Details

Autor(en) / Beteiligte

• Cook, Taylor B

Titel

Expressing and Engineering Natural Product Enzymes in Bacterial Hosts

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2021

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Drug discovery has depended primarily on the isolation of natural products from living organisms because these compounds often have inherent properties applicable to modern medicine. Microorganisms from the environment proved to be rich sources of natural products with antibiotic, anticancer, and immunosuppressant activities, but as this resource was exhausted over the last century, researchers have had to resort to alternative strategies for developing novel drugs. One such strategy is to engineer microorganisms to produce non-native natural products of interest. This is a multi-disciplinary effort that applies synthetic biology and metabolic engineering towards the manipulation of natural product chemistry. The biosynthetic pathways for two natural product classes, polyketides and nonribosomal peptides, are particularly challenging to manipulate due to their reliance on large, modular enzymes. In this dissertation, we identified Pseudomonas putida as an ideal host for expressing and engineering these enzymes. To facilitate strain engineering of this bacterial host, we developed a genome editing toolkit that enabled chromosomal deletions, integrations, and point mutations. We then applied these methods towards expressing heterologous pathways in P. putida to produce polyketides and nonribosomal peptides. Chromosomal integration of a heterologous gene cluster responsible for prodigiosin in biosynthesis resulted in a strain capable of producing 1.1 g/L of product. P. putida has a relatively high-GC chromosome, but we found that expression of a low-GC gene cluster for glidobactin A biosynthesis resulted in higher heterologoustiters compared to a related high-GC gene cluster. Further strain and pathway engineering, including improvements to pathway expression and deletion of a native carbon sink, resulted in a strain capable of producing 470 mg/L glidobactin A. P. putida natively produces a siderophore called pyoverdine through a nonribosomal peptide synthetase(NRPS). We utilized this pathway to explore subdomain substitutions for altering substrate specificity in NRPSs. Subdomain boundaries for creating functional chimeras were identified, but the most active variants still performed poorly compared to the wild-type enzyme. The findings discussed here will enable the production of novel compounds through the heterologous expression of uncharacterized pathways and the manipulation of key catalytic domains in modular megaenzymes.