Characterization of NonR, an esterase that confers nonactin resistance
Nonactin is the parent compound of the macrotetrolide class of antibiotics, atypical cyclic polyethers, consisting of both enantiomers linked via four ester linkages in a (+)(-)(+)(-) manner. The higher macrotetrolide homologs of nonactin are made up of successive substitutions of nonactic acid with homononactate or bishomononactate. The macrotetrolides act as ionophores, forming complexes with both monovalent and divalent ions. Organisms that produce antibiotics as a defense mechanism need to protect themselves from their own biosynthesis products. Self-protection is achieved using many methods such as modification of the antibiotic itself, modification of the cellular target, removal of the produced antibiotic to specific binding proteins, or changes in the cell wall. Many species employ several of these methods simultaneously, incorporating antibiotic modification steps into the biosynthesis pathway and using the other methods to offer layers of resistance. A genetic resistance element conferred tetranactin resistance to a macrotetrolide sensitive host. The genetic element was cloned from the producer, Streptomyces griseus subsp. griseus. By sequence analysis the protein product, NonR appeared to be an esterase. This work describes the subcloning of the gene nonR, its expression in a heterlogous host, and examination of the activity of the expressed protein. Expression and purification of the protein NonR, was accomplished using an affinity tag; NonR proved to be labile, losing activity throughout the purification process. NonR hydrolyzes the the ester bonds of nonactin stereoseletively, cleaving between the acid of the (-)-nonactate and the alcohol of the (+)-nonactate. The order of hydrolysis is first the closed chain parent, nonactin, is cleaved to an open chain tetramer, followed by cleavage of the tetramer to two dimer species. Under conditions of high protein concentration, NonR will hydrolyze the dimer species to the monomer, nonactic acid. This hydrolysis causes nonactin to lose its biological activity. To meet our synthesis need we set out to increase the yields of nonactin that is produced by fermentation. By a mix of strain selection, recipe and process improvement, nonactin production was improved from 0.1 g/L in stirred tank reactors to 4 g/L.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:nonr esterase self resistance nond nonl protein expression stereoselectivity nonactin
Date of Publication:01/01/2004