Stabilizing porcine interferon-α production byPichia pastoriswith an ethanol on-line measurement based DO-Stat glycerol feeding strategy

Abstract

BACKGROUND Achievement of very high cell concentration is the prerequisite for enhanced porcine interferon‐α (pIFN‐α) production by Pichia pastoris, but it is closely associated with severe ethanol accumulation, leading to instability in pIFN‐α expression. RESULTS By analyzing the transcriptional levels of genes encoding the key enzymes in methanol metabolism, it was found that high (more than 6 g L−1) and long term (more than 4 h) ethanol accumulation in the late glycerol feeding cultivation phase irreversibly repressed alcohol oxidase (AOX) promoter, leading to instability in pIFN‐α production. A novel improved DO‐Stat glycerol feeding strategy based on on‐line ethanol measurement was thus proposed to control ethanol concentration at a low level of 2 g L−1, while maintaining cell growth at comparably high level. With the aid of the proposed strategy, maximum pIFN‐α concentration increased 29.8–75.5% compared with the best one adopting the ‘traditional’ DO‐Stat strategy, when the same methanol induction strategy (methanol/sorbitol co‐feeding ratio of 1:1) was applied. CONCLUSION The proposed improved DO‐Stat strategy is effective in simultaneously achieving very high concentration of the cells with functional cellular skeletons and repressing ethanol accumulation in the glycerol feeding phase, which enhanced and stabilized pIFN‐α production during the induction condition in turn. © 2013 Society of Chemical Industry

Improvement of poly-γ-glutamic acid biosynthesis in a moving bed biofilm reactor by Bacillus subtilis NX-2

“…Various researchers have adopted the DO-stat feeding strategy to supplement substrate, especially for aerobic fermentation of an oxygen-sensitive and high-density cell culture (Ding et al, 2014). The basic principle is that the rise of DO concentration caused by substrate deficiency signals addition of the substrate (Sun et al, 2006).…”

Enhancement of pullulanase production from recombinant Bacillus subtilis by optimization of feeding strategy and fermentation conditions

“…Concerning the feeding mode, we used two methods for comparison, DO-Stat feeding and constant-rate feeding (Ding et al 2014). Both feeding strategies were conducive to the accumulation of fermentation products.…”

Effect of Ethanol Accumulation on Porcine Interferon-α Production by Pichia pastoris and Activities of Key Enzymes in Carbon Metabolism

“…Measurement methods of cell density, ethanol, and methanol concentrations were the same as those described in the previous report [9]. Total protein concentration was detected by the method of Coomassie brilliant blue G-250 [11].…”

“…The effect of high ethanol concentration could not be removed although ethanol had been depleted before induction, because high and long-term ethanol concentration environment could damage the fluidity of membrane protein and irreversibly destroy cytoskeleton of recombinant P. pastoris [9]. As shown as Fig.…”

“…It was reported that glycerol in excess and oxygen limited environment in glycerol feeding phase caused ethanol accumulation, which decreased targeted protein production [9]. To further investigate the impact of ethanol accumulation on the following induction, several fermentations were performed with identical induction operation, but with different ethanol concentration control patterns in their growth phase.…”