Development And Application Of An L-Tryptophan Biosensor Based On The Violacein Biosynthesis Pathway

L-Tryptophan is an important aromatic amino acid with broad applications in medicine,food,feed,and as a precursor for synthesizing many high value-added compounds.Rational design of metabolic engineering can increase L-Tryptophan production in Escherichia coli（E.coli）by conventional modification of known gene targets.By establishing a library and combining it with a biosensor strategy,potential gene targets,enzyme mutants,and promoter sequences that further affect its production can be screened in a high-throughput manner.Currently developed L-Tryptophan biosensors have a generally low operational range and dynamic range,limiting the practical application of this strategy.L-Tryptophan is the only substrate in the violacein biosynthesis pathway,and four substances with different colors can be synthesized by the enzyme Vio ABCDE.This characteristic can be used to transform it into an enzyme-coupled L-Tryptophan biosensor.This study tested different sources of the Vio A enzyme,combined with RBS（ribosome binding site）engineering to develop a novel L-Tryptophan biosensor,optimized the process of establishing a genome library,and conducted a preliminary screening of potential targets that affect L-Tryptophan production.（1）Knocking out the Dug.B2＿vio D gene enabled the enzyme Dug.B2＿Vio ABCE to catalyze L-Tryptophan to deoxyviolacein.The fermentation endpoint was selected at 24 h by analyzing the growth curve.By adjusting the translation initiation rate（TIR）through RBS engineering to adjust the expression level of the Dug.B2＿Vio A enzyme,the developed biosensor p BL＿406 carried the Dug.B2＿vio ABCE gene cluster.When the TIR of RBSb was 4941.17,the dynamic range reached 3 times under the condition of adding 2-7 g·L^-1 L-Tryptophan externally.（2）Analyzing the homologous sequence of Dug.B2＿vio A,a phylogenetic tree was constructed,and five Vio A sequences with distant relationships were selected for testing.Directly replacing the Dug.B2＿vio A sequence would affect the expression of the downstream Dug.B2＿vio BCE gene cluster.Therefore,Dug.B2＿vio BCE and Dug.B2＿vio A were isolated,and two T7 promoters and terminators were used to control their transcription,and Dug.B2＿vio A was replaced.The results showed that Jan＿Vio A and Chr＿Vio A expressed in E.coli by this method were active at 30°C,but were inactive at 37°C.Further adjustments were made to the RBSa sequence of their vio A,and it was found that the biosensor p BL＿439 carrying Chr＿vio A reached a dynamic range of 55 times under the condition of adding 0-10 g·L^-1 of L-Tryptophan externally when the TIR of RBSa was 2 004.59.Moreover,E.coli strains with different L-Tryptophan production could be directly distinguished by naked eye observation.（4）Gradient dilution was used to test the restriction endonucleases Alu I,Afa I,Mlu C I,and Nla III,and it was determined that 100-fold dilution of Alu I,10-fold dilution of Afa I,100-fold dilution of Mlu C I,and 10-fold dilution of Nla III were optimal for incomplete digestion of the E.coli genome.The number of transformants was increased from both the perspective of improving electroporation conversion efficiency and ligation efficiency,while the library quality was enhanced by reducing self-ligation rate and increasing the length of random fragments.By improving the library construction process in the aforementioned ways,the number of transformants per transformation was increased from 50 CFU to 9.0×10⁴ CFU,an1,800-fold improvement.Through four repeated experiments,an E.coli genomic library with a capacity of 3.6×10⁵ was established.The library plasmids were then introduced into E.coli B8 containing the biosensor p BL＿439,and preliminary screening was conducted to identify genes that could potentially affect L-tryptophan production.In this study,we successfully engineered the violacein biosynthetic pathway and developed an enzyme-coupled L-tryptophan biosensor with a dynamic range of 55-fold within the working range of 0-10 g·L^-1 L-tryptophan.This biosensor,in combination with high-throughput screening of the library,can be used to identify genes,enzyme variants,or promoters that affect L-tryptophan production,which is of significant importance for further enhancing the production of L-tryptophan and its high-value derivatives.Additionally,we optimized the construction process of the genomic library and,in conjunction with the biosensor,conducted preliminary screening to identify genes that influence L-tryptophan production.