| Popis: |
Microbial platforms as a means of chemical production present a cost effective, environmentally friendly, and dynamic strategy for the synthesis of biofuels, pharmaceuticals, and food products. Most bacterial chemical production platforms utilize plasmid vectors for gene expression due to the ease of cloning and optimization. However, these vector-based systems necessitate costly antibiotics for genetic stability, making many processes less financially viable and potentially increasing the prevalence of antibiotic resistance in the environment. Therefore, it is desired to utilize gene expression systems integrated into the genomic DNA of the microbe, eliminating the antibiotic selection requirement. Here, we present a system for high efficiency pseudorandom integration of large metabolic pathways using a hyperactive Tn5 transposase coupled with a Cas9/λ-red recombinase-based method for small, site-specific genomic modifications to enable rapid in vivo transcriptional optimization. The norbaeocystin and psilocybin biosynthetic pathways will be utilized as proofs-of-concept. The process presented herein is designed to be agnostic to the pathway of interest and therefore widely applicable to enhance production of microbial products ranging from commodity chemicals to pharmaceuticals. Here, I show a screening, characterization, and validation pipeline which will result in transcriptionally balanced mutants with production comparable to that achieved through plasmid-based optimization approaches. |
