Heterologous Expression Of Key Enzyme Genes Encoding The Glycosylation Module For Biosynthesis Of Doxorubicin,An Anthrac Ycline Anticancer Medicine

Doxorubicin is a popular non-specific antitumor medicine which shows broad and high anti-tumor activity.There is a strong demand for doxorubicin in the market,however,it has not yet been industrialized.Currently,the yield of doxorubicin remains to be improved,which has led to high price.Fortunately,with the development of genetic engineering and other technologies,there is a new breakthrough in the synthesis of doxorubicin.That is,microbial factory is engineered to heterologously reconstruct the synthetic pathway of doxorubicin.Not only is this method safe and environment-friendly,it overcomes the production bottleneck of native host,and thereby holds potential for large-scale production of doxorubicin.The biosynthesis of doxorubicin can be divided into three modules:aglycone synthesis module,glycosylation module,and post-modification module.In this article,experiments include cloning and heterologous expression of the crucial enzyme genes for sugar production,bioinformatics analysis and heterologous expression of glycosyltransferases DnrS and accessory proteins DnrQ.The experimental results are shown as follows:First,the key enzyme genes of sugar synthesis including RmbA,RmbB,DnmU,DnmT,DnmJ,DnmV were codon-optimized.The individual gene expression vectors were constructed,and these enzymes were stably expressed in recombinant bacteria.This work proceeds the lab mission of cloning and heterologous expression of the genes for doxorubicin biosynthesis and thereby has provided basis for subsequent doxorubicin synthesis.Second,considering yeast expression system enables post-translational modification,it was thus employed as a host for heterologous expression of DnrS and DnrQ.The expression vectors of DnrS and DnrQ were constructed in Saccharomyces cerevisiae and Pichia pastoris,respectively.The corresponding proteins were finally expressed in P.pastoris,and verified at transcription and protein levels.To overexpress them,the protein expression conditions were optimized.In this section,proteins were heterologously expressed,which provides the basis for their forthcoming functional identification.Third,the DnrS and DnrQ were subjected to bioinformatics analysis to understand their basic physical and chemical properties and structures,which may provide insight for future experiments.