Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces cerevisiae

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 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. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.

Introducing an Artificial Deazaflavin Cofactor in Escherichia coli and Saccharomyces cerevisiae

“…In a recent study, use of chemically synthesized FO for tetracycline biosynthesis in S. cerevisiae was demonstrated. 42 Although FO can be used for some F 420 -dependent conversion, in vivo FOP production can expand the reaction scope owing to the phosphate group offering better binding to more F 420 -dependent enzymes and less leakage from the cell. In this study, we show that it is possible to produce FOP in S. cerevisiae using the heterologously expressed Sp RFK and FO supplemented in the media.…”

“…To the extent of our knowledge, in vivo production of F 420 or other deazaflavins in yeast have not been reported so far. In a recent study, use of chemically synthesized FO for tetracycline biosynthesis in S. cerevisiae was demonstrated . Although FO can be used for some F 420 -dependent conversion, in vivo FOP production can expand the reaction scope owing to the phosphate group offering better binding to more F 420 -dependent enzymes and less leakage from the cell.…”

High-Titer Production of the Fungal Anhydrotetracycline, TAN-1612, in Engineered Yeasts

“…cerevisiae. , Our laboratory successfully achieved the final steps of tetracycline biosynthesis using S. cerevisiae with anhydrotetracycline supplementation, an oxytetracycline biosynthesis intermediate . However, total biosynthesis of tetracyclines in yeasts has yet to be demonstrated.…”

“…Nevertheless, because of its chemical structural similarity to the tetracycline core and its fungal origins, TAN-1612 represents an excellent biosynthetic scaffold for novel tetracycline analogue production in heterologous yeast hosts to combat tetracycline-resistant bacteria. For example, heterologous production of TAN-1612 in yeasts does not require the challenging expression or exogenous supplementation of unique bacterial tetracycline biosynthesis cofactors that are non-native to yeasts like F 420 or F O . , Indeed, important steps toward tetracycline analogues production in yeasts were made by Tang and co-workers, who reconstituted the TAN-1612 biosynthetic pathway from A. niger in S.…”

Hot off the press

“…The final steps of tetracycline biosynthesis have been reconstituted in Saccharomyces cerevisiae , using three heterologous genes and the exogenous supply of cofactor intermediate. 28 The conversion of anhydrotetracycline to tetracycline was achieved using the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM and the F 420 reductase FNO (Scheme 2).…”