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High-intensity green fluorescence was observed in the transformants by using fluorescence and laser confocal microscope.

Plasmid extraction was performed from each qnr-positive isolate and its corresponding transformants by using the Kieser technique (22 ).

Additional β-lactamase genes carried in parental strains and transformants were identified by using the Check-MDR CT101 microarray assay (23 ) (Table 4).

Putative transformants were identified by using blue-white screening of XL1-Blue Cells (Stratagene, La Jolla, CA, USA), grown overnight in 5 mL LB medium with ampicillin (100 μg/μL) and purified by using Wizard Plus Miniprep DNA Purification System (Promega).

The Δ gng1 transformant was confirmed by using primers gng1cDNA1F/gng1cDNA1R.

Electroporation and selection of transformants were carried out by using MD and G418.

Selected colonies were picked off and confirmed as positive transformants by colony PCR using gene-specific primers.

Transformants generated using the CRISPR Cas9 system were screened by colony PCR using the primer pair P_Km01-010 + P_Km01-010-011 (Additional file 1: Table S1).

Recombined plasmids were harvested from yeast by pooling all transformant colonies using a Qiagen miniprep kit modified by including a lysis step in which the harvested yeast were vortexed for 5 min the presence of glass beads.

Compared to the transformant TYEGLAC3-1 expressing the lac3 gene by the constitutive GPD promoter, transformants using an inducible GAL1 promoter revealed higher levels of laccase activity.

Inserts were amplified by colony PCR of transformants using vector-specific primers (p-topo2.1).