industries Chemical platform products.At present,most of the synthesis of ?,?-unsaturated fatty acids adopts the chemical synthesis method.Among them,the synthesis process of medium-chain ?,?-unsaturated fatty acids has a certain bottleneck due to the low reaction activity.Saturated fatty acids are difficult to break throkh.Trans 2-decenoic acid,as a type of medium-chain ?,?-unsaturated fatty acid,can be used as an intermediate for important products such as neuropharmaceuticals,spices,and health products.Its application value is very wide,so further research on trans The efficient and green synthesis of 2-decenoic acid is of great significance for the further exploration of its application value.In this thesis,we designed based on the ?-oxidation pathway of E.coli fatty acids,modified key enzymes,and constructed a metabolic pathway for the biosynthesis of trans 2-decenoic acid.The metabolic pathway designed in this study uses decanoic acid as a raw material.After the decanoic acid is activated by the action of the ester acyl Co A synthase Fad D,the unsaturated double bond between specific carbon atoms is formed under the oxidation of the ester acyl Co A dehydrogenase Fad E.Finally,under the action of the thioesterase Ydi I,the coenzyme A of decenoyl Co A is removed,thereby releasing the target product trans 2-decenoic acid.The synthesis pathway designed in this study successfully expressed the three key enzymes of ester acyl-Co A synthetase Fad D,ester acyl-Co A dehydrogenase Fad E,and ester acyl-Co A thioesterase Ydi I,which ensured the expression of key enzymes in the ?-oxidation pathway while benefiting accumulation of trans decenoyl Co A,and the production of decenoic acid reach to 23 mg/L;at the same time,this study successfully knocked out the enoyl Co A hydratase Fad B,which effectively inhibited the consumption of product branch pathways and the formation of by-products;successfully knocked out the protein operon Fad R inhibits the regulation of local fatty acid degradation-related genes and promotes the process of ? oxidation.The production of this recombinant can reach to 39 mg/L.In the end,we successfully constructed a metabolic pathway for the biosynthesis of trans 2-decenoic acid,and realized the biosynthesis of trans 2-decenoic acid for the first time.On this basis,conditions were optimized to further increase the yield of the target product.After the optimization of the whole cell catalytic conditions,the yield showed an upward trend,and the final yield of trans 2-decenoic acid could reach 111 mg/L.The construction of this metabolic pathway provides a platform and theoretical basis for the subsequent synthesis of 10-HDA and ?,?-unsaturated fatty acids with different carbon chain lengths.