Chassis strain engineering for heterologous production of cyclic lipopeptides (CLiPs)

Cyclic lipopeptides (CLiPs) represent a unique and major class of microbial amphiphilic secondary metabolites produced by bacteria and fungi. Unlike typical peptides, CLiPs are synthesized by mega-modular Nonribosomal Peptide Synthetases (NRPSs). These enzymes are encoded by large biosynthetic gene clusters (BGCs) found within extensive segments of microbial genomes. CLiPs exhibit vast potential as bioactive compounds with diverse applications, mainly as biocontrol agents. However, their production by microorganisms is often constrained, resulting in the synthesis of limited quantities, often as mixtures of two or three structurally distinct CLiPs, or some of them remain cryptic. To address these challenges, cell-bioengineering techniques are employed to enhance expression rates, optimize production and purification processes, and obtain pure CLiPs, enabling the elucidation of their mechanisms of action and the utilization of their properties. One effective approach to overcome these challenges involves heterologous production, where BGCs containing NRPS biosynthesis genes are introduced into a host strain capable of robust expression. In this study, the BGCs responsible for producing putisolvin CLiP (40.5 kb) from the native strain Pseudomonas ekonensis COR58 and bananamide CLiP (27.7 kb) from the native strain Pseudomonas azadiae SWRI103 were integrated into a conjugative plasmid, which includes a strong constitutive promoter (pEforRed) and flanking regions of the pyoverdine (PVD) gene cluster for future heterologous expression in the host Pseudomonas fluorescence 2TR3. The resulting plasmids, namely pPso and pBan, contained the full BGCs of putisolvin and bananamide, respectively. The antimicrobial and enzymatic activity of the Pseudomonas strains were also tested to understand their biocontrol potential. Pseudomonas ekonensis COR58 showed higher inhibition activity than Pseudomonas azadiae SWRI103 and Pseudomonas fluorescence 2TR3, while also producing antimicrobial hydrogen cyanide.