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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.

The objectives of this work are the optimization of the codons of xylA gene from Thermus thermophilus to enhance the production of recombinant glucose isomerase (rGI) in P. pastoris and to investigate the effects of feeding strategies on rGI production. Codons of xylA gene from T. thermophilus were optimized, ca. 30 % of the codons were replaced with those with higher frequencies according to the codon usage bias of P. pastoris, codon optimization resulted in a 2.4-fold higher rGI activity. To fine-tune bioreactor performance, fed-batch bioreactor feeding strategies were designed as continuous exponential methanol feeding with pre-calculated feeding rate based on the pre-determined specific growth rate, and fed-batch methanol-stat feeding. Six feeding strategies were designed, as follows: (S1) continuous exponential methanol- and pulse- sorbitol feeding; (S2) continuous exponential methanol- and peptone- feeding; (S3) continuous exponential methanol- and pulse- mannitol feeding; (S4) continuous exponential methanol- and peptone- feeding and pulse-mannitol feeding; (S5) methanol-stat feeding by keeping methanol concentration at 5 g L(-1); and, (S6) methanol-stat feeding by keeping methanol concentration at 5 g L(-1) and pulse-mannitol feeding. The highest cell and rGI activity was attained as 117 g L(-1) at t = 66 h and 32530 U L(-1) at t = 53 h, in strategy-S5. The use of the co-substrate mannitol does not increase the rGI activity in methanol-stat feeding, where 4.1-fold lower rGI activity was obtained in strategy-S6. The overall cell yield on total substrate was determined at t = 53 h as 0.21 g g(-1) in S5 strategy.

BACKGROUND The effects of designed methanol and co‐substrate feeding strategies on fed‐batch recombinant human growth hormone (rhGH) production by Pichia pastoris hGH‐Mut+ were investigated. RESULTS The highest cell concentration was obtained as 157 g L−1 with the exponential feeding strategy where methanol was fed at a pre‐calculated feeding rate based on pre‐determined µ0 = 0.03 h−1, and mannitol concentration was kept constant at 50 g L−1 for t=0–6 h. The highest rhGH concentration was obtained as 1.3 g L−1 at t=54 h with the methanol‐stat feeding strategy, where no co‐substrate was used and the methanol concentration was kept at 5 g L−1 in the medium during the production phase. Thereafter, in another methanol‐stat feeding strategy with pulse‐fed sorbitol, the highest rhGH concentration was obtained as 1.2 g L−1 at t=42 h. CONCLUSION The methanol‐stat fed‐batch method keeping methanol concentration constant at 5 g L−1 in the production medium increased rhGH production at least 2‐fold compared with that of the exponential feeding strategies. In the designed methanol‐stat feeding strategies, use of sorbitol as co‐substrate decreased methanol demand of the cells 2‐fold, increased the overall product yield on the substrate 1.6‐fold, shortened the cultivation time by 12 h and increased the productivity. Thus, methanol‐stat feeding with co‐substrate sorbitol was found to be a favorable fed‐batch strategy for rhGH production by Pichia pastoris under alcohol oxidase I promoter. © 2014 Society of Chemical Industry

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