Journal: ACS Synthetic Biology

Article Title: PYR1 Biosensor-Driven Genome-Wide CRISPR Screens for Improved Monoterpene Production in Kluyveromyces marxianus

doi: 10.1021/acssynbio.4c00797

Figure Lengend Snippet: Biosensor-driven genome-wide screens reveal knockouts that improve geraniol production. Hits from the growth-based screen were investigated and evaluated for terpene production. (a) A 10-fold coverage genome-wide library was transformed into our growth-based screening strain with HAB1/PYR1 Geraniol on a plasmid. If a knockout improves terpene production, strong growth will be observed on selective plates. (b) Geraniol production of gene knockout hits. Six hits exhibited significantly ( p < 0.05) more geraniol production than the base strain, while four hits (* represents p < 0.05) produced less geraniol. Samples were taken after 16 h of growth in 250 mL shake flasks with 50 mL YPD and 2 mL dodecane overlay grown at 220 rpm, 30 °C. The dodecane layer was extracted and ran on GC-FID to extrapolate geraniol production in the aqueous phase.

Article Snippet: Strains without plasmids were grown in YPD media (10 g L –1 yeast extract, 5 g L –1 peptone; DB Difco, Becton-Dickinson, 20 g L –1 glucose).

Techniques: Genome Wide, Transformation Assay, Plasmid Preparation, Knock-Out, Gene Knockout, Produced