Methanol addition after glucose depletion improves rPOXA 1B production under the pGap in P. pastoris X33: breaking the habit

The purpose of this study was to demonstrate that methanol addition after glucose depletion has a positive effect on improving rPOXA 1B production under the control of pGap in P. pastoris. Four different culture media (A, B, C and D) were used to culture P. pastoris X33/pGapZαA-LaccPost-Stop (clone 1), containing a previously optimized POXA 1B synthetic gene coding for P. ostreatus laccase, which after glucose depletion was supplemented or not with methanol. Enzyme activity in culture media without methanol (A, B, C and D) was influenced by media components, presenting activity of 1254.30 ± 182.44, 1373.70 ± 182.44, 1343.50 ± 40.30 and 8771.61 ± 218.79 U L⁻¹, respectively. In contrast, the same culture media (A, B, C and D) with methanol addition 24 h after glucose depletion attained activity of 4280.43 ± 148.82, 3339.02 ± 64.36, 3569.39 ± 68.38 and 14,868.06 ± 461.58 U L⁻¹ at 192 h, respectively, representing an increase of approximately 3.9-, 2.4-, 3.3- and 1.6-fold compared with culture media without methanol. Methanol supplementation had a greater impact on volumetric enzyme activity in comparison with biomass production. We demonstrated what was theoretically and biochemically expected: recombinant protein production under pGap control by methanol supplementation after glucose depletion was successful, as a feasible laboratory production strategy of sequential carbon source addition, breaking the habit of utilizing pGap with glucose.