Establishment of recombineering genome editing system in Paraburkholderia megapolitana empowers activation of silent biosynthetic gene clusters

Abstract

The Burkholderiales are an emerging source of bioactive natural products. Their genomes contain a large number of cryptic biosynthetic gene clusters (BGCs), indicating great potential for novel structures. However, the lack of genetic tools for the most of Burkholderiales strains restricts the mining of these cryptic BGCs. We previously discovered novel phage recombinases Redab7029 from Burkholderiales strain DSM 7029 that could help in efficiently editing several Burkholderiales genomes and established the recombineering genome editing system in Burkholderialse species. Herein, we report the application of this phage recombinase system in another species Paraburkholderia megapolitana DSM 23488, resulting in activation of two silent non-ribosomal peptide synthetase/polyketide synthase BGCs. A novel class of lipopeptide, haereomegapolitanin, was identified through spectroscopic characterization. Haereomegapolitanin A represents an unusual threonine-tagged lipopeptide which is longer than the predicted NRPS assembly line. This recombineeringmediated genome editing system shows great potential for genetic manipulation of more Burkholderiales species to activate silent BGCs for bioactive metabolites discovery.

Genomics-Driven Activation of Silent Biosynthetic Gene Clusters in Burkholderia gladioli by Screening Recombineering System

“…During our ongoing genome mining efforts to discover new bioactive compounds from Burkholderia sensu lato, the model plant pathogen Burkholderia gladioli ATCC 10248, which potentially produces new structures based on bioinformatic analysis, attracted our interests [ 4 , 13 , 14 , 15 , 16 ]. Its genome size is estimated to be 8.9 Mbp with 68% of the G + C content, harboring two chromosomes (NZ_CP009323.1, NZ_CP009322.1) and three free plasmids (NZ_CP009321.1, NZ_CP009320.1, NZ_CP009319.1) [ 17 ].…”

“…Due to the limits of the Red/ET recombination system in the other microorganisms, our group recently established two other recombination techniques based on the homology to the recombinases for Burkholderia genus and Pseudomonas genus, respectively [ 13 , 24 ]. One is Redγ-Redαβ7029, which was discovered from Schlegelella brevitalea DSM 7029 (previously known as Burkholderiales strain) and is workable in several Burkholderiales strains [ 13 , 15 , 25 ]. The other is a lambda Red-like recombination system BAS from Pseudomonas aeruginosa phage Ab31 that was established to carry out genome editing in four Pseudomonas species [ 24 ].…”

Mining for novel antibiotics in the age of antimicrobial resistance

“…For example, recent advances have been focussed on microbial co‐cultivation, high‐throughput elicitor screenings, synthetic biology strategies, the development of new chassis microorganisms for heterologous expression or paired metatranscriptomics and metabolomics of native microbial communities (Rutledge and Challis, 2015; Matilla et al ., 2018; Zhang et al ., 2019; van Bergeijk et al ., 2020). In this regard, a recent report published in Microbial Biotechnology used a novel bacteriophage recombinase system, Redαβ7029, to efficiently exchange the natural promoters of the seven cryptic polyketide synthase/non‐ribosomal peptide synthetase BGCs present in the genome of the antimicrobial producer and plant‐growth‐promoting bacterium Paraburkholderia megapolitana DSM 23488 (Zheng et al ., 2020). This elegant genome editing strategy resulted in the successful activation of two silent BGCs, BGC5 and BGC9.…”

“…These molecules belong to a novel class of lipopeptides and, based on the domain organization of the core biosynthetic proteins encoded in BGC9, the authors proposed a model for the biosynthesis of both haereomegapolitanins. Although the identified haereomegapolitanins did not show antimicrobial properties against the tested bacteria and fungi, the real impact of the study lies in the enormous potential of the Redαβ7029‐based expression strategy for the efficient manipulation of Burkholderiales strains (Zheng et al ., 2020). Burkholderiales are emerging as rich sources of natural products (Blin et al ., 2019).…”

“…Burkholderiales are emerging as rich sources of natural products (Blin et al ., 2019). However, the majority of the BGCs present in their genomes seem to be cryptic (Zheng et al ., 2020) and the lack of efficient genetic tools for the manipulation of native Burkholderiales species is currently hindering the identification of bioactive natural products with potential medical and agricultural importance.…”