Metabolic Engineering Of Yarrowia Lipolytica For GABA Production

?-Aminobutyric acid is a naturally occurring non-proteinogenic amino acid,which is an important inhibitory neurotransmitter in the mammalian central nervous system.It is widely distributed in the human cerebral cortex,hippocampus,thalamus,basal ganglia and cerebellum,and has a regulating effect on various functions of the body.About 30% of central nervous system synapses use GABA as the transmitter.GABA also has the functions of delaying brain aging,lowering blood pressure,promoting renal function improvement,and inhibiting fatty liver and obesity.In view of its various pharmacological effects,domestic and foreign scholars have a lot of studies on GABA.GABA is produced by chemical synthesis and biosynthesis.The chemical synthesis method is very polluting to the environment,and the reaction is accompanied by a large number of by-products.The biosynthesis method includes plant enrichment method and microbial synthesis method.The raw materials are safe and easy to obtain,but the yield is low,so we used the microbial synthesis method to produce chemicals.The use of microbial synthesis to produce chemicals is of great significance to the sustainable development of and the improvement of the quality life.In this study,we constructed a pathway for the synthesis of ?-aminobutyric acid in the TCA cycle of Yarrowia lipolytica.The main research contents and conclusions are as follows:(1)In order to construct the heterogenous synthetic pathway,the intermediate metabolite ?-ketoglutaric acid in the TCA cycle was utilized to catalyze ?-ketoglutaric acid to form glutamate by heterogenous expression of glutamate synthase(gltB),and then glutamate decarboxylase(gadA)was expressed to catalyze glutamate translate to ?-aminobutyric acid.Firtly,the glutamate synthase,glutamate decarboxylase genes from Escherichia coli K-12 were amplified by PCR,and these two genes were separately constructed into plasmid pINA1312.The recombinant plasmid were constructed by One Step Cloning Kit,namely pINA1312-gltB-gadA.The recombinant plasmid was ntroduced into Yarrowia lipolytica.The recombinant strain after transformation and screening was Po1f-gltB-gadA Po1f-gltB-gadA,and the cells were fermented for 108 hours under shake flask culture conditions.The ?-aminobutyric acid accumulated in the strain of express the glutamic acid synthetase and glutamate decarboxylase genes(Po1f-gltB-gadA)reached 0.74 g/L.(2)In order to further increase the yield of the product,we expressed the ?-aminobutyric acid transporter gene(gltB)based on the Po1f-gltB-gadA strain.The glutamate synthase,glutamate decarboxylase and?-aminobutyric acid transporter genes from Escherichia coli K-12 were amplified by PCR,and then the three genes were constructed on the plasmid pINA1312.The recombinant plasmid were constructed by One Step Cloning Kit,namely pINA1312-gltB-gadC-gadA.The recombinant plasmid was ntroduced into Yarrowia lipolytica.The recombinant strains after transformation and screening was Po1f-gltB-gadC-gadA,.Under flask culture condition,the accumulation of ?-aminobutyric acid by the strain(Po1f-gltB-gadC-gadA)which expressed the glutamate synthetase,glutamate decarboxylase and gamma-aminobutyric acid transporter genes reached 0.86 g/L for 108 hours,indicating that the ?-aminobutyric acid transporter gene was expressed can increase the yield of the product yield by 0.12g/L(about 16.2%).(3)Study on the tolerance of the strain,we found that the Po1f-gltB-gadC-gadA strain we constructed can still grow under the concentration of 25g/L ?-aminobutyric acid,and has the potential of large-scale industrial fermentation;By selecting carbon sources with different concentrations,we found that this strain has a product yield of 1.03 g/L when 4% of glycerol was used as a carbon source.