Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris

Abstract

Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing.

Improved yield of recombinant merozoite Surface protein 3 (MSP3) from Pichia pastoris using chemically defined media

“…These findings are in good agreement with those observed by others that high biomass production does not always translate to a high yield of secreted recombinant proteins. Therefore, critical factors in addition to or other than methanol feeding strategies, as probed by Trinh et al (2002) and Zhang et al (2000), need to be scrutinized in order to improve product yield.…”

“…Although it is easy for P. pastoris cells to achieve high biomass with these medium recipes, it is not always straightforward when it comes to recombinant protein synthesis in fermentors. Trinh et al (2002), during their production of mouse endostatin, have compared unlimited with limited methanol feeding strategies for P. pastoris fed-batch process. The unlimited methanol feeding strategies, in which two-to three-fold more methanol was used, resulted in a two-fold higher biomass production but a two-fold decline in specific productivity of endostatin.…”

A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

“…There are two different techniques for using an organic vapor sensor to methanol monitoring in fermentation broth. First, detecting methanol vapor that permeates from the culture broth across a silicone tube (Guarna et al, 1997;Trinh et al, 2003), or by using a probe inserted into the bioreactor (Hellwig et al, 2001;Mayson et al, 2003). Second, detecting directly the methanol vapor in stream gas outlet (Curvers et al, 2002;Katakura et al, 1998;Zhang et al, 2000a).…”

“…This approach is based on simple cell growth models without any online information about the system (d' Anjou and Daugulis, 2001;Sinha et al, 2003;Trinh et al, 2003;Zhang et al, 2000a).…”

Developing high cell density fed‐batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source‐regulated FLD1 Promoter

“…Although several fed-batch cultures control strategies for PAOX-based P. pastoris systems have been described, e.g., those based on the on-line measurement of the residual methanol concentration in the cultivation broth (Chung, 2000;Trinh et al, 2003;Zhang et al, 2002), or those based on the dissolved oxygen (DO) spike method or preprogrammed linear feed rates (Rodríguez-Jiménez et al, 1997), to our knowledge, there are no substrate addition strategies for heterologous protein production fed-batch processes using PFLD-based P. pastoris systems.…”