Studies On The Synthesis Of Aromatic Amino Acids In Escherichia Coli

Aromatic amino acids include L-phenylalanine,L-tryptophan and L-tyrosine,and their synthesis uses phosphoenolpyruvate and erythrose-4-phosphate as precursors.Among them,L-tryptophan and L-phenylalanine are both essential amino acids for the human body and can only be supplemented from the diet.These three aromatic amino acids can also be used as precursors for many high value-added compounds.At present,the main production methods of aromatic amino acids include extraction method,chemical synthesis method,enzymatic method and microbial fermentation method.Among them,microbial production from renewable resources as a carbon source has many advantages such as environmental friendliness,low cost,and uniform product configuration.Acoordingly,it is widely used in the synthesis of a variety of high value-added chemical compounds.As a model microorganism,Escherichia coli has been widely used in the production of aromatic amino acids because of its ability to grow rapidly in cheap media and easy genetic modification.However,the complex regulation mechanism of E.coli itself severely restricts its ability to accumulate aromatic amino acids.Based on the preliminary work in the laboratory,this thesis has carried out three researches on the synthesis of E.coli aromatic amino acids:First,we constructed a base strain for the synthesis of aromatic amino acids,and then we used basic elements of synthetic biology to assemble a regulatory switch with multiple regulatory functions.The key genes phe A^FBR and trp E^FBR in the branching pathway of L-phenylalanine and L-tryptophan were placed under the control of P_{acu R} and P_{lac UV5} promoters,respectively.Then,this metabolic toggle switch was transferred to the aromatic amino acid base strain to obtain the recombinant strain GRE-8.By supplement of IPTG,acrylate or no inducer,GRE-8 showed three different production modes.When no inducer was added,the concentrations of L-tryptophan,L-phenylalanine and L-tyrosine in batch fermentation of GRE-8 were 0.34,0.65 and 0.59 g/L,respectively.When IPTG was added to the medium,the concentration of L-phenylalanine increased to 1.22 g/L,while the concentration of L-tryptophan and L-tyrosine decreased by 1.83 and 1.46 times,respectively.When acrylate was added to the medium,the concentration of L-tryptophan in GRE-8 increased to 1.02 g/L,while the concentration of L-phenylalanine and L-tyrosine were reduced to 0.16and 0.28 g/L,respectively.These results showed that the regulatory switch could regulate the final products distribution in engineered strains.In addition,this regulatory switch can also be employed to perform complex regulatory functions other branch pathways in E.coli or other microorganisms.Second,in order to construct a plasmid-free L-tryptophan producing E.coli,we used previously constructed E.coli MG1655（Δtrp RΔtna A）as the starting strain.By inactivating L-tryptophan permease Mtr and Tna B,the PTS glucose transport system EI protein Pts I and the L-tryptophan attenuator leader peptide gene Trp L,and the wild-type aro G in the genome was replaced with a aro G^FBR that released feedback inhibition of L-phenylalanine.As a result,recombinant E.coli GP-6 was obtained.In the batch fermentation,the L-tryptophan production of the GP-6 strain was 398.42 mg/L,which was 23.38 times higher than that of the initial strain.In the 5-L fed-batch fermentation,the L-tryptophan production can reach 1735.62 mg/L.These results indicate that the constructed plasmid-free recombinant Escherichia coli can synthesize a certain amount of L-tryptophan and has the potential for further engineering.Third,to further screen L-tryptophan high-producing strains and discover unknown L-tryptophan regulatory factors,,four different L-tryptophan biosensors were constructed based on two L-tryptophan sensing elements Trp70-727 and tna C.Among them,p LXZ-1 and p LXZ-2 are positively regulated biosensors.As the concentration of L-tryptophan increases,the expression levels of the reporter genes rfp and tet A also increase,leading to improved tetracycline tolerance of recombinant strains;p LXZ-3 and p LXZ-4 are negatively regulated L-tryptophan biosensors.As the concentration of L-tryptophan increases,the expression levels of the reporter genes rfp and tet A decrease,resulting improved resistant to Ni²⁺for recombinant strains.In order to reduce the L-tryptophan consumption of host strain,tryptophanase leader peptide and tryptophanase were inactivated in MG1655（Δtna CΔtna A）.The reporter genes rfp and tet A were employed in biosensors to detect the concentration response range to L-tryptophan and the activation intensity of the reporter gene.Until now,four biosensors that can be regulated by the concentration of L-tryptophan have been obtained,and their response concentration range,threshold,response time and other parameters are being optimized to prepare for the next step of pressure-induced evolution.