Glycosyltransferase cascades for natural product glycosylation: Use of plant instead of bacterial sucrose synthases improves the UDP-glucose recycling from sucrose and UDP

Alexander Gutmann, Alexander Lepak, Margo Diricks, Tom Desmet, Bernd Nidetzky

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Natural product glycosylations by Leloir glycosyltransferases (GTs) require expensive nucleotide-activated sugars as substrates. Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. Coupling of SuSy and GT reactions in one-pot cascade transformations creates a UDP cycle, which regenerates the UDP-glucose continuously and so makes it an expedient donor for glucoside production. Here we compare SuSys with divergent kinetic characteristics for UDP-glucose recycling in the synthesis of the natural C-glucoside nothofagin. Development of a fast reversed-phase ion-pairing HPLC method, quantifying all relevant reactants from the coupled conversion in a single run, was key to dissect the main factors of recycling efficiency. Limitations due to high KM, both for UDP and sucrose, were revealed for the bacterial SuSy from Acidithiobacillus caldus. The L637M-T640V double mutant of this SuSy with a 60-fold reduced KM for UDP substantially improved UDP-glucose recycling. The SuSy from Glycine max (soybean) was nevertheless the most active enzyme at the UDP (≤ 0.5 mM) and sucrose (≤ 1 M) concentrations used. It was also unexpectedly stable at up to 50°C where spontaneous decomposition of UDP-glucose started to become problematic. The herein gained in-depth understanding of requirements for UDP-glucose regeneration supports development of efficient GT-SuSy cascades.
Original languageEnglish
JournalBiotechnology Journal
DOIs
Publication statusE-pub ahead of print - 2017

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Uridine Diphosphate Glucose
Glycosyltransferases
Uridine Diphosphate
Recycling
Biological Products
Glycosylation
Sucrose
Glucosides
Soybeans
Acidithiobacillus
sucrose synthase
Regeneration
Nucleotides
High Pressure Liquid Chromatography
Ions
Enzymes

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Glycosyltransferase cascades for natural product glycosylation: Use of plant instead of bacterial sucrose synthases improves the UDP-glucose recycling from sucrose and UDP. / Gutmann, Alexander; Lepak, Alexander; Diricks, Margo; Desmet, Tom; Nidetzky, Bernd.

In: Biotechnology Journal, 2017.

Research output: Contribution to journalArticleResearchpeer-review

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abstract = "Natural product glycosylations by Leloir glycosyltransferases (GTs) require expensive nucleotide-activated sugars as substrates. Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. Coupling of SuSy and GT reactions in one-pot cascade transformations creates a UDP cycle, which regenerates the UDP-glucose continuously and so makes it an expedient donor for glucoside production. Here we compare SuSys with divergent kinetic characteristics for UDP-glucose recycling in the synthesis of the natural C-glucoside nothofagin. Development of a fast reversed-phase ion-pairing HPLC method, quantifying all relevant reactants from the coupled conversion in a single run, was key to dissect the main factors of recycling efficiency. Limitations due to high KM, both for UDP and sucrose, were revealed for the bacterial SuSy from Acidithiobacillus caldus. The L637M-T640V double mutant of this SuSy with a 60-fold reduced KM for UDP substantially improved UDP-glucose recycling. The SuSy from Glycine max (soybean) was nevertheless the most active enzyme at the UDP (≤ 0.5 mM) and sucrose (≤ 1 M) concentrations used. It was also unexpectedly stable at up to 50°C where spontaneous decomposition of UDP-glucose started to become problematic. The herein gained in-depth understanding of requirements for UDP-glucose regeneration supports development of efficient GT-SuSy cascades.",
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AU - Lepak, Alexander

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AU - Desmet, Tom

AU - Nidetzky, Bernd

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AB - Natural product glycosylations by Leloir glycosyltransferases (GTs) require expensive nucleotide-activated sugars as substrates. Sucrose synthase (SuSy) converts sucrose and uridine 5'-diphosphate (UDP) into UDP-glucose. Coupling of SuSy and GT reactions in one-pot cascade transformations creates a UDP cycle, which regenerates the UDP-glucose continuously and so makes it an expedient donor for glucoside production. Here we compare SuSys with divergent kinetic characteristics for UDP-glucose recycling in the synthesis of the natural C-glucoside nothofagin. Development of a fast reversed-phase ion-pairing HPLC method, quantifying all relevant reactants from the coupled conversion in a single run, was key to dissect the main factors of recycling efficiency. Limitations due to high KM, both for UDP and sucrose, were revealed for the bacterial SuSy from Acidithiobacillus caldus. The L637M-T640V double mutant of this SuSy with a 60-fold reduced KM for UDP substantially improved UDP-glucose recycling. The SuSy from Glycine max (soybean) was nevertheless the most active enzyme at the UDP (≤ 0.5 mM) and sucrose (≤ 1 M) concentrations used. It was also unexpectedly stable at up to 50°C where spontaneous decomposition of UDP-glucose started to become problematic. The herein gained in-depth understanding of requirements for UDP-glucose regeneration supports development of efficient GT-SuSy cascades.

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