Workflows for optimization of enzyme cascades and whole cell catalysis based on enzyme kinetic characterization and pathway modelling

Kuschmierz, Laura; Shen, Lu; Bräsen, Christopher; Snoep, Jacky L.; Siebers, Bettina

doi:10.1016/j.copbio.2021.10.020

Cited by 10 publications

(7 citation statements)

References 38 publications

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“…As several studies have pointed out (e.g. [ 15 , 56 – 60 ]), the optimization of cascade reactions involves the challenging task of catalyst design according to the complex max-min criterion that the composition of individual enzymes should enable maximum overall flux through the multi-step cascade at minimum total mass of catalytic protein used. Based on earlier work done with cell-free enzyme preparations [ 41 ], the approach used here was systematic kinetic model-based prototyping of the enzyme composition in order to inform differential protein co-expression in E. coli for experimental development of a whole cell catalyst consistent with the stated max–min principle.…”

Section: Resultsmentioning

confidence: 99%

“…Subdivision of enzyme cascades into compact functional modules offers flexibility for rapid prototyping of whole-cell catalysts [ 9 – 13 ]. Compared to fermentation, realization of the bioconversion as decoupled from the cell growth opens up a larger space of engineering parameters (e.g., catalyst loading) amenable to process intensification and optimization [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase

Schelch,

Eibinger,

Zuson

et al. 2023

Microb Cell Fact

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Background In whole-cell bio-catalysis, the biosystems engineering paradigm shifts from the global reconfiguration of cellular metabolism as in fermentation to a more focused, and more easily modularized, optimization of comparably short cascade reactions. Human milk oligosaccharides (HMO) constitute an important field for the synthetic application of cascade bio-catalysis in resting or non-living cells. Here, we analyzed the central catalytic module for synthesis of HMO-type sialo-oligosaccharides, comprised of CMP-sialic acid synthetase (CSS) and sialyltransferase (SiaT), with the specific aim of coordinated enzyme co-expression in E. coli for reaction flux optimization in whole cell conversions producing 3′-sialyllactose (3SL). Results Difference in enzyme specific activity (CSS from Neisseria meningitidis: 36 U/mg; α2,3-SiaT from Pasteurella dagmatis: 5.7 U/mg) was compensated by differential protein co-expression from tailored plasmid constructs, giving balance between the individual activities at a high level of both (α2,3-SiaT: 9.4 × 102 U/g cell dry mass; CSS: 3.4 × 102 U/g cell dry mass). Finally, plasmid selection was guided by kinetic modeling of the coupled CSS-SiaT reactions in combination with comprehensive analytical tracking of the multistep conversion (lactose, N-acetyl neuraminic acid (Neu5Ac), cytidine 5′-triphosphate; each up to 100 mM). The half-life of SiaT in permeabilized cells (≤ 4 h) determined the efficiency of 3SL production at 37 °C. Reaction at 25 °C gave 3SL (40 ± 4 g/L) in ∼ 70% yield within 3 h, reaching a cell dry mass-specific productivity of ∼ 3 g/(g h) and avoiding intermediary CMP-Neu5Ac accumulation. Conclusions Collectively, balanced co-expression of CSS and SiaT yields an efficient (high-flux) sialylation module to support flexible development of E. coli whole-cell catalysts for sialo-oligosaccharide production.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase

Schelch,

Eibinger,

Zuson

et al. 2023

Microb Cell Fact

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“…As for any catalytic process, the cost and stability of the catalyst are crucial for the economic application in chemical manufacturing. Employing the whole cell as a biocatalyst circumvents the need for cell lysis and enzyme purification, thus greatly reducing the cost [ 34 , 35 ]. Advances in system biology and metabolic engineering techniques have led to the rational design of whole-cell catalysts for the bioconversion of C1 compounds.…”

Section: Whole-cell Biotransformation Of C1 Compoundsmentioning

confidence: 99%

Artificial multi-enzyme cascades and whole-cell transformation for bioconversion of C1 compounds: Advances, challenge and perspectives

Qiao,

Ma,

Zhang

et al. 2023

Synthetic and Systems Biotechnology

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“…The idea of an organic synthesis, realized by conversion of expedient substrates in several chemical steps without the isolation of intermediates, has strong appeal (Kara & Rudroff, 2021; Rosenthal & Lütz, 2018; Ruales‐Salcedo et al, 2019; Schrittwieser et al, 2018; Sheldon & Brady, 2019). The view of biochemical engineering on enzyme cascades is however mixed (Burek et al, 2022; Kuschmierz et al, 2022; Nazor et al, 2021; Teshima et al, 2023). Advantage of economy in the processing steps used must be balanced against the drawback of expanse in process system complexity.…”

Section: Introductionmentioning

confidence: 99%

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development

Sigg,

Klimacek,

Nidetzky

2023

Biotech & Bioengineering

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One‐pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α‐d‐glucose 1‐phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step‐by‐step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial‐rate models of the two‐substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two‐site glucose substrate inhibition of the cellobiose phosphorylase (fromCellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.

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Workflows for optimization of enzyme cascades and whole cell catalysis based on enzyme kinetic characterization and pathway modelling

Cited by 10 publications

References 38 publications

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase

Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase

Artificial multi-enzyme cascades and whole-cell transformation for bioconversion of C1 compounds: Advances, challenge and perspectives

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development

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