High-throughput screening of catalytically active inclusion bodies using laboratory automation and Bayesian optimization

Microbial Cell Factories, Journal Year: 2024, Volume and Issue: 23(1)

Published: Feb. 24, 2024

In recent years, the production of inclusion bodies that retain substantial catalytic activity was demonstrated. These catalytically active (CatIBs) are formed by genetic fusion an aggregation-inducing tag to a gene interest via short linker polypeptides. The resulting CatIBs known for their easy and cost-efficient production, recyclability as well improved stability. Recent studies have outlined cooperative effects on CatIB activities. However, no priori prediction is possible so far indicate best combination thereof. Consequently, extensive screening required find performing variant.

Language: Английский

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Analytical and Numerical Approaches to the Analysis of Progress Curves: A Methodological Comparison

Process Biochemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 1, 2025

Language: Английский

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Development of a multi-enzyme cascade for 2′3′-cGAMP synthesis from nucleosides

Catalysis Science & Technology, Journal Year: 2024, Volume and Issue: 14(12), P. 3335 - 3345

Published: Jan. 1, 2024

A five-enzyme cascade was developed for the synthesis of cyclic dinucleotide 2′3′-cGAMP from nucleosides in seven reaction steps.

Language: Английский

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

Biotechnology and Bioengineering, Journal Year: 2023, Volume and Issue: 121(2), P. 580 - 592

Published: Nov. 20, 2023

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

Language: Английский

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Pushing the boundaries of phosphorylase cascade reaction for cellobiose production II: Model‐based multiobjective optimization

Biotechnology and Bioengineering, Journal Year: 2023, Volume and Issue: 121(2), P. 566 - 579

Published: Nov. 21, 2023

Abstract The inherent complexity of coupled biocatalytic reactions presents a major challenge for process development with one‐pot multienzyme cascade transformations. Kinetic models are powerful engineering tools to guide the optimization towards performance suitable scale up an actual production. Here, we report kinetic model‐based window operation analysis cellobiose production (≥100 g/L) from sucrose and glucose by indirect transglycosylation via 1‐phosphate as intermediate. two‐step transformation is catalyzed phosphorylase in presence substoichiometric amounts phosphate (≤27 mol% substrate). modeling was instrumental uncover hidden effect bulk microviscosity due high sugar concentrations on decreasing rate specifically. mechanistic‐empirical hybrid model thus developed gives comprehensive description reaction at industrially relevant substrate conditions. Model simulations serve unravel opposed relationships between efficient utilization enzymes maximized concentration (or yield) product within given time, dependence initial used. Optimum balance these competing key metrics suggested model‐calculated verified experimentally. evidence shown highlights important use characterization ways that appear be inaccessible purely data‐driven approaches.

Language: Английский

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

Microbial Cell Factories, Journal Year: 2023, Volume and Issue: 22(1)

Published: Nov. 27, 2023

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

Language: Английский

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Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems

ChemElectroChem, Journal Year: 2023, Volume and Issue: 10(22)

Published: Oct. 5, 2023

Abstract Adenosine‐5’‐triphosphate (ATP) plays a crucial role in many biocatalytic reactions and its regeneration can influence the performance of related enzymatic reaction significantly. Here, we established electrochemically coupled ATP by pyruvate oxidase acetate kinase (ACK) for phosphorylation mevalonate catalyzed kinase. A yield 84 % product phosphate was reached total turnover number ADP 68. These metrics are promising development an economic feasible bioprocess surpass other systems. comparison made to polyphosphate kinases (PPKs), ACK, kinase, creatine terms donor properties exemplary reactions. Furthermore, our system expanded PPK that enables AMP, which broaden spectrum applications even further.

Language: Английский

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Calibration design for pharmaceutical bioprocesses: A novel approach for knowledge transfer in Design of Experiments

Authorea (Authorea), Journal Year: 2024, Volume and Issue: unknown

Published: March 29, 2024

Modern machine learning methods, and their use alongside established paradigms such as Quality by Design, have the potential to fundamentally change way bioprocesses are developed. In particular, horizontal knowledge transfer which seek exploit data from historical processes facilitate process development for a new product, provide an opportunity rethink workflows. this work, we firstly assess of two approaches, meta one-hot encoding, in combination with Gaussian (GP) models. We compare performance GPs developed only on process. Using simulated mammalian cell cultivation data, observe that both approaches outperform individual-product approach. second part, address question whether experiments product could be designed more effectively exploiting existing knowledge. suggest specifically design few runs novel calibrate models, task coin calibration design. propose novel, customised metric identify set runs, exploits differences evolutions products. case studies, observed training designs yields similar test errors compared common Design Experiments. However, much fewer needed former, suggesting interesting alternative future bioprocess development. Overall, results significantly streamlined when systematically carrying one next.

Language: Английский

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Boosting of Enzymatic Cascades by Intermediates: Theoretical Analysis and Model-Based Optimization

Published: Jan. 1, 2024

In an earlier work we demonstrated that adding intermediates of enzymatic cascade reaction at the start process can increase space-time yield significantly. This method was coined boosting by (BBI) and potentially productivity systems. BBI remains largely unexplored, its mechanism, potential, limitations are not fully understood. present work, provide a theoretical background studying four chemical networks (CRN) exhibit intermediates. We study dynamics these via quasi steady-state approximation numerical solutions. novel graphical be used to deduce which boost linear, redox neutral networks. Further, demonstrate how such designed using model-based optimization.

Language: Английский

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Novel calibration design improves knowledge transfer across products for the characterization of pharmaceutical bioprocesses

Biotechnology Journal, Journal Year: 2024, Volume and Issue: 19(7)

Published: July 1, 2024

Modern machine learning has the potential to fundamentally change way bioprocesses are developed. In particular, horizontal knowledge transfer methods, which seek exploit data from historical processes facilitate process development for a new product, provide an opportunity rethink current workflows. this work, we first assess of two approaches, meta and one-hot encoding, in combination with Gaussian (GP) models. We compare their performance GPs trained only on process, that is, local Using simulated mammalian cell culture data, observe both approaches exhibit test set errors approximately halved compared those models when two, four, or eight experiments product used training. Subsequently, address question whether could be designed more effectively by exploiting existing knowledge. suggest specifically design few runs novel calibrate models, task coin calibration design. propose customized objective function identify runs, exploits differences evolution products. case studies, observed training designs yields similar common approaches. However, former requires four times fewer experiments. Overall, results significantly streamlined systematically carrying one next.

Language: Английский

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