SignificanceNatural products biosynthesized by cryptic gene clusters represent a largely untapped source for drug discovery. However, mining of these products by promoter engineering is restricted by the lack of streamlined genetic tools, especially in nonmodel biosynthetic gene cluster (BGC)-rich bacteria. Here, we describe the discovery of a pair of bacteriophage recombinases and application of recombinase-assisted promoter engineering to rapidly identify and activate several cryptic biosynthetic gene clusters in two Burkholderiales strains that currently lack effective genetic tools. Construction of an efficient genome engineering platform in a natural product producer expedites mining of cryptic BGCs in their native backgrounds, and host melioration for yield or structure optimization. This strategy enables potentially scalable discovery of novel metabolites with intriguing bioactivities from many other bacteria.

Summary The limited efficiency of the available tools for genetic manipulation of Pseudomonas limits fundamental research and utilization of this genus. We explored the properties of a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (RecTE Psy ) from Pseudomonas syringae pv. syringae B728a. Compared with RecTE Psy , the BAS operon was functional at a higher temperature indicating potential to be a generic system for Pseudomonas . Owing to the lack of RecBCD inhibitor in the BAS operon, we added Redγ or Pluγ and found increased recombineering efficiencies in P . aeruginosa and Pseudomonas fluorescens but not in Pseudomonas putida and P . syringae . Overexpression of single-stranded DNA-binding protein enhanced recombineering in several contexts including RecET recombination in E . coli . The utility of these systems was demonstrated by engineering P. aeruginosa genomes to create an attenuated rhamnolipid producer. Our work enhances the potential for functional genomics in Pseudomonas .

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

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.

The lambda phage Red proteins Reda/Redb/Redc and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecEThe BDU8 , RecETh TJI49 and RecETh1h2e YI23 , which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecETh TJI49 and RecETh1h2e YI23 showed higher recombination efficiency compared to RecEThe BDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2e YI23, RecETh TJI49 and RecEThe BDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2e YI23 . Additionally, RecET YI23 combined with exonuclease inhibitors Pluc or Redc exhibited equivalent recombination efficiency compared to Redcba in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.