(32b) Genetic Engineering of Pseudomonas Putida for the Heterologous Production of Polyketides and Non-Ribosomal Peptides

Pseudomonas putida has shown promise as a heterologous host for the production of polyketides and non-ribosomal peptides, natural products that commonly have medically-relevant bioactivities. Unfortunately, titers of polyketides and non-ribosomal peptides produced heterologously in P. putida are often low compared to native hosts. Recent reports were potentially hindered by poorly characterized genetic tools, such as weak synthetic promoters and ribosome binding sites and unstable replicative vectors. However, there has recently been substantial progress in the development of genetic tools for P. putida, including characterized plasmids, constitutive and inducible promoters, and several CRISPR-based genome-editing methods. To demonstrate the utility of our own Cas9-assisted genome-editing method for integrating heterologous gene clusters into P. putida, we have introduced a biosynthetic gene cluster (BGC) responsible for the synthesis of prodigiosin, a hybrid polyketide/non-ribosomal peptide with antibacterial and antitumor properties. Prodigiosin is a red pigment that is easily detectable and provides a facile screen for identifying mutants of P. putida with altered production of prodigiosin. We are actively screening for mutants that improve or reduce prodigiosin production to better understand how to engineer P. putida for the heterologous production of polyketides and non-ribosomal peptides. We have also targeted other BGCs from other proteobacteria for heterologous expression, including a BGC encoding the biosynthesis of the yellow pigments, anthraquinones.