No abstract

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

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q O/V = 3 and 10 vvm, and agitation rate was N = 900 min(-1); while the other one was based on constant dissolved oxygen concentrations, C DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ o = 0.15 h(-1). The highest cell concentration was obtained as 44 g L(-1) at t = 9 h of the glucose fed-batch phase at C DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L(-1) and 126 U g(-1) cell, respectively at C DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K L a = 0.045 s(-1) and OUR = 8.91 mmol m(-3) s(-1), respectively.