*4.5. Cloning, Heterologous Expression and Purification of Recombinant GOX Proteins*

The obtained *cp*-*gox* gene was subsequently cloned without a stop codon into the expression vector pASG-IBA43plus. The resulting strep-tagged fusion proteins were generated in *E. coli* strain BL21 GOLD and purified using the Strep-tactin matrix, according to the supplier's protocol (IBA Bio technology, Göttingen, Germany). The *gox* gene of *Spirogyra* and the gene encoding the ancestral GOX protein were also cloned into pASG-IBA43plus. However, the N-terminal His-tag was used for purification with Ni-NTA Sepharose, according to the supplier's protocol (Invitrogen, Karlsruhe, Germany).

For the production of Sp-GOX and Cp-GOX, the recombinant *E. coli* strain was grown in an LB medium to an OD750 of 0.6. The gene expression was initiated with the addition of 200 μg/L anhydrotetracycline for 4 to 16 h at 20 to 30 ◦C. A soluble protein of the ancestral N3-GOX could be only obtained when cultivating *E. coli* cultures at 18 ◦C for 16 h after induction with anhydrotetracycline. *E. coli* cells were harvested by centrifugation and re-suspended in buffer A (20 mM Tris-HCl, 500 mM NaCl, 1 mM dithiothreitol (DTT), and 0.1 mM Flavin mononucleotide (FMN)) in case of His-tag fusion proteins or buffer W (100 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.1 mM FMN) in case of Strep-tag proteins. The protein extraction was done by ultrasonic treatments (4 times 30 s, 90 W) on ice. After centrifugation (20,000 g, 20 min, 4 ◦C, Sorvall SS34 rotor), the cell-free protein extract was directly loaded onto Ni-NTA or Strep-tactin columns. The His-tagged proteins were washed using buffer A supplemented with 40 to 80 mM imidazole, whereas His-tagged GOX was eluted with buffer A supplemented with 200 mM imidazole. For Strep-tagged proteins, the washing steps were performed using the buffer W, and the proteins were eluted by buffer W, supplemented with 2.5 mM desthiobiotin. Subsequently, the eluted proteins were desalted using PD-10 columns (GE Healthcare, Schwerte, Germeny), and finally dissolved in 20 mM Tris/HCl pH 8.0 containing 1 mM DTT and 0.1 mM FMN. The purity of the eluted proteins was checked by SDS-PAGE and staining with Coomassie Brilliant Blue (Figure S8).

#### *4.6. Enzyme Activity Assays*

The GOX or LOX activity was measured by the detection of O2 consumption in the presence of different concentrations of l-lactate and glycolate using Hansatech oxygen electrodes (Oxygraph), as described by Hackenberg et al. [30]. One unit of enzyme activity was defined as the consumption of 1 μmol O2 in 1 min at 30 ◦C. The protein concentrations were estimated according to the literature [73].
