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Wild-type cells (JY450) were transformed with the resulting DNA fragments, which carried no annotated neighbouring gene, and transformants resistant to G418 (indicating the gain of the kanamycin cassette) were isolated at 25°C.

Thereby, a total of 60 P. pastoris transformants resistant to G418 of 4.0 mg mL−1, separately containing Aoxyn11A G21I, Aoxyn11A Y13F and Aoxyn11A G21I–Y13F, were picked out for expression tests.

Several transformants resistant to both gentamicin and tetracycline, and exhibiting a disperse colony morphology were isolated (Fig. 1D).

More than ten transformants resistant to hygromycin B were obtained and verified by PCR (data not shown).

Therefore, all P. pastoris transformants, resistant to 1.0, 2.0, and 4.0 mg mL-1 of G418, were picked out for flask expression tests.

Transformants resistant to chloramphenicol and sensitive to sucrose were selected on LB agar media containing chloramphenicol and sucrose and grown on LB agar media without NaCl or antibiotics to select a strain resistant to sucrose and sensitive to chloramphenicol.

The pRS314, pRS314DNA2, pRS314dna2K1080E, and pRS314dna2K1080E,E675A transformants were resistant to IR, whereas the transformant containing the pRS314dna2E675A plasmid was sensitive to IR.

Five transformants remained resistant to aztreonam, indicating that an additional resistance mechanism (e.g., AmpC or ESBL) was also carried on the NDM-encoding plasmid because MBLs do not independently hydrolyze aztreonam.

Most transformants are resistant to drugs for other (unknown) reasons [ 13].

Transformants were resistant to ampicillin, cefazolin, and pipericillin/tazobactam but susceptible to third- and fourth-generation cephalosporins.

The transformants were resistant to 2 mg ml-1 G418, with no lag phase, indicative that the rRNA promoter was driving high level expression of the neo gene.