Fig. 3: Xylose utilization, antibiotic susceptibility, plasmid transformation, and CRISPR-Cas9 editing of adipic acid tolerant Pichia.
a Xylose utilization, antibiotic susceptibility and transformation efficiency of adipic acid tolerant Pichia spp. Strains are sorted by adipic acid tolerance (AUC in YPD containing 20 g L−1 adipic acid) and xylose utilization and susceptibility to four common yeast antibiotics is shown. Transformation efficiency using a plasmid conferring HygR is provided rounded to the nearest order of magnitude. Transformants of P. kudriavzevii 2658 NCYC and P. membranifaciens NCYC55 were not detected (ND). Repeating plasmid transformation and antibiotic susceptibility assays routinely yielded similar results. b ADE2 deletion strategy utilized in adipic acid tolerant Pichia. Large homology regions (~800 bp) were designed flanking the ADE2 coding sequence. c Structure of ADE2 pCas CRISPR-Cas9 vectors containing precloned ade2Δ donor cassettes. Donor cassettes were precloned into the BglII site of pCas vectors. d Gap repair strategy of pCas vector assembly in adipic acid tolerant Pichia. pCas vectors harboring precloned ade2Δ donor cassettes were linearized by digestion with BsaI or NotI and used to transform Pichia species along with an overlapping ADE2 gRNA PCR product. e Deletion of ADE2 yields pink colonies in adipic acid tolerant Pichia. Transformants were plated onto YPD agar plates containing hygromycin without adenine supplementation. A representative YPD agar plate containing P. occidentalis Y-7552 is shown. f Screening ADE2 deletion in adipic acid tolerant Pichia. A pCas-Hyg-CEN6ARS4 vector possessing an ADE2 gRNA and repair donor was transferred to P. kluyveri and three strains of P. occidentalis. The ADE2 locus was screened for deletion in randomly selected HygR transformants prior to color development. Source data are provided as a Source Data file.
