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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/ pGapZα A- 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.

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