Exploring Beta-Lactones as an Unexpected Link Between Hydrocarbon and Natural Product Biosynthesis in Bacteria

Abstract

Recent decades have seen a great interest in using biological systems as sources for the diverse chemicals used in modern society. The production of long-chain olefins (alkenes) from two fatty acids by diverse bacterial species has been well reported in literature and patented for the production of hydrocarbons. A four-gene cluster, oleABCD, encodes the enzymes responsible for this conversion; however, only two of the four enzymatic steps had been elucidated previously. Here, the third enzyme of the pathway, OleC, was characterized and found to convert beta-hydroxy acids to beta-lactones in an ATP-dependent reaction. beta-Lactone-containing natural products show great potential in clinical settings and are known for their potent inhibition of proteases and esterases. OleC is the first beta-lactone synthetase ever characterized, and sequence analysis suggests that the biosynthesis of other beta-lactone natural products is carried out by OleC homologues. The final enzymatic step of olefin biosynthesis was found to be the decarboxylation of the beta-lactone to an olefin by OleB. This enzymatic reaction was not known to exist in nature, making OleB the first known beta-lactone decarboxylase. OleB is frequently annotated as a haloalkane dehalogenase, but we find no dehalogenase activity, suggesting an incorrect annotation of OleB sequences. Mechanistic studies of OleB suggest a similar mechanism to haloalkane dehalogenases that relies on the same Asp-His-Asp catalytic triad and proceeds via an acyl-enzyme intermediate. Finally, the hydrophobic nature of the ole-pathway metabolites and the toxicity of the beta-lactone intermediate were hypothesized to require a direct transfer between the Ole enzymes. Accordingly, the four pathway enzymes, OleABCD, were recombinantly expressed in Escherichia coli, and OleBCD were found to form an enzyme complex. Immunoblots of OleC from the native Xanthomonas campestris on non-denaturing gels were found to migrate similarly to the recombinantly purified OleBCD complex and much slower than OleC by itself, suggesting formation of a complex in the native X. campestris host. Electron microscopy images showed distinct, circular assemblies of OleBCD between 24 and 40 nm in size, with an average size of 32 nm. The OleBCD complex functions to efficiently traffic the hydrophobic metabolites and sequester the toxic beta-lactone intermediate.