| 4-Hydroxycinnamaldehydes and 4-hydroxycinnamyl alcohols are key intermediates in the biosynthesis of lignins through the phenylpropanoid pathway,which are significant and valuable not only for biology study but also for economic and biomedical science.However,the extraction method of these natural resources from plants has some limitations,such as the long growth time and environmental impacts.Traditional chemical synthetic methods involve many complicated steps,excessive by-products and environment contaminations.Therefore,the research and development of microbial transformation for the production of 4-hydroxycinnamaldehydes and 4-hydroxycinnamyl alcohols has important theoretical and practical value.This dissertation aims to modify the structure of phenylpropanoic acids using the microbial transformation technique,thus producing valuable 4-hydroxycinnamaldehydes and 4-hydroxycinnamyl alcohols.The results can be summarized as the following four aspects:(1)In the experiment,four kinds of genetically engineered Escherichia coli(E.coli)strains have been successfully constructed,including E.coli M15-4CL1+M15-CCR,M15-4CL1-CCR,BL21-4CL1+CCR and BL21-4CL1-CCR.The SDS-PAGE profiles revealed that the target proteins were efficiently expressed in E.coli.Comparison of genetic engineering bacteria was discussed in order to provide an effectively novel method for 4-hydroxycinnamaldehydes synthesis.E.coli M15-4CL1-CCR was chosen as the optimum whole-cell biocatalyst.The bifunctional enzyme 4CL1-CCR can catalyze sequential multistep reactions,thereby avoiding the permeability problem of intermediates.Furthermore,the fusion expression system has significantly higher enzymatic activity than the co-expression system.(2)An HPLC-PDA-ESI-MSn detection method was established to provide a more effective analytical tool for separation and identification of phenylpropanoic acids and their corresponding cinnamaldehydes and alcohols.The method includes crude extraction with ethyl acetate,pre-purification with a SPE cartridge,separation by HPLC and verification by an ESI-MSn system.(3)The bioconversion experiment was carried out using the p-coumaric acid,caffeic acid and ferulic acid as substrate.The results of HPLC-PDA-ESI-MSn analysis proved the existence of p-coumaraldehyde,caffeldehyde and coniferaldehyde.Through quantitative analysis,we calculate the conversion ratio and yield.p-Coumaric acid is the most favorable substrate for the bioconversion.The yield of the p-coumaraldehyde,caffeldehyde and coniferaldehyde was 34%,11%and 19%,respectively.(4)Using immobilized cells of E.coli M15-4CL1-CCR and M15-CAD as the biocatalyst,a three-step biocatalytic cascade reaction for the production of 4-hydroxycinnamyl alcohols from inexpensive phenylpropanoic acids was developed.There was no additional cofactor added during the biotransformation process.The bioconversion experiment was carried out using the p-coumaric acid,caffeic acid and ferulic acid as substrate.The results of HPLC-PDA-ESI-MSn analysis proved the existence of 4-hydroxycinnamyl alcohols.The optimal transforming condition was investigated:the optimum reaction temperature was 30?,the buffer pH was 7.0 and the bioconversion time was 6?8 h.Under these conditions,the yield of the p-coumaryl alcohol,the caffeyl alcohol and the coniferyl alcohol was 58%,24%and 60%,respectively.Microbial transformation is a new kind of green synthesis technology,which has the characteristics of simple,effective,economical and environmentally-friendly,and plays an irreplaceable role in producing natural products.The application of fusion enzyme can not only avoid the permeability problem of intermediates but also improve the enzymatic activity.Overall,this study offers a bioconversion pathway for the microbial production of valuable 4-hydroxycinnamaldehydes and 4-hydroxycinnamyl alcohols from inexpensive phenylpropanoic acids,which provides a foundation for the biosynthesis of these aromatic polymers,and also provides new insights into the synthesis of other natural products. |
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