Expression And Optimization Of Acyl Ligase And Transferase And Their Application In Ester Synthesis

Ester compounds are easily soluble in organic solvents and play an important role in the structure of living cells.They are widely used in chemistry,food and medicine.Therefore,the establishment of an efficient and green ester synthesis method has always been a research focus.Esterification by microorganisms is a new synthetic pathway based on metabolic engineering,and screening acyl ligase and transferase which can be expressed efficiently is the key to constructing this pathway.This paper takes Escherichia coli as the chassis,and a joint optimization strategy based on genetic engineering and fermentation engineering,explores the different sources of acyl ligase and transferase adapter expression mechanism,and combined with whole-cell catalysis and gene expression strategy,can be screened in the catalytic growth chain fatty acid ester,structural analogues of benzoates products such as two key enzymes.In this paper,by expressing acyl ligase and transferase from different sources,the adaptive expression mechanism of the two enzymes was explored,and the efficient expression of acyl ligase and transferase in Escherichia coli was obtained.By regulating the induction temperature and the concentration of isopropyl-beta-D-thiogalactosyl pyranosid（IPTG）during the expression process,optimizing the medium,expressing strain and cell fragmentation parameters,the acyl ligase Baf X and Orf3 from Streptomyces Griseus,ligase4CL from Arabidopsisa,ligase Ict A from Aspergillus terreus,and transferase Atf A from Acinetobacter baylyi,transferase Orf2 from Streptomyces Griseus,transferase Pha C from Allochromatium vinosum were screened and determined the optimal expression conditions.The protein expression levels of acyl ligase and transferase were measured after nickel column purification.Based on 5,5’-Dithiobis-（2-nitrobenzoic acid）（Ellman）reagent,a method for acyl ligase analysis was established.The enzyme activities of Baf X,Orf3,4CL and Ict A were 1.53×10^-3U/mg,6.06×10^-3U/mg,0.79×10^-3U/mg and3.40×10^-3U/mg.In this paper,two key enzymes that catalyze the synthesis of long-chain fatty acid ester and dibutanol phthalate were screened by gene co-expression and whole-cell catalysis strategy.The highly expressed acyl ligase and transferase genes were co-transformed into Escherichia coli for expression optimization.By establishing the whole-cell catalytic system and analyzing the products catalyzed by different combinations,it was found that the single expression of transferase Atf A from Acinetobacter baylyi could catalyze the formation of butyl hexadecenoate and butyl octadecenoate.And it was proved that acyltransferase Atf A has the ability to synthesize long-chain fatty acids.The combination of acyl ligase 4CL from Arabidopsisa and transferase Atf A from Acinetobacter baylyi can catalyze the formation of structural analogues of benzoates,which proves that the combination of acyl ligase 4CL and transferase Atf A has the characteristics of catalyzing the synthesis of aromatic diacid alcohol esters.The extensive catalytic substrate of Atf A was revealed,which laid a foundation for the further study of catalytic synthesis of short-chain diacid alcohol esters.