Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Zhang, Wenhui; Potter, Karen J.; Plantz, Bradley A.; Schlegel, Vicki; Smith, Leonard A.; Meagher, Michael M.

doi:10.1007/s10295-003-0035-3

Cited by 94 publications

(61 citation statements)

References 24 publications

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“…For methanol consumption capability, the result showed m was independent of cell density within the range 200-415 g l . This discovery supports that a constant l can be realized in a fed-batch phase by simply carrying out the feeding strategy that is based on the model m=al+m, as observed previously [9][10][11]. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.…”

Section: Discussionsupporting

confidence: 84%

“…13 is about the same as that obtained from fed-batch fermentations of the same strain [8] and also quite similar to other P. pastoris strains [9][10][11]. Based on these results, we can express the relationship as m=al+m, with a=0.73-0.84 and m=0.003-0.013.…”

Section: Discussionsupporting

confidence: 80%

“…Although the actual X c and D obtained from the runs did not exactly match the design due to the reasons discussed above, the models of Q and q corresponding to X c and D were successfully established (Eqs. 10,11) through the ANOVA significance test of the regressions [7]. From the models, we found the optimal X c (328.9 g l )1 ) for the maximum Q was higher than the optimal X c (287.7 g l )1…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Optimization of cell density and dilution rate in Pichia pastoris continuous fermentations for production of recombinant proteins

Zhang

Liu²,

İnan

et al. 2004

J IND MICROBIOL BIOTECHNOL

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This paper provides an approach for optimizing the cell density (X c ) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon s (INF-s). The objective was to maximize the volumetric productivity (Q, mg INF-s l )1 h )1 ), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X c and D within the ranges 150 £ X c £ 450 g cells (wet weight) l )1 and 0.1 l m £ D £ 0.9 l m (l m =0.0678 h )1 , the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X c and D were determined based on the mass balance. From the RSM model, the optimal X c and D were 328.9 g l )1 and 0.0333 h )1 for a maximum Q of 2.73 mg l )1 h )1. The model of specific production rate (q, mg INF-s g)1 cells h )1) was also established and showed the optimal X c =287.7 g l )1 and D=0.0361 h )1 for the maximum q (predicted to be 8.92·10)3 mg )1 g )1 h )1 ). The methanol specific consumption rate (m, g methanol g)1 cells h )1) was calculated and shown to be independent of the cell density. The relationship between m and l (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Optimization of cell density and dilution rate in Pichia pastoris continuous fermentations for production of recombinant proteins

Zhang

Liu²,

İnan

et al. 2004

J IND MICROBIOL BIOTECHNOL

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“…The fourth phase, production, typically requires a continuous feed of methanol. While fermentation methods have been devel oped with glycerol/methanol mixed feeds (Katakura et al 1998;Zhang et al 2003), the typical fermentation strategy uses methanol as the sole source for carbon and energy (Zhang et al 2000a). …”

Section: Introductionmentioning

confidence: 99%

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Plantz

Sinha

Villarete³

et al. 2006

Appl Microbiol Biotechnol

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“…To accomplish this goal, we hypothesized that varying the composition of the rich medium (18) and the fermentation carbon source (27) would attenuate the toxicity of the immunotoxin for host cells. We further hypothesized that a reduction in the temperature (9, 10) during induction would have an enhancing effect on immunotoxin secretion.…”

mentioning

confidence: 99%

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

Woo

Liu

Stavrou

et al. 2004

Appl Environ Microbiol

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The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut ؉ ) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G 4 S) was developed for treatment of T-cell leukemia and autoimmune diseases and tolerance induction for transplantation. This immunotoxin selectively kills human T cells and is the most efficacious of a variety of anti-T-cell immunotoxins that have been developed (20). The bivalent immunotoxin contains the first 390 amino acid residues of diphtheria toxin (DT) and two tandem sFv molecules that are responsible for binding the immunotoxin to the CD3ε␥ subunit of the T-cell receptor complex on human T cells. The first 390 amino acid residues of DT (DT390) contain the catalytic domain or A chain of DT that inhibits protein synthesis by ADP ribosylation of EF-2 and the translocation domain that translocates the catalytic domain to the cytosol by interactions with cytosolic Hsp90 and thioredoxin reductase (17).The inhibition of protein synthesis by the catalytic domain makes it difficult to produce the toxin-related proteins in most eukaryotic cells (e.g., wild-type CHO cells, insect cells, and yeasts). The use of toxin-resistant eukaryotic cells can overcome the immunotoxin toxicity. However, selection and characterization of toxin-resistant eukaryotic cells are tedious, labor-intensive, ...

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Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Cited by 94 publications

References 24 publications

Optimization of cell density and dilution rate in Pichia pastoris continuous fermentations for production of recombinant proteins

Optimization of cell density and dilution rate in Pichia pastoris continuous fermentations for production of recombinant proteins

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

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