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 weightat 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.
authors: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010, Graz, Austria.
authors: Katharina
authors: K
authors: Schmölzer
authors: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010, Graz, Austria.
authors: Martin
authors: M
authors: Lemmerer
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12/I, 8010, Graz, Austria.
authors: Alexander
authors: A
authors: Gutmann
authors: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010, Graz, Austria.|Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12/I, 8010, Graz, Austria.
authors: Bernd
authors: B
authors: Nidetzky
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D009711:Nucleotides; D011994:Recombinant Proteins / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000032:analysis* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C022699:sucrose synthase; D014532:Uridine Diphosphate Glucose
keywords: Leloir glycosyltransferases; glycobiotechnology; integrated process development; nucleotide sugars; sucrose synthase; whole-cell biocatalyst