Bacterial sialyltransferases and their use in biocatalytic cascades for sialo-oligosaccharide production

Schelch, Sabine; Zhong, Chao; Petschacher, Barbara; Nidetzky, Bernd

doi:10.1016/j.biotechadv.2020.107613

Cited by 31 publications

(37 citation statements)

References 254 publications

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“…Hydrolysis of CMP-Neu5Ac by PdST is shown. 3SL, 3ʹ-sialyllactose; CMP-Neu5Ac, cytidine 5ʹ-monophosphate-N-acetyl-D-neuraminic acid; ManNAc, N-acetyl-D-mannosamine; PdST, α2,3sialyltransferase from Pasteurella dagmatis relatively high K M for ManNAc of this (~160 mM) and other NAL enzymes (Schelch et al, 2020), we examine an alternative cascade reaction in which sialic acid synthase (SiaC; EC 2.5.1.56) is used. The SiaC requires PEP instead of PYR as the substrate and shows a lower K M for ManNAc (9.4 mM) than NAL.…”

Section: Introductionmentioning

confidence: 99%

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Schelch

Eibinger

Belduma

et al. 2021

Biotech & Bioengineering

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Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialooligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3ʹ-sialyllactose (3SL) synthesis from N-acetyl-D-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ-triphosphate, via the reactions of cytidine 5ʹ-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl-Dmannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase.We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h).Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.

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Section: Introductionmentioning

confidence: 99%

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Schelch

Eibinger

Belduma

et al. 2021

Biotech & Bioengineering

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“…Lack of enzyme recycling is another limitation. Using glycosyltransferases for oligosaccharide synthesis in which the issue of substrate solubility does not arise in general, the TTN values are higher (≤10 3 g/g), as might be expected (for reviews, see: Nidetzky et al, 2018;Schelch et al, 2020). For batch synthesis of UDP-glucose from sucrose and UDP by GmSuSy, a TTN of 1440 was obtained Schmölzer et al, 2017).…”

Section: Continuous Production Of Nothofaginmentioning

confidence: 77%

Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural C‐glycoside nothofagin

Liu

Nidetzky

2021

Biotech & Bioengineering

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C-glycosyltransferase (CGT) and sucrose synthase (SuSy), each fused to the cationic binding module Z basic2 , were co-immobilized on anionic carrier (ReliSorb SP400) and assessed for continuous production of the natural C-glycoside nothofagin. The overall reaction was 3ʹ-C-β-glycosylation of the polyphenol phloretin from uridine 5ʹ-diphosphate (UDP)-glucose that was released in situ from sucrose and UDP.Using solid catalyst optimized for total (∼28 mg/g) as well as relative protein loading (CGT/SuSy = ∼1) and assembled into a packed bed (1 ml), we demonstrate flow synthesis of nothofagin (up to 52 mg/ml; 120 mM) from phloretin (≥95% conversion) solubilized by inclusion complexation in hydroxypropyl β-cyclodextrin. About 1.8 g nothofagin (90 ml; 12-26 mg/ml) were produced continuously over 90 reactor cycles (2.3 h/cycle) with a space-time yield of approximately 11 mg/(ml h) and a total enzyme turnover number of up to 2.9 × 10 3 mg/mg (=3.8 × 10 5 mol/mol). The coimmobilized enzymes exhibited useful effectiveness (∼40% of the enzymes in solution), with limitations on the conversion rate arising partly from external liquid-solid mass transfer of UDP under packed-bed flow conditions. The operational half-life of the catalyst (∼200 h; 30°C) was governed by the binding stability of the glycosyltransferases (≤35% loss of activity) on the solid carrier. Collectively, the current study shows integrated process technology for flow synthesis with coimmobilized sugar nucleotide-dependent glycosyltransferases, using efficient glycosylation from sucrose via the internally recycled UDP-glucose. This provides a basis from engineering science to promote glycosyltransferase applications for natural product glycosides and oligosaccharides.

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“…All mammalian SiaTs have a GT-A fold. Although GT-A enzymes typically require divalent cations (e.g., Mn 2+ or Mg 2+ ) for activity and contain a DxD motif to coordinate their binding, SiaT enzymes are peculiar in that they lack a DxD motif and have a metal-independent activity [ 63 ]. Functionally, bSiaTs are either α2,3 or α2,6 SiaTs or pSiaTs, although some are also promiscuous and capable of performing multiple types of reaction.…”

Section: Manipulating Sialyltransferases At a Molecular Levelmentioning

confidence: 99%

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

2021

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Glycans have been shown to play a key role in many biological processes, such as signal transduction, immunogenicity, and disease progression. Among the various glycosylation modifications found on cell surfaces and in biomolecules, sialylation is especially important, because sialic acids are typically found at the terminus of glycans and have unique negatively charged moieties associated with cellular and molecular interactions. Sialic acids are also crucial for glycosylated biopharmaceutics, where they promote stability and activity. In this regard, heterogenous sialylation may produce variability in efficacy and limit therapeutic applications. Homogenous sialylation may be achieved through cellular and molecular engineering, both of which have gained traction in recent years. In this paper, we describe the engineering of intracellular glycosylation pathways through targeted disruption and the introduction of carbohydrate active enzyme genes. The focus of this review is on sialic acid-related genes and efforts to achieve homogenous, humanlike sialylation in model hosts. We also discuss the molecular engineering of sialyltransferases and their application in chemoenzymatic sialylation and sialic acid visualization on cell surfaces. The integration of these complementary engineering strategies will be useful for glycoscience to explore the biological significance of sialic acids on cell surfaces as well as the future development of advanced biopharmaceuticals.

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Bacterial sialyltransferases and their use in biocatalytic cascades for sialo-oligosaccharide production

Cited by 31 publications

References 254 publications

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Leloir glycosyltransferases enabled to flow synthesis: Continuous production of the natural C‐glycoside nothofagin

Cellular and Molecular Engineering of Glycan Sialylation in Heterologous Systems

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