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Batch-wise sorbitol addition as a co-substrate at the induction phase of methanol fed-batch fermentation by Pichia pastoris (Mut + ) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch-wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag-phase for the cells and reaching 'high cell density production' at t = 24 h of the process (C X = 70 g CDW/l); (b) achieving 1.8-fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2-fold; (d) eliminating the lactic acid build-up period; (e) lowering the oxygen uptake rate two-fold; and (f) obtaining 1.4-fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut + strains of P. pastoris for the production of any bioproduct, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris.

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BACKGROUND: Effects of co-substrate sorbitol different feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut + were investigated by eight designed experiments grouped as: (i) fed-batch methanol feeding without the co-substrate; (ii) fed-batch methanol feeding with pulse sorbitol feeding; (iii) fed-batch methanol feeding together with fed-batch sorbitol feeding at t = 0-15 h, followed by fed-batch methanol feeding; and (iv) fed-batch methanol and sorbitol feeding at t = 0-30 h, followed with fed-batch methanol feeding. RESULTS:The highest rhGH and cell concentrations were achieved, respectively, as 0.64 g L −1 and 105 g L −1 at t = 42 h of induction phase, with the strategy where methanol was fed to the system at a pre-determined feeding rate of µ M0 =0.03 h −1 , and sorbitol concentration was kept at 50 g L −1 at t = 0-15 h of the rhGH production phase where the specific growth rate on sorbitol was µ S0 =0.025 h −1 . The overall cell and product yield on total substrate were found as 0.26 g g −1 and 2.26 mg g −1 , respectively. CONCLUSION: This work demonstrates that co-carbon source, sorbitol, feeding strategy is as important as methanol feeding strategy in recombinant protein production by Mut + strains of P. pastoris.