Project description:Polyketide synthases (PKSs) are modular enzymes with exceptional potential as biocatalysts for producing non-native compounds. Here, we report the first PKS-based pathway for adipic acid (AA), an industrial monomer for nylon production, by engineering one of the most extensively hybridized PKS systems to date. Using a retrobiosynthetic approach, we identified EtnB, a succinyl-CoA loading module that uniquely retains the terminal carboxyl group, enabling access to dicarboxylic polyketide products, rarely produced by canonical PKSs. EtnB was coupled to a fully reducing extension module through an engineered communication linker, which improved ACP–KS interactions, enhanced titers, and demonstrated selective S-CoA loading in vivo. This construct integrates genes from five organisms, seven PKS modules joined across six non-natural junctions, and functions in both Escherichia coli and Pseudomonas putida. Additional engineering that included AT domain swaps, optimization of extender unit supply, and host strain metabolic rewiring further improved AA titers into the low mg/L range. Together, this work demonstrates that highly chimeric PKSs can be rendered functional through rational design, expands the PKS toolkit with a carboxyl-retaining loading module, and establishes a versatile platform for engineering diacids and other non-canonical products through PKS pathways.
Project description:ErfA is a transcription factor of Pseudomonas aeruginosa. We here define the genome-wide binding sites of ErfA by DAP-seq in Pseudomonas aeruginosa PAO1 and IHMA87, Pseudomonas chlororaphis PA23, Pseudomonas protegens CHA0 and Pseudomonas putida KT2440.
Project description:Various species of the intestinal microbiota have been associated with the
development of colorectal cancer (CRC), yet a direct role of bacteria in the
occurrence of oncogenic mutations has not been established. Escherichia coli can
carry the pathogenicity island pks, which encodes a set of enzymes that
synthesize colibactin. This compound alkylates DNA on adenine residues and
induces double strand breaks in cultured cells. Here, we exposed human intestinal
organoids to genotoxic pks+ Escherichia coli by repeated luminal injection over a
period of 5 months. Whole genome sequencing (WGS) of clonal organoids before
and after this exposure reveals a distinct mutational signature, absent from
organoids injected with isogenic pks-mutant bacteria. The same mutational
signature is detected in a subset of 3668 human metastatic cancer genomes,
predominantly in a subset of CRC cases. Our study describes a distinct mutational
signature in CRC and implies that the underlying mutational process directly
results from past exposure to bacteria carrying the colibactin-producing pks
pathogenicity island.
Project description:We observed that deletion of polyketide synthase (pks) from E. coli NC101 reduces its ability to induce tumors in interleukin-10 knockout (Il10-/-) mice injected with azoxymethane (AOM), without altering histologic inflammation. The goal of this experiment is to assess inflammatory cytokine levels in colonic tissue of these mice. 2 germ-free Il10-/- mice were assayed and used as controls. 3 E. coli NC101 and 3 E. coli NC101-delta-pks monoassociated mice were experimental samples.
Project description:The goals of this dual-seq experiment were to 1) identify transcriptional changes between mono-species and dual-species biofilms of Candida albicans and Pseudomonas aeruginosa and 2) identify transcriptional changes within mono- or dual-species P. aeruginosa biofilm cells in response to meropenem treatment.
Project description:We observed that deletion of polyketide synthase (pks) from E. coli NC101 reduces its ability to induce tumors in interleukin-10 knockout (Il10-/-) mice injected with azoxymethane (AOM), without altering histologic inflammation. The goal of this experiment is to assess inflammatory cytokine levels in colonic tissue of these mice.
