Genetic and Biochemical Characterization of Natural Tetracycline Biosynthesis

Tetracyclines are a group of natural products produced by soil-borne Actinobacteria. Their broad-spectrum biological activities such as antibiotic, anticancer, and novel activity against tetracycline resistant bacteria are attributed to their signature linearly fused four ring structure. However, the extensive use of tetracyclines during the last sixty years has led to emergence of resistance mechanisms among microorganism communities, resulting in dramatically decreased effectiveness of tetracyclines as first line antibiotic agents. Therefore, new generation of tetracyclines is highly demanded to overcome current resistance mechanisms. To obtain new generation of tetracyclines, fundamental understanding of tetracycline biosynthesis and establishment of robust manipulation platform are required. The biosynthetic pathways of three natural tetracycline, including oxytetracycline, SF2575, and dactylocycline, were investigated by using genetic, biochemical, and protein structure based analysis.Oxytetracycline represents an important example of natural tetracyclines featuring a signature C5 hydroxyl group. Unveiling of oxytetracycline biosynthetic pathway will establish a cornerstone to understand and engineer tetracycline biosynthesis. After a decade investigations, the biosynthetic pathway of a key intermediate anhydrotetracycline has been revealed; however, the longstanding missing link involved in the final transformations from anhydrotetracycline to oxytetracycline remains elusive since the first discovery of oxytetracycline in 1950. Two redox enzymes OxyS and OxyR were unravelled to catalyze the mysterious final transformations by using flavin adenine dinucleotide (FAD) and F420 as coenzymes respectively. The protein structure of OxyS has been determined and provided valuable insights into the enzymology unique to tetracycline biosynthesis. SF2575 stands out from tetracycline family due to its fully substituted tetracycline aglycone and novel potent anticancer activity. Cascade transformations are catalyzed by a set of new tetracycline tailoring enzymes involved in SF2575 biosynthesis, including four methyltransferases, a glycosyltransferase, and three redox enzymes. To decipher the biosynthesis and understand the chemical reactions catalyzed by these dedicated tailoring enzymes, the corresponding encoding genes were inactivated in heterologous expression host Streptomyces lividans K4-114. The functions of the tailoring enzymes were elucidated by isolation and structural characterization of intermediates accumulated from the corresponding gene-inactivation mutants. The production of fourteen tetracycline analogs from ten mutants led to functional assignment of SF2575 tailoring enzymes and elucidation of SF2575 biosynthetic pathway. Most interestingly the redundancy of the methyltransferase SsfM3 demonstrates an evolutionary event in which combinatorial biosynthesis strategy has been employed by nature to generate novel tetracycline compounds.Dactylocycline offers us the third example of natural tetracycline with novel activity against tetracycline resistant bacteria. This promising activity is due to a unique hydroxylamino sugar modification at C6 hydroxyl group of dactylocyclinone which shows cross resistance with tetracycline. Identification of dactylocycline biosynthetic gene cluster led to a proposed biosynthetic pathway and expanded enzymatic tools to synthesize both dactylocycline aglycone and the hydroxylamino sugar moiety. To validate this gene cluster, the dac gene cluster was heterologously expressed in Streptomyces lividans K4-114 resulting in the production of dactylocyclinone. Given three natural tetracycline biosynthetic pathways and an accommodating heterologous host, we are able to generate new tetracycline analogs by using combinatorial biosynthesis approaches.