Effects of oxygen and ethanol on recombinant yeast fermentation for hepatitis B virus surface antigen production: Modeling and simulation studies

Shi, Yijun; Ryu, Dewey D. Y.; Yuan, Wenjie

doi:10.1002/bit.260410108

“…Cutinase has been cloned in Saccharomyces cerevisiae as this yeast has become an important host in industrial biotechnology (Barthel and Kula, 1993;Shi et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Calado¹,

Mannesse²,

Egmond³

et al. 2002

Biotechnol. Bioeng.

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This study focused on the growth of Saccharomyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP) 2 and (WP) 4 , respectively. Different cultivation and strategies were tested in a 2-L shake¯ask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (speci®c cell activity) of 4.1 Umg )1 with activity per protein broth (speci®c activity) of 266 Umg )1 , whereas cutinase-(WP) 2 was secreted with a speci®c cell activity of 2.1 Umg )1 with a speci®c activity of 200 Umg )1 , and cutinase-(WP) 4 with a speci®c cell activity of 0.7 Umg )1 with a speci®c activity of 15 Umg )1 . The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake¯ask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture). ã

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“…Cutinase from Fusarium solani pisi is a versatile enzyme showing several noteworthy properties for industrial applications (Carvalho et al, 1999). Cutinase has been cloned in Saccharomyces cerevisiae as this yeast has become an important host in industrial biotechnology (Barthel and Kula, 1993;Shi et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Calado

¹

,

Mannesse

²

,

Egmond

³

et al. 2002

Biotech & Bioengineering

View full text Add to dashboard Cite

This study focused on the growth of Saccharomyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP) 2 and (WP) 4 , respectively. Different cultivation and strategies were tested in a 2-L shake¯ask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (speci®c cell activity) of 4.1 Umg )1 with activity per protein broth (speci®c activity) of 266 Umg )1 , whereas cutinase-(WP) 2 was secreted with a speci®c cell activity of 2.1 Umg )1 with a speci®c activity of 200 Umg )1 , and cutinase-(WP) 4 with a speci®c cell activity of 0.7 Umg )1 with a speci®c activity of 15 Umg )1 . The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake¯ask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture). ã

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Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

Fu

¹

,

VanDusen

²

,

Kolodin

³

et al. 2000

Biotechnol. Bioeng.

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We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24s monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose‐limited continuous culture. The hepatitis B virus S gene, which encodes the p24s monomer, is transcribed under the control of the GAL 10p on a chimeric 2‐μm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host‐cell‐derived components. Steady states were evaluated in a range from 0.015 h−1 to washout (0.143 h−1). Both p24s monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24s monomer to HBsAg particle, estimated at five‐ to tenfold by mass, was found at all dilution rates. The average copy number of the 2‐μm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h−1. Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h−1), even though HBsAg expression was maximal. Total p24s monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h−1. © 1996 John Wiley & Sons, Inc.

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Effects of oxygen and ethanol on recombinant yeast fermentation for hepatitis B virus surface antigen production: Modeling and simulation studies

Cited by 14 publications

References 34 publications

Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae

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