Functional characterization of the dfn polyketide synthase gene cluster in Bacillus velezensis ATCC 39374

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Robert Kenneth Tikkanen III (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Jason Reddick

Abstract: The polyketide macrolactone, difficidin, is a secondary metabolite of B. velezensis ATCC 39374 and 39320 which has been found to exhibit broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. While the majority of the difficidin structure follows canonical polyketide biosynthesis, it contains an unprecedented exomethylene “ß-branch” which is found in only a small number of other polyketide natural products. In past work conducted by the Reddick lab, it had been hypothesized that baeC, a malonyl-CoA-acyl carrier protein transacylase from B. velezensis strain ATCC 39374, loaded a malonyl acyl group from malonyl CoA onto the free thiol group of the phosphopantetheine arm of holo-DfnX acyl carrier protein from the dfn biosynthetic gene cluster. While data from these initial experiments appeared to support this initial hypothesis, it was revealed that the promiscuous 4’- phosphopantetheinyl transferase encoded by sfp from strain OKB105 used in this in vitro system was capable of independently loading the entire, malonyl-containing, phosphopantetheine prosthetic group from malonyl-CoA directly to the apo form of DfnX. Therefore, the data produced from these experiments were not reflective of the true malonyl acyl transferase activity of baeC. The goal of the research presented in this thesis was to redesign an in vitro system that does not require the addition of the 4’- phosphopantetheinyl transferase to activate apo-dfnX. The characterization and assignment “ß-branching” activity of genes within the dfn and bae gene clusters in the B. subtilis ATCC 39374 strain had been an ongoing project conducted by Dr. Brittany Kiel formerly of the Reddick Lab. By modeling the biochemical insights gleaned from the biosynthesis of bacillaene by the pksX gene cluster from B. subtilis 168, previously characterized by Walsh and Kelleher, and the biosynthesis of bacillaene and difficidin produced by the respective bae and dif gene clusters in B. velezensis (previously classified as B. amyloliquefaciens FZB42) characterized by Chen, Piel, and Borris, an on-going long-term goal of this project is to assign the activity of the genes responsible for the incorporation of the “ß-branches” in the polyketide synthase gene clusters of B. subtilis ATCC 39374. The overall objective of the work presented in this thesis is to characterize the genes within the dfn gene cluster which we hypothesize to constitute the biosynthetic pathway for the formation of the exomethylene ß-branch in the difficidin structure by the analysis of polyketide intermediates via tandem electrospray ionization mass spectrometry analysis (ESI-MS/MS). The central hypothesis is that the enzyme encoded by bae C catalyzed the transfer of a malonyl acyl group from malonyl-CoA to holo form of the acyl carrier protein dfnX, which is subsequently decarboxylated by the enzyme encoded by yhdS to yield acetyl-S-DfnX (Ac-S-DfnX). What has previously not been shown in preliminary data involves the gene product of dfnL hypothesized to catalyze the reaction of acetoacetyl-S-DfnJ-T2 (Acac-S-DfnJ-T2) and Ac-S-DfnX to yield a 3-hydroxymethylglutaryl-S-DfnJ-T2-like (HMG-S-DfnJ-T2) intermediate. The subsequent dehydration and the decarboxylation of HMG-S-DfnJ-T2 is catalyzed by the enzymes encoded by baeH and dfnM respectively, which is hypothesized to produce an external olefin containing isopentenyl derivative resulting in a difficidin-like “ß-branch”. Whereas, the catalysis of this reaction by the genes baeH and baeI is hypothesized to produce an internal olefin containing isopentenyl derivative bacillaene-like “ß-branch”. The scope of this project only encompasses the assignment of activity to the genes responsible for the formation of this internal or external olefinic isopentenyl product, however further structural characterization of the gene-specific isopentenyl derivatives beyond analysis of MS3 fragmentation patterns are a future goal for the lab. The rationale that underlies the research proposed here is that the characterization of the activity of the dfn gene cluster in B. subtilis ATCC 39374 would give a much deeper and strain specific understanding of the formation of the polyketide “ß-branch” in difficidin, rather than a strict reliance on the sequencing data and proposed order of PKS modules within the dif cluster of B. velezensis. [This abstract has been edited to remove characters that will not display in this system. Please see the PDF for the full abstract.]

Additional Information

Language: English
Date: 2020
Beta-branching, Biosynthetic Gene Clusters, Difficidin, Polyketide Biosynthesis
Polyketides $x Synthesis
Bacillus (Bacteria) $x Genetics

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