Substreate Specificities Of Polyketide Synthases From Sargassum Fusiforme And Gegetically Engineered Escherichia Coli Construction

This thesis mainly studied the enzymatic properties of recombinant polyketide synthesis from Sargassum fusiforme,which were heterologously expressed in Escherichia coli,and explored the essential amino acid residues.Finally,a genetically engineered E.coli containing PKS of S.fusiforme was construct to produce flavonoid compound-naringenin.There are three type III polyketide synthase(PKS)genes in S.fusiforme,designated as Sfu PKS1,Sfu PKS2 and Sfu PKS3,which encode414,484 and 367 amino acids,respectively,resulting in the protein size of40-50 k Da.Homology comparison revealed that Sfu PKS1-3 has high homology similarities with Sb PKS from Sargassum binderi and Esi PKS from Ectocarpus siliculosus,e.g.Sfu PKS1 shows 98%and 92.4%homology to Sb PKS and Esi PKS1,respectively.Besides the catalytic triad(Cys-His-Asn),Sfu PKSs share quite conservative amino acids at the catalytically active site and the substrate binding site.PKSs can use aliphatic Co A and aromatic Co A as the starting substrates.For example,Esi PKS1 can use lauroyl Co A as the starting unit for Claisen condensation with three molecules of malonyl Co A,resulting in phloroglucinol-containing aliphatic chain products.Sb PKS can use various long-chain aliphatic Co A(C₆-C₁₄)to generate triketone or tetraketopyran ring products.Sfu PKS1-3 accept a variety of long-chain aliphatic Co A(C₂-C₁₄),resulting in triketone or tetraketopyran ring products.Moreover,Sfu PKS1 can also use aromatic Co A(coumaroyl-Co A),to generate naringenin.During the analysis of the protein tertiary structure,the amino acid residues in the 6(?)range around the substrate were selected for comparison,where the natural vibration of the residues around 1(?)were filtered out.Subsequently,these amino acids were mutated in Sfu PKSs,whose substrate specificities were analyzed.Finally,a genetically engineered Escherichia coli strain containing4cl,mat BC and Sfu PKS1 was constructed.In the medium with coumaric acid and malonic acid,naringenin was produced.With condition optimized,a yield of 4.911 mg/L had been obtained.Research of the PKS will help further explore on the polyketide compound and lay the foundation for in-depth study and utilization of natural products from S.fusiforme.