Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Abstract

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.

Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate

“…Although the former protein is encoded by a putative ldhA gene of P. pastoris (not annotated in S. cerevisiae), considering the fact that ethanol does not accumulate in P. pastoris medium (Cereghino and Cregg, 2000), a direct reduction of the intracellular pyruvate to lactate could be the alternative pathway for the NAD þ regeneration. Thus, lactate was included in the model, since its accumulation was previously observed in P. pastoris extracellular medium (Ç elik et al, 2009;Xie et al, 2005).…”

Fermentation and oxygen transfer characteristics in recombinant human growth hormone production by Pichia pastoris in sorbitol batch and methanol fed‐batch operation

“…Thereafter, Jungo et al 4 performed methanol/sorbitol mixed fed-batch bioreactor experiments for recombinant avidin production using P. pastoris Mut + phenotype and reported that accumulation of sorbitol in the medium does not affect the specific recombinant protein productivity. In a recent study Ç elik et al 5 investigated the effect of batch-wise sorbitol addition in the fed-batch production phase for glycosylated protein, recombinant human erythropoietin (rHuEPO) production by P. pastoris-EPO-Mut + strain, and concluded that the simultaneous utilization of sorbitol and methanol not only decreased the process time but also increased the product yield on substrate.…”

“…2 -5 Typically, either a three-or four-stage fed-batch operating strategy is utilized for the production of r-proteins. 6,2,5 In a three-stage process, first, in order to accumulate cells, the engineered strain is cultured in defined medium containing glycerol, a non-fermentable but repressing carbon source (Phase I). Second, a fed-batch transition phase is applied in which glycerol is fed to the bioreactor at limiting levels to further increase cell concentration (Phase II).…”

High Expression of Human Cathepsin S by Recombinant <b><i>Pichia pastoris</i></b> with Cod Skin as an Organic Co-Nitrogen Source

“…Most studies have reported the mixture of multicarbon substrates for P. pastoris growth and for foreign protein expression [Celik et al, 2009;Wang et al, 2010]. Few reports can be found on alternative convenient organic nitrogen sources for P. pastoris to express heterologous proteins.…”