Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase

Alexander Gutmann

Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

Abstract

Nucleotide sugar-dependent (“Leloir”) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5′-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct/L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc/gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924–928. © 2016 Wiley Periodicals, Inc.
Originalspracheenglisch
Seiten (von - bis)924-928
FachzeitschriftBiotechnology and Bioengineering
DOIs
PublikationsstatusVeröffentlicht - 2017

Fingerprint

Uridine Diphosphate Glucose
Glycosyltransferases
Sugar (sucrose)
Biotransformation
Glucose
Process design
Diphosphates
Uridine
Acidithiobacillus
Escherichia coli
Weights and Measures
Uridine Diphosphate
Bioreactors
Enzymes
Glycosylation
Recombinant Proteins
Chromatography
Nucleotides
Efficiency
Recombinant proteins

Fields of Expertise

  • Human- & Biotechnology

Dies zitieren

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase. / Gutmann, Alexander.

in: Biotechnology and Bioengineering, 2017, S. 924-928.

Publikation: Beitrag in einer FachzeitschriftArtikelForschungBegutachtung

@article{f628ca4939264d4897dbe7a4fae10c7e,
title = "Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase",
abstract = "Nucleotide sugar-dependent (“Leloir”) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5′-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct/L, 86{\%} yield (based on UDP), and a total turnover number of 103 gUDP-glc/gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90{\%} purity and in 73{\%} isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924–928. {\circledC} 2016 Wiley Periodicals, Inc.",
author = "Alexander Gutmann",
year = "2017",
doi = "10.1002/bit.26204",
language = "English",
pages = "924--928",
journal = "Biotechnology and Bioengineering",
issn = "0006-3592",
publisher = "Wiley-VCH",

}

TY - JOUR

T1 - Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP-glucose production with sucrose synthase

AU - Gutmann, Alexander

PY - 2017

Y1 - 2017

N2 - Nucleotide sugar-dependent (“Leloir”) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5′-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct/L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc/gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924–928. © 2016 Wiley Periodicals, Inc.

AB - Nucleotide sugar-dependent (“Leloir”) glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5′-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct/L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc/gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924–928. © 2016 Wiley Periodicals, Inc.

U2 - 10.1002/bit.26204

DO - 10.1002/bit.26204

M3 - Article

SP - 924

EP - 928

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

ER -