Pseudomonas putida has shown promise as a heterologous host for the production of polyketides and nonribosomal peptides, natural products that commonly have bioactivities relevant to medicine. Like other heterologous hosts, titers of polyketides and nonribosomal peptides produced heterologously in P. putida remain low compared to the native organism. Recent projects were likely hindered by nonoptimal genetic manipulation to introduce an active gene cluster to the host, such as weak synthetic promoters and ribosome binding sites, unstable episomal vectors, and non-specific chromosomal integration methods. 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 CRISPR/Cas9-assisted genome-editing method for integrating heterologous gene clusters into P. putida, we are introducing the gene cluster expressing a nonribosomal peptide synthetase (NRPS) that produces prodigiosin, a red pigment with antibacterial and antitumor properties. Prodigiosin production yields a deep-red color that is easily detectable and provides an opportunity for rapidly screening mutants of P. putida that improve the production of prodigiosin. We are actively screening for mutants that improve or abolish prodigiosin production to better understand how to engineer P. putida for the heterologous production of polyketides and nonribosomal peptides.

Genetic engineering of Pseudomonas putida for the heterologous production of polyketides and nonribosomal peptides

2019 Spring National Meeting

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