Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

Murray, William D.; Duff, Sheldon J.B.; Lanthier, Patricia

doi:10.1007/bf00164829

Cited by 31 publications

(23 citation statements)

References 13 publications

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“…Cell proliferation was not inhibited until the methanol concentration reached >3% (Murray et al, 1989). Our study also indicated that growth was not impaired with 1.0% methanol compared to 0.5%, and the cells grew twice as fast with 1.0% (l max 0.026 h )1 ) as with 0.5% methanol (l max 0.012 h )1 ) at 20°C ( Fig.…”

Section: In¯uence Of Methanol Concentration On Recombinant Laccase Exsupporting

confidence: 60%

“…The in¯uence of two dierent methanol concentrations, 1.0% (v/v) and 0.5% (v/v) on recombinant laccase expression, was investigated at 20°C by comparing the feeding strategies B and C. A direct positive correlation between the amount of methanol consumed and the quantity of protein produced has been observed (Murray et al, 1989). In the present study, a positive correlation between total soluble protein and methanol consumption was evident.…”

Section: In¯uence Of Methanol Concentration On Recombinant Laccase Exmentioning

confidence: 73%

“…The use of 1.0% methanol to induce the expression of foreign genes with P. pastoris has been recommended (Murray et al, 1989;JimeÂ nez et al, 1997). Cell proliferation was not inhibited until the methanol concentration reached >3% (Murray et al, 1989).…”

Section: In¯uence Of Methanol Concentration On Recombinant Laccase Exmentioning

confidence: 99%

“…However, high concentrations of methanol are toxic to P. pastoris because of the accumulation of formaldehyde and hydrogen peroxide inside the cells (Couderc and Baratti, 1980;Murray et al, 1989). Therefore, accurate regulation of the methanol concentration in P. pastoris cultures is necessary not only to maintain the induction of genes under the control of the AOX1 promoter but also to prevent accumulation of methanol to levels that are toxic to the cells.…”

Section: Introductionmentioning

confidence: 99%

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Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Hong

Meinander

Jönsson

2002

Biotech & Bioengineering

199

104

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Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.

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Section: In¯uence Of Methanol Concentration On Recombinant Laccase Exsupporting

confidence: 60%

Section: In¯uence Of Methanol Concentration On Recombinant Laccase Exmentioning

confidence: 73%

Section: In¯uence Of Methanol Concentration On Recombinant Laccase Exmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Hong

Meinander

Jönsson

2002

Biotech & Bioengineering

199

104

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“…Acetaldehyde formation results in the net production of 1 mol of NADH per mole and, thus, represents another means of balancing the NADH requirement for glycerol production in response to osmotic stress. Consequently, off-gas trapping was performed with 0.5 M TRIS-HCl (pH 9.0) (15,37), and a flux of approximately 1.0 mmol acetaldehyde g Ϫ1 h Ϫ1 was observed. This represents a 2.4% increase in carbon recovery.…”

Section: Vol 74 2008 Yeast Responses To Lactic Acid 5761mentioning

confidence: 99%

Physiological and Transcriptional Responses to High Concentrations of Lactic Acid in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae

Abbott

Suir

Maris

et al. 2008

Appl Environ Microbiol

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Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1⌬ mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.

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Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris

Adrio

Velasco

Soler

et al. 2001

Biotech & Bioengineering

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We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.

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Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

Cited by 31 publications

References 13 publications

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Physiological and Transcriptional Responses to High Concentrations of Lactic Acid in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris

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