Molecular Biology

First site-saturation libraries were prepared using NNK primer-based mutagenesis. Subsequently, the small-intelligent library creation method⁵³ was applied for obtaining site-saturation libraries with a more homogeneous amino acid distribution. To this end, a set of four mutagenesis primers was used to represent each amino acid by only one codon without stop codons. All primers were designed using the NEBaseChanger online tool. The forward primer was designed to carry the mismatch codon for mutagenesis. Further procedures were done according to the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). The polymerase chain reaction (PCR) program comprised an initial denaturation step at 98 °C for 30 s, followed by 35 cycles of 10 s denaturation at 98 °C, 30 s annealing at the respective annealing temperature given by the NEBaseChanger online tool, and 3 min elongation at 72 °C, terminated by 5 min of final elongation at 72 °C. Template DNA was used at 1 ng together with 10 μM of each forward and reverse primer and the CutSmart buffer provided by the kit at the recommended concentrations. For defined amino acid exchanges, one colony of Escherichia coli (E. coli) transformants, obtained following the manufacturer’s instructions, was used to inoculate 3 mL of lysogeny broth (LB, 10 g L^–1 peptone from casein, 5 g L^–1 yeast extract, 5 g L^–1 NaCl) liquid culture containing 0.025 mg mL^–1 Zeocin. The plasmid was harvested after 16–24 h incubation at 37 °C using the Monarch Plasmid Miniprep Kit (New England Biolabs). For site-saturation libraries, all picked colonies were resuspended in 3 mL of LB medium containing 0.025 mg mL^–1 Zeocin. This resuspension was subjected directly to plasmid preparation following the kit instruction. Plasmids were sequenced to confirm mutagenesis using a commercial sequencing service (Microsynth Austria). Sequences were analyzed using the Benchling online tool (Benchling). Combinatorial libraries of the ChCDH variant gene mentioned above were provided by SESAM Biotech. These included the NAGL loop combinatorial library, shuffled at three positions (A322, G323, L324, each position substituted by A, C, F, G, I, L, M, and V, in a total 511 variants excluding the WT), and the oxygen channel combinatorial library, shuffled at three positions (F326, substituted by C, D, F, H, K, M, N, Q, V, W, and Y; Q597, substituted by C, E, F, H, K, M, N, Q, S, T, W, and Y; T747, substituted by the same amino acids as Q597; in total 1583 variants excluding the WT).

The parent CDH sequence used as a control is consistently referred to as WT regardless of the mutations introduced to improve glucose turnover (C291Y, W295R) and the presence of a C-terminal hepta-histidine tag.

Selected variant-carrying Komagataella phaffii (formerly Pichia pastoris) cells confirmed by the rescreening were streaked out on yeast extract-peptone-dextrose (YPD, 20 g L^–1 peptone from casein, 10 g L^–1 yeast extract, 4 g L^–1 glucose) agar plates containing 0.1 mg mL^–1 Zeocin. After incubation at 30 °C for 3 days, cells were transferred into 20 μL of sterile H₂O using a toothpick, heated to 98 °C for 10 min, then spun down at 16 000g. The supernatant (5 μL) was used as template DNA for the following PCR, further comprising 10 μM of forward and reverse primer, each. The colony PCR primers were designed to have annealing temperatures of 72 °C. Apart from that, the same PCR program as described for the mutagenesis was used. The PCR products were purified using the Monarch PCR and DNA Cleanup Kit (New England Biolabs) according to the manufacturer’s instructions. The purified PCR products were sequenced by a commercial provider (Microsynth Austria). Sequences were evaluated using the Benchling online tool (Benchling).

The library plasmid mixtures were transformed into K. phaffii cells via electroporation after linearization for 1 h by PmeI in CutSmart Buffer (New England Biolabs). To this end, 5 μL of linearized plasmids were added to aliquots of 50 μL electrocompetent ATUM PPS 9011 MutS cells. The device used was the MicroPulser electroporator with Gene Pulser/MicroPulser Electroporation Cuvettes and an electrode gap of 0.1 cm (Bio-Rad Laboratories). The voltage was set to 1.5 kV applied for 3 ms. The transformed cells were resuspended immediately in 500 μL of 1 M ice-cold sorbitol followed by 500 μL of ice-cold YPD. The transformant suspension was incubated at 30 °C for 3 h, then applied to selective YPD agar containing 0.1 mg mL^–1 Zeocin by using sterile glass beads to obtain single colonies. The agar plates were incubated at 30 °C for 3 days.