Optimization of the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system using mixed substrates on controlled fed-batch bioprocess.

In this work a systematic study of the influence of methanol set-point and sorbitol feeding rate in fed-batch operation with a Pichia pastoris Mut(s) strain producing Rhizopus oryzae lipase is presented. Different experiments were made at a constant methanol set-point of 0.5, 2 and 4gl(-1), controlled by a predictive algorithm at two different sorbitol feeding rates to assure a constant specific growth rate of 0.01 and 0.02h(-1), by means of a pre-programmed exponential feeding rate strategy. Lipolytic activity, yields, productivity and specific productivity, but also specific growth, consumption and production rates were analyzed showing that the best values were reached when the methanol set-point was 2gl(-1) with a low influence of the constant specific growth rate tested. The sorbitol addition as a co-substrate during the induction phase avoids the severe decrease of the specific production rate obtained when methanol was used as a sole carbon source and it permits to achieve higher ROL production.

[1]  Francisco Valero,et al.  Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter. , 2004, Journal of biotechnology.

[2]  R. Schmid,et al.  Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. , 2001, Journal of biotechnology.

[3]  A. Subramaniam,et al.  Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. , 1997, Gene.

[4]  J. Cregg,et al.  Heterologous protein expression in the methylotrophic yeast Pichia pastoris. , 2000, FEMS microbiology reviews.

[5]  M. Sauer,et al.  Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris. , 2006, FEMS microbiology letters.

[6]  Qin Ye,et al.  Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production , 2005 .

[7]  Stephen G Oliver,et al.  Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol , 2009, Yeast.

[8]  J. Cregg,et al.  High–Level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast, Pichia Pastoris , 1987, Bio/Technology.

[9]  Urs von Stockar,et al.  A quantitative analysis of the benefits of mixed feeds of sorbitol and methanol for the production of recombinant avidin with Pichia pastoris. , 2007, Journal of biotechnology.

[10]  M. Meagher,et al.  High cell-density fermentation. , 1998, Methods in molecular biology.

[11]  Francisco Valero,et al.  Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review , 2006, Microbial cell factories.

[12]  Francisco Valero,et al.  Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris. , 2007, Journal of biotechnology.

[13]  X. Liu,et al.  Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris , 1997, Applied Microbiology and Biotechnology.

[14]  L. Harvey,et al.  Heterologous protein production using the Pichia pastoris expression system , 2005, Yeast.

[15]  M. Inan,et al.  Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. , 2001, Journal of bioscience and bioengineering.

[16]  Francisco Valero,et al.  A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess , 2006, Biotechnology and bioengineering.

[17]  Brigitte Gasser,et al.  Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter. , 2009, New Biotechnology.

[18]  S. Baldwin,et al.  A Pichia pastoris fermentation process for producing high-levels of recombinant human cystatin-C , 2001 .

[19]  Francisco Valero,et al.  Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 Promoter. , 2005, Biotechnology and bioengineering.

[20]  N. K. Khatri,et al.  Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction , 2007, Microbial cell factories.

[21]  Urs von Stockar,et al.  A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy. , 2007, Journal of biotechnology.

[22]  A. Daugulis,et al.  Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris , 1999, Biotechnology Letters.

[23]  B. Hélène,et al.  High-level secretory production of recombinant porcine follicle-stimulating hormone by Pichia pastoris , 2001 .

[24]  J. Cregg,et al.  Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. , 2005, Journal of biotechnology.

[25]  R. Siegel,et al.  Fermentation Development of Recombinant Pichia pastoris Expressing the Heterologous Gene: Bovine Lysozyme , 1990, Annals of the New York Academy of Sciences.