Methanol feeding strategy design enhances recombinant human growth hormone production by Pichia pastoris

Abstract

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

Hybrid fed‐batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high‐cell‐density fermentations

“…and SDS‐PAGE analysis. Protease activities and concentrations were determined as described elsewhere . All samples were analysed three times and the average values were given in the figures.…”

“…Protease activities and concentrations were determined as described elsewhere. 23,24 All samples were analysed three times and the average values were given in the figures. The significance of differences were calculated using Student's t-test (P-values <0.07), and shown in the figures.…”

Feeding strategy design for recombinant human growth hormone production by Bacillus subtilis

“…By E. coli [28], 63.2 g L -1 cell and 4.9 g L -1 rhGH concentrations were obtained in a fed-batch cultivation. Recently, a methanol-stat feeding strategy was designed in our research group by r-P. pastoris [29], and the highest rhGH production was reported as C rhGH = 1.3 g L -1 when the cell concentration reached to C X = 82 g L -1 . The Table 2 Characteristics and performances of the designed feeding strategies Strategy Batch operation cultivation time prior to fed-batch operation, t (h) Initial glucose for batch operation at t = 0, m G,0 (g)…”

Comparative study of μ‐stat methanol feeding control in fed‐batch fermentation of Pichia pastoris producing HBsAg: an open‐loop control versus recurrent artificial neural network‐based feedback control

“…For P. pastoris with P AOX1 , various methanol feeding strategies have been proposed. These strategies are mainly performed by controlling one of the important key factors, including the specific growth rate ( μ ‐stat), the dissolved oxygen (DO‐stat), the methanol concentration (methanol‐stat), the biomass (biomass‐stat) and limited temperature, reviewed extensively in many previous studies . Among the mentioned feeding strategies, μ ‐stat feeding under the optimal value of specific growth rate has shown promising results in terms of process efficiency …”

“…These strategies are mainly performed by controlling one of the important key factors, including the specific growth rate ( -stat), the dissolved oxygen (DO-stat), the methanol concentration (methanol-stat), the biomass (biomass-stat) and limited temperature, reviewed extensively in many previous studies. 6,19,34 Among the mentioned feeding strategies, -stat feeding under the optimal value of specific growth rate has shown promising results in terms of process efficiency. 6,35 For the industrial strain of recombinant P. pastoris with P AOX1 producing intracellular HBsAg, -stat feeding had already been established as one of the suitable methanol feeding strategies.…”