Alternative Synthetic Biology Bacterial Host Development

Herein is described efforts towards developing an alternative bacterial host (synthetic biology chassis) based on Burkholderia sp. FERM BP-3421. As described in Chapter 2, genomic, epigenomic, and metabolomic data sets were used to guide the discovery of a novel nonribosomal peptide from Burkholderia sp. FERM BP-3421. The novel lipopeptide and its corresponding biosynthetic gene cluster were named selethramide, based upon the structure of the molecule mostly consisting of iterative serines, leucines, and a single threonine. Knockout of the selethramide biosynthesis gene selB effectively halted production of selethramide. The selethramide knockout mutant displayed a phenotypic defect in swarming motility. To unequivocally link selethramide to the swarming ability of Burkholderia sp. FERM BP-3421, chemical complementation assays were performed in which swarming was restored in the selethramide deletion mutant when agar was supplemented with phenotypic concentrations of selethramide. The identification of selethramide expands knowledge of autologous natural product biosynthesis. Additionally, as described in Chapter 3, transcriptomic data sets were used to guide genome minimization. Transcriptomic data sets were collected on the Burkholderia sp. FERM BP-3421 wild type as well as two mutants in which production of the previously known, endogenous compound spliceostatin was halted by either 1) deletion of polyketide synthase biosynthesis genes (fr9DEF), or 2) deletion of a transcriptional activator gene (fr9A). Transcriptomic data sets were analyzed to determine how deletion of the genes affected transcription across the Burkholderia sp. FERM BP-3421 genome. fr9A-, fr9DEF-, and selB- mutants were then used to test heterologous expression capacity in comparison to the wild type. The results reported here set the stage for continued Burkholderia sp. FERM BP-3421 host development.