Abstract
The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.
Highlights
A limited number of species belonging to the genera Hansenula, Candida, Turolopsis and Pichia are capable of growing on methanol [1,2,3,4]
In the early 70's, the interest in the production of single cell protein (SCP) from methanol as the sole carbon and energy source intensified the studies of these specific strains for two reasons: to explore their possible commercial applications and to study the specific cell compartments which are abundantly present in methanol-growth cells, namely peroxisomes [5]
P. pastoris emerged as important yeast in biotechnology one decade later when Phillips Petroleum together with the Salk Institute Biotechnology/Industrial Associates Inc (SIBIA, La Jolla, CA, USA) used this methylotrophic yeast as a system for heterologous protein expression becoming a successful choice both for academic and industrial purposes [7,8]
Summary
A limited number of species belonging to the genera Hansenula, Candida, Turolopsis and Pichia are capable of growing on methanol [1,2,3,4]. The strategy used in the induction phase was to maintain a constant methanol concentration of 5 g/L and using DO concentration as indicator to avoid over feeding of glycerol, or a preprogrammed exponential feeding rate when sorbitol was used They suggested that lactic acid is a potential nonrepressive carbon source for expression of foreign genes in P. pastoris because the highest angiostatin production level was reached with this substrate, even with lactic acid accumulation up to 6.3 g/L along the fermentation [74]. Glycerol and sorbitol are the most commonly used co-substrates jointly with methanol With both substrates, but specially with glycerol, where the repression effect of foreign protein production is well known, the continuous strategy is to select a dilution rate far enough from μmax to ensure that no glycerol is allowed to accumulate in the broth, and methanol concentrations have to be maintained at levels sufficient to fully induced heterologous protein production, yet no so high as to be inhibitory to cell growth or heterologous protein expression [48]. No bioreactor experiments have been reported and further studies must be necessary to optimize the promoter and the fermentation conditions
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