title: Functional modular dissection of DEBS1-TE changes triketide lactone ratios and provides insight into Acyl group loading, hydrolysis, and ACP transfer.
abstract: The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products.
authors.affiliation: Department of Chemistry, Portland State University, Portland, Oregon 97201, United States.
authors.forename: John
authors.initials: J
authors.lastname: Yan
authors.forename: Christopher
authors.initials: C
authors.lastname: Hazzard
authors.forename: Shilah A
authors.initials: SA
authors.lastname: Bonnett
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Reynolds
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D005285:Fermentation; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry*; D013058:Mass Spectrometry; D010957:Plasmids; D048630:Polyketide Synthases / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D016930:Saccharopolyspora / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D007783:Lactones; D048630:Polyketide Synthases