Abstract:
Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key A- and C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature’s biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Here, we describe the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts. Additionally, our collaborators in the Tang laboratory reported the heterologous biosynthesis of a non-antibiotic fungal anhydrotetracycline derivative, TAN-1612, in Saccharomyces cerevisiae from Aspergillus niger. We have built upon this system, allowing for the high-titer production of TAN-1612 in yeasts. Here, we propose to convert TAN- 1612 into a high titer tetracycline- and analog-producer by modifying the 2-, 4-, and 6-positions, proven critical for antibiotic activity. By hijacking biosynthetic hydroxylating and reducing enzymes, we will modify the 6α-position, dearomatizing the C-ring. We also expressed heterologous enzymes within the TAN-1612 pathway that could furnish the 2-position with a carboxamido group instead of its natural hydrogen groups. By taking advantage of yeast’s natural biosynthetic pathways, we will create inexpensive, single-dose antibiotics, setting the stage to pursue yeast as a novel therapeutic. These state-of-the-art synthetic biology technologies will create entirely new paradigms, leading the charge against infections and diseases.
