Heterologous Expression And Biosynthesis Of Anthracycline Anticancer Medicines Adriamycin And Aclarubicin Synthesis Genes

As the anthracycline anti-cancer antibiotics,both doxorubicin and acrylamycin are widely used in clinic due to their broad-spectrum,non-specificity and high efficiency.The two compounds in the form of hydrochloride inhibit and destroy tumor cells by inhibiting intracellular macromolecules especially DNA.To date,the production of doxorubicin relies mainly on semi-chemical method and biological method,while production of acrylamycin is realized only by biological method.The semi-chemical method for doxorubicin refers to pure chemical catalysis from precursor daunorubicin.This approach shows flaws of stringent temperature control,high toxicity of catalytic reagents,long time-consuming,and thereby deviation from environmental protection and sustainable development.In addition,the final conversion rate and yield of raw materials are low.In contrast,bio-production of doxorubicin is accomplished by large-scale cultivation and fermentation of Streptomyces peucetius ATCC 27952.In particular,the strain Streptomyces sp.NO.MA 144-MI ATCC 31133 can naturally produce acrylamycin.Despite this advantage,this strategy shows drawbacks of long fermentation time,low yield,and high cost,which restrict the large-scale clinical applications of doxorubicin and acrylamycin.To overcome aforementioned shortcomings,the multiple pathways were divided into several modules based on their distinct functions.This gigantic gene pools would be transformed into host strains.The genes in this study involves 9 key synthetase genes as fundamental module,the genes for modification of doxorubicin side chain groups,15 key enzyme genes for generation of glycosyl ligands and glycosylation module,the genes for modification of acrylamycin side chain groups,and 7 key enzyme genes for glycosylation module.To begin with,these reported key genes were subjected to bioinformatics analysis with emphasis on the physical and chemical properties,active center sites,and two-dimensional and three-dimensional structure upon transcription and translation.This provided valuable insights for subsequent stable heterologous expression and catalysis in host cells.The premise of heterologous gene expression is construction of corresponding vectors that can harbor,replicate,transcribe and translate.Next,the key synthesis genes of doxorubicin and acrylamycin are independently cloned into the modified expression vectors,and the recombinant vectors were transformed into host cells,leading to recombinant bacteria.Considering the high GC base content of the partial reported original gene and likely failure in stable expression,the codon was optimized in view of the original sequence.Next,molecular cloning was carried out to screen recombinant strains that could be successfully cloned and stably expressed.As a consequence,all key synthesis genes were independently cloned into vectors and heterologously expressed in host strains.Finally,the genes in each functional module were individually subcloned into vectors and expressed according to their distinct functions.Subsequent transformation led to recombinant strains,and these engineered bacteria were co-cultured for large-scale production of two antibiotics.