Metabolic And Enzymatic Engineering Of Saccharomyces Cerevisiae To Efficiently Biosynthesize The(2s)-naringenin

Flavonoids have a variety of physiological functions and are widely used in food,pharmaceuticals,health care products,and other industries.（2S）-Naringenin is the skeleton of flavonoids,and can derive multiple high value-added flavonoids after modification.At present,（2S）-naringenin is mainly obtained by plant extraction.Its yield is affected by the quality of raw materials,season and extraction process,resulting in many problems such as low yield and difficult quality control.Microbial synthesis has multiple advantages and is a potential production method that can effectively solve the above-mentioned difficulties.However,the conversion rate of biosynthesis of（2S）-naringenin is low,and it is still unable to compete with the plant extraction method.In this study,chalcone synthase（CHS）was proved to be the main rate-limiting enzyme in the（2S）-naringenin biosynthesis pathway,and higher-active CHS isozymes from plants rich in flavonoids were screened.Through the high-throughput screening technology constructed in this study,CHS mutants with increased enzyme activity were obtained.Finally,the yield of（2S）-naringenin biosynthesis was improved by fermentation optimization.The main results of this study are summarized as follow:1.Identification of key enzymes in the（2S）-naringenin biosynthesis pathway and optimization of CHS sources.Four genes（tyrosine ammonia lyase（TAL）,4-hydroxycinnamoyl-Co A ligase（4CL）,chalcone synthase（CHS）,and chalcone isomerase（CHI））in the（2S）-naringenin biosynthesis pathway were overexpressed in Saccharomyces cerevisiae.Overexpression of CHS can significantly increase the yield of（2S）-naringenin with growth inhibition of S.cerevisiae,which proves that CHS is a key rate-limiting enzyme in the（2S）-naringenin biosynthesis pathway,and overexpression of this enzyme causes a metabolic burden on the host strain.Then,12 CHS genes were screened from 9 plants with high flavonoid content,and it was confirmed that Sj CHS1 derived from Sophora japonica had the highest transformation efficiency.Compared with the commonly used Ph CHS,Sj CHS1 increased the（2S）-naringenin yield by 48.38%.Subsequently,Sj CHS1 was purified,and enzymatic properties were identified.It was found that the optimum temperature of Sj CHS1 was 50^oC,the optimum p H was 7,and its affinity for p-coumaroyl-Co A was stronger than that for malonyl-Co A.Subsequently,fusion expression of Pc4CL and Sj CHS1 was performed,demonstrating that the distance between enzymes is not a key factor affecting the efficiency of synergistic catalysis of 4CL and CHS.2.Establishment of biosensor-based high-throughput screening methods.（2S）-Naringenin biosensors were constructed based on the Ttg R operon and the Fde R operon in S.cerevisiae and Escherichia coli,respectively.Both biosensors performed better in E.coli than in S.cerevisiae,while the Ttg R operon-based biosensor performed better than the Fde R operon.In E.coli,the Ttg R operon-based biosensor has a signal-to-noise ratio of 77.12,which is 55.29times higher than that in S.cerevisiae,and the detection limit of（2S）-naringenin is 0-100 mg/L.Subsequently,the expression level of Ttg R was optimized in E.coli,and the detection limit was extended to 0-300 mg/L.Subsequently,the specificity and in vivo efficiency of the biosensor were verified.Results showed that the biosensor has high specificity for（2S）-naringenin,and can accurately reflect the（2S）-naringenin yield in vivo.Combined with the high-throughput screening platform built by the laboratory,a high-throughput screening scheme based on biosensors was established to provide rapid screening for the mutation library obtained in this study.3.Catalytic performance enhancement and mechanism analysis of Sj CHS1 based on directed evolution.Directed evolution of Sj CHS1 through a Ttg R-based（2S）-naringenin biosensor and error-prone PCR resulted in higher-active mutants Sj CHS1^A308R,Sj CHS1^S208Cand Sj CHS1^E61K.Subsequently,A308,S208 and E61 sites of Sj CHS1 were saturated mutated in S.cerevisiae.Compared to Sj CHS1,the yield of（2S）-naringenin of recombinant strains harboring Sj CHS1^A308K,Sj CHS1^A308R,Sj CHS1^S208N,Sj CHS1^S208C,Sj CHS1^E61K,Sj CHS1^E61R,Sj CHS1^E61A,Sj CHS1^E61T and Sj CHS1^E61C was increased by 1.41,1.24,1.44,1.28,1.30,1.10,1.17,1.09 and 1.11 times,respectively.However,stacking the best mutations could not improve（2S）-naringenin production.The enzyme activity of the three best mutants,Sj CHS1^E61K,Sj CHS1^S208N and Sj CHS1^A308K,were 1.34,2.34 and 1.63 times higher than that of the wild-type Sj CHS1,respectively.The three mutants were more stable at p H 5.0-8.0 and the affinity for malonyl-Co A was higher than Sj CHS1.MD simulation analysis found that the decreased binding free energy and the increased salt bridge and electrostatic potential on the surface of the Co A-binding channel in the mutants were responsible for the increased affinity of the enzyme for malonyl-Co A.4.Metabolic engineering of S.cerevisiae to improve the precursor supply of Sj CHS1^S208Nto enhance（2S）-naringenin biosynthesis.Based on overexpressing of Sj CHS1^S208N,the supply of p-coumaroyl-Co A and malonyl-Co A were improved by enhancing the phenylpropane metabolic pathway and the malonyl-Co A biosynthesis pathway.Overexpression of ARO1,ARO2 and TYR1 in the phenylpropane metabolic pathway,the yield of（2S）-naringenin was increased by 17.67%,7.26%and 22.90%,respectively.Proved that increasing the supply of p-coumaroyl-Co A can increase the（2S）-naringenin production.Overexpression of ACC1^S659A,S1157A,Se ACS^L641P,PEX10,PDC1 and ALD6 in the malonyl-Co A biosynthesis pathway,the yield of（2S）-naringenin was increased by 23.60%.Increasing the supply of malonyl-Co A was proved to increase the（2S）-naringenin production.When the p-coumaroyl-Co A and malonyl-Co A biosynthesis pathways were co-strengthened,the yield of（2S）-naringenin was finally increased to 434.65 mg/L.The type and concentration of carbon sources,nitrogen sources and metal ions in the fermentation medium were optimized.The optimal medium formula was 38.75g/L sucrose,18.20 g/L soybean peptone,and 12.01 m M Fe Cl₂.After optimization,the yield of（2S）-naringenin was achieved at 1016.8 mg/L,and increased by 2.34 times.By optimizing the inoculum amount and dissolved oxygen in the 5-L bioreactor,the maximum yield of（2S）-naringenin was 2513.27 mg/L.