Harnessingin vivoSynthesis of Bioactive Multiarylmethanes inEscherichia colivia Oxygen-Mediated Free Radical Reaction Induced by Simple Phenols

Abstract

ABSTRACTBackgroundXanthenes and multi-aryl carbon core containing compounds represent different types of complex and condensed architectures that have impressive wide range of pharmacological, industrial and synthetic applications. Moreover, indoles as building blocks were only found in naturally occurring metabolites with di-aryl carbon cores and in chemically synthesized tri-aryl carbon core containing compounds. Up to date, rare xanthenes with indole bearing multicaryl carbon core have been reported in natural or synthetic products. The underlying mechanism of fluores-cein-like arthrocolins with tetra-arylmethyl core were synthesized in an engineeredEscherichia colifed with toluquinol remained unclear.ResultsIn this study, the Keio collection of single gene knockout strains of 3901 mutants ofE. coliBW25113, together with 14 distinctE. colistrains, was applied to explore the origins of endoge-nous building blocks and the biogenesis for arthrocolin assemblage. Deficiency in bacterial res-piratory and aromatic compound degradation genesubiX,cydB,sucAandssuEinhibited the mu-tant growth fed with toluquinol. Metabolomics of the cultures of 3897 mutants revealed that only disruption oftnaAinvolving in transforming tryptophan to indole, resulted in absence of arthro-colins. Further media optimization, thermal cell killing and cell free analysis indicated that a non-enzyme reaction was involved in the arthrocolin biosynthesis inE. coli. Evaluation of redox potentials and free radicals suggested that an oxygen-mediated free radical reaction was respon-sible for arthrocolins formation inE. coli. Regulation of oxygen combined with distinct phenol derivatives as inducer, 31 arylmethyl core containing metabolites including 13 new and 8 biolog-ical active, were isolated and characterized. Among them, novel arthrocolins withp-hydroxylbenzene ring from tyrosine were achieved through large scale of aerobic fermentation and elucidated x-ray diffraction analysis. Moreover, most of the known compounds in this study were for the first time synthesized in a microbe instead of chemical synthesis. Through feeding the rat with toluquinol after colonizing the intestines of rat withE. coli, arthrocolins also ap-peared in the rat blood.ConclusionOur findings provide a mechanistic insight intoin vivosynthesis of complex and condensed ar-throcolins induced by simple phenols and exploits a quinol based method to generate endoge-nous aromatic building blocks, as well as a methylidene unit, for the bacteria-facilitated synthesis of multiarylmethanes.