Discovery of megapolipeptins by genome mining of a Burkholderiales bacteria collection

Abstract

Burkholderiales bacteria have emerged as a promising source of structurally diverse natural products that are expected to play important ecological and industrial roles. This order ranks in the top three in terms of predicted natural product diversity from available genomes, warranting further genome sequencing efforts. However, a major hurdle in obtaining the predicted products is that biosynthetic genes are often 'silent' or poorly expressed. Here we report complementary strain isolation, genomics, metabolomics, and synthetic biology approaches to enable natural product discovery. First, we built a collection of 316 rhizosphere-derived Burkholderiales strains over the course of five years. We then selected 115 strains for sequencing using the mass spectrometry pipeline IDBac to avoid strain redundancy. After predicting and comparing the biosynthetic potential of each strain, a biosynthetic gene cluster that was silent in the native Paraburkholderia megapolitana and Paraburkholderia acidicola producers was cloned and activated by heterologous expression in a Burkholderia sp. host, yielding megapolipeptins A and B. Megapolipeptins are unusual polyketide, nonribosomal peptide, and polyunsaturated fatty acid hybrids that show low structural similarity to known natural products, highlighting the advantage of our Burkholderiales genomics-driven and synthetic biology-enabled pipeline to discover novel natural products.

Fig. 1. Overview of the approach used in this study. (A) Environmental samples (rhizosphere) were collected from British Columbia, Canada. Burkholderiales strains were then isolated from the rhizosphere of root samples using selective media. In the first cycle, 230 isolated strains were analyzed by MALDI-TOF MS/IDBac and 100 strains were selected for genome sequencing based on the analysis of metabolite association networks; the intent of this step was to avoid strain redundancy while maximizing metabolite diversity entering sequencing efforts (ESI Fig. S1†). One hundred draft genome sequences were obtained. Biosynthetic gene clusters (BGCs) were predicted using antiSMASH and the biosynthetic potential of strains was compared in terms of BGC numbers and biosynthetic class. Informed by the predicted biosynthetic potential, cycle 2 targeted any newly isolated genera not included in cycle 1 and species determined to be the most ‘talented’ in cycle 1, resulting in 15 additional strains sequenced from 86 analyzed. See ESI Tables S1–S3 for details on the strains sequenced. (B) Phylogenomic and gene cluster family (GCF) analyses were performed on a total of 115 strains to gain insight into GCF distribution and to prioritize BGCs for discovery. (C) A prioritized BGC with clade-specific distribution that was silent in the native strains was cloned and heterologously expressed in Burkholderia sp. FERM BP-3421. (D) Natural product isolation and structure elucidation yielded megapolipeptins A (1) and B (2).

Fig. 2. Genome library metrics. (A) Genome size by number of BGCs, color coded according to the clades attributed in Fig. 3. R² = 0.18. The top 10% strains most prolific in terms of number of BGCs are highlighted in yellow (P. megapolitana/acidicola) and purple (P. sediminicola/fungorum). (B) Donut charts depicting the total number of either BGCs or (C) GCFs subdivided by biosynthetic class.

Fig. 3. Phylogenomic analysis and BGC distribution. (A) Phylogenomic tree of Burkholderiales strains based on 49 genes within cluster of orthologous groups (ESI Table S6†). The tree was constructed using the neighbor-joining method. Select Paraburkholderia, Herbaspirillum and Caballeronia genomes available in public databases were included in addition to the 115 strains sequenced in this study which are shown with our internal strain numbering scheme. The Paraburkholderia clade was further subdivided into seven monophyletic groups as highlighted. See also ESI Fig. S4 and S5. (B) BiG-SCAPE BGC Sequence Similarity Network within the 115 Burkholderiales genomes (distance cutoff = 0.4). A total of 1388 BGCs are displayed, color-coded according to the clades in panel A. Node shape indicates BGC class according to BiG-SCAPE classification. Known and orphan BGCs described in the text are highlighted. (C) Example of a widely distributed BGC that is part of the core genome of Paraburkholderia strains. BGCs in this family contain three core genes and varying gene neighborhoods. The core genes are predicted to encode the biosynthesis of 2-aminoethyl phosphonate (2-AEP) from phosphoenolpyruvate (PEP) via phosphonopyruvate (PnPy) and phosphonoacetaldehyde. (D) The clade-specific mgp GCF and BGC investigated in this work from genome #76 (ESI Table S1†). See ESI Table S7 for gene details.

Fig. 4. Heterologous expression of the mgp BGC from P. megapolitana RL18-039-BIC-B. (A) Genome map of P. megapolitana RL18-039-BIC-B with the two chromosomes oriented to replication gene dnaA. BGCs are color-coded according to biosynthetic class and numbered in clockwise order from the replication gene (lane 1 from the outside in). Predicted open reading frames (ORFs) on the leading and lagging strands are shown on lanes 2 and 3, respectively. A normalized and skewed plot of guanosine + cytosine (G + C) content (yellow/orange) is depicted in lanes 4 and 5, respectively. (B) Heterologous expression of mgp (BGC 2.11) in Burkholderia sp. FERM BP-3421 Δfr9A. LC-MS analysis of strains containing either the empty vector (pBS003, bottom trace) or the vector containing the mgp BGC (pBS001, top trace). Extracted ion chromatogram (EIC) of region m/z 950–1000.

Fig. 5. Structure elucidation of megapolipeptins from P. megapolitana RL18-039-BIC-B. (A) The structures of megapolipeptin A (1) and megapolipeptin B (2). (B) Key MS/MS fragments showing neutral losses of threonine amino acid residues on the peptidic terminus of 1 and 2. (C) Reaction scheme with TMS diazomethane in DCM/MeOH affording two diastereotopic epoxide-containing products via the Büchner–Curtius–Schlotterbeck reaction. (D) Structure of partial hydrolysis product used to determine configuration of threonine residues. (E) EIC traces of Marfey's derivative of threonine (m/z = 400.15) for partial hydrolysis product, and l-threonine and l-allo-threonine standards.

Fig. 6. Biosynthetic proposal for mgp BGC from P. megapolitana RL18-039-BIC-B. (A) Megapolipeptin biosynthetic gene cluster from P. megapolitana RL18-039-BIC-A (genome #76). (B) Biosynthetic hypothesis based on gene/domain content and the observed structures. The configuration of chiral centers containing hydroxyl groups was predicted based on KR domain type (ESI Fig. S43†). The KR type is indicated with red A, B letters.