In Vivo Assay Reveals Microbial OleA Thiolases Initiating Hydrocarbon and β-Lactone Biosynthesis

Abstract

OleA, a member of the thiolase superfamily, is known to catalyze the Claisen condensation of long-chain acyl coenzyme A (acyl-CoA) substrates, initiating metabolic pathways in bacteria for the production of membrane lipids and β-lactone natural products. OleA homologs are found in diverse bacterial phyla, but to date, only one homodimeric OleA has been successfully purified to homogeneity and characterized in vitro. A major impediment for the identification of new OleA enzymes has been protein instability and time-consuming in vitro assays. Here, we developed a bioinformatic pipeline to identify OleA homologs and a new rapid assay to screen OleA enzyme activity in vivo and map their taxonomic diversity. The screen is based on the discovery that OleA displayed surprisingly high rates of p-nitrophenyl ester hydrolysis, an activity not shared by other thiolases, including FabH. The high rates allowed activity to be determined in vitro and with heterologously expressed OleA in vivo via the release of the yellow p-nitrophenol product. Seventy-four putative oleA genes identified in the genomes of diverse bacteria were heterologously expressed in Escherichia coli, and 25 showed activity with p-nitrophenyl esters. The OleA proteins tested were encoded in variable genomic contexts from seven different phyla and are predicted to function in distinct membrane lipid and β-lactone natural product metabolic pathways. This study highlights the diversity of unstudied OleA proteins and presents a rapid method for their identification and characterization. IMPORTANCE Microbially produced β-lactones are found in antibiotic, antitumor, and antiobesity drugs. Long-chain olefinic membrane hydrocarbons have potential utility as fuels and specialty chemicals. The metabolic pathway to both end products share bacterial enzymes denoted as OleA, OleC, and OleD that transform acyl-CoA cellular intermediates into β-lactones. Bacteria producing membrane hydrocarbons via the Ole pathway additionally express a β-lactone decarboxylase, OleB. Both β-lactone and olefin biosynthesis pathways are initiated by OleA enzymes that define the overall structure of the final product. There is currently very limited information on OleA enzymes apart from the single representative from Xanthomonas campestris. In this study, bioinformatic analysis identified hundreds of new, putative OleA proteins, 74 proteins were screened via a rapid whole-cell method, leading to the identification of 25 stably expressed OleA proteins representing seven bacteria phyla.

p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA

“…p -Nitrophenyl esters react rapidly with OleA to release p -nitrophenol and this has been used to identify OleA enzymes in dozens of different bacteria and to probe OleA substrate specificity [29] , [30] . Here, we wished to produce Claisen condensation products using p -nitrophenyl esters.…”

“…Progress on OleA had initially been slowed by a cumbersome and time-demanding assay and was recently accelerated by the development of a high throughput readout of OleA activity, both in vitro and in vivo [29] ( Fig. 1 C).…”

“…This assay has paved the way to conduct a broader palette of in vitro experiments with OleA. The method can also be used in vivo since p -nitrophenyl hexanoate diffuses into recombinant E. coli cells expressing OleA enzymes, while other cellular enzymes have relatively low activity with this substrate [29] .…”

“…The new assay was leveraged, along with bioinformatics identifying thousands of putative OleA orthologs, to broaden OleA enzyme studies [29] , [30] . The method allowed identification of 72 additional OleA enzymes based on both bioinformatics criteria and p -nitrophenyl hexanoate hydrolysis activity ( Fig.…”

Substrate promiscuity of xenobiotic-transforming hydrolases from stream biofilms impacted by treated wastewater

“…Activity screening for the 64 metagenomic DH homologs and the P. putida reference sequence was performed using an established whole-cell colorimetric screening protocol for hydrolase activity (Robinson et al, 2020; Smith et al, 2020). Briefly, individual transformants were inoculated in 5 mL lysogeny broth (LB, final concentration 50 mg/L) supplemented with spectinomycin (final conc.…”