(160al) Medium Throughput Combinatorial Gene Knockout Strategy to Improve Polyketide Production

Knocking out competing pathway genes is a proven method to direct metabolite flux through a desired pathway. Selection of genes by rational design limits knockout options as it is difficult to fully predict the effects of a gene deletion on various metabolic pathways. Additionally, knocking out genes one at a time or sequentially is time-consuming without knowing if the selected knock out will be advantageous or not. In contrast, utilizing a combinatorial knockout method greatly allows for quick determination of advantageous genes or combinations of genes to delete. Our method uses CRISPR/Cas9 to knock out all combinations of a group of genes, opening up the possibility to discover unexpected advantageous pathway modifications. The method employs a simple medium-throughput cultivation using 48 well plates, screening 3x coverage of possible knockout combinations. The cultivated stains are then screened for product production using a plate reader. After identification of improved strains, the knockouts are identified and confirmed by PCR. This method was tested using production of the polyketide triacetic acid lactone (TAL) but can be adapted for use with any product that can be analyzed by absorbance. We have also adapted the method gene downregulation via CRIPSRi that can be utilized when gene knock outs are detrimental to growth. The simple medium-throughput cultivation is the same and gene knock downs are determined by gRNA plasmid extraction and Sanger sequencing.