Fig. 4
From: Metabolic engineering of Ashbya gossypii for limonene production from xylose
CRISPR/Cas9 genomic edition of ERG20. A A synthetic sgRNA-dDNA fragment was designed to introduce the selected nucleotide substitutions (red). The ERG20 genomic locus is shown and both the target sequence and the PAM sequences are indicated. The donor DNA (dDNA) is shown with the designed mutations. The sequences corresponding to the F95W and N126W amino acid changes are highlighted in red. The sequence of the primer used for the analytical PCR is shown in blue. B The effect of the overexpression of the native ERG20 gene and the mutant alleles erg20F95W and erg20N126W were evaluated in the parental strain A1206. The production of limonene in the engineered strains was measured in flask cultures grown for 72 h with 0.5% glucose plus 2% xylose as the carbon sources. The results are the means of two independent experiments performed in duplicate