3.2. Random Mutagenesis

A mutagenic library (~20,000 mutants) was generated with a GeneMorph II Random Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) using αlacA as the template. The library was constructed at a mutation rate of between 0 to 4.5 mutations per 1000 bp. Error-prone PCR (epPCR) was performed in a T100 thermocycler (Bio-Rad Laboratories, Woodinville, WA, USA) at a final volume of 50 µL containing 500 nM each primer, 814 ng template, dNTPs (0.2 mM each), and 2.5 units of Mutazyme II DNA polymerase. PCR was performed under the following conditions: 95 °C for 2 min (1 cycle); 95 °C for 0.5 min, 50 °C for 0.5 min, and 72 °C for 2 min (30 cycles); and, 72 °C for 10 min (1 cycle). The primers OL_pYEα-F and OL_lac-R (Table S2) used for amplification had overhangs of 44 and 66 bp homologous to the vector pYES2 to enable in vivo ligation. PCR products and the linearized pYES2 (linearized by EcoRI and XbaI) were purified and concentrated after electrophoresis by using a gel extraction kit (Omega Bio-tek, Norcross, GA, USA). Subsequently, 300 ng of PCR fragments were mixed with the linearized vector (100 ng) and transformed into S. cerevisiae BJ5465 competent cells by using the Gene Pulser Xcell System (Bio-Rad Laboratories, Woodinville, WA, USA). All of the transformed cells were spread onto selection plates and incubated for three days at 30 °C.