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

MBio
Megan D SmithLawrence P Wackett

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 test...Continue Reading

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Citations

Jan 31, 2021·Biotechnology Advances·Pei XuWen-Yong Lou
Jun 19, 2021·Computational and Structural Biotechnology Journal·Megan D SmithLawrence P Wackett

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Methods Mentioned

BETA
X-ray
lipase
electrophoresis
glycosylation

Software Mentioned

Coffee Expresso
BLAST
HMMER3
RODEO
CD
FastTree
DECIPHER
Hit

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