Cascade enzymatic synthesis of <scp>l</scp>-homoserine – mathematical modelling as a tool for process optimisation and design

Česnik, Morana; Sudar, Martina; Hernández, Karel; Charnock, Simon J.; Vasić-Rački, Đurđa; Clapés, Pere; Blažević, Zvjezdana Findrik

doi:10.1039/c9re00453j

Cited by 15 publications

(39 citation statements)

References 31 publications

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“…This rough model was sufficient in order to explore the overall system behavior and to identify bottlenecks as well as the most sensitive parameters of the reaction cascade consisting of 27 enzymes. Another kinetic model was developed for an L-homoserine synthesis cascade in order to improve the volumetric productivity and final titer with regard to an industrial application [104]. The development of a kinetic model based on insights of homologous enzymes led to a more systematic reaction optimization and reduced the amount of experimental effort.…”

Section: Mechanistic Based Modelling Of Enzyme Cascadesmentioning

confidence: 99%

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

et al. 2021

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In vitro enzyme cascades possess great benefits, such as their synthetic capabilities for complex molecules, no need for intermediate isolation, and the shift of unfavorable equilibria towards the products. Their performance, however, can be impaired by, for example, destabilizing or inhibitory interactions between the cascade components or incongruous reaction conditions. The optimization of such systems is therefore often inevitable but not an easy task. Many parameters such as the design of the synthesis route, the choice of enzymes, reaction conditions, or process design can alter the performance of an in vitro enzymatic cascade. Many strategies to tackle this complex task exist, ranging from experimental to in silico approaches and combinations of both. This review collates examples of various optimization strategies and their success. The feasibility of optimization goals, the influence of certain parameters and the usage of algorithm-based optimizations are discussed.

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Section: Mechanistic Based Modelling Of Enzyme Cascadesmentioning

confidence: 99%

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

et al. 2021

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“…A series of the batch reactor experiments were carried out for that purpose, in which the change in product concentration was monitored by HPLC. Considering the previous knowledge on this particular enzymatic cascade system, 18 it was not necessary to investigate the influence of all compounds, as it was expected that certain compounds will have no influence on the reaction rate. For example, this was the case for methanol, which was found to inhibit transaminase in forward and reverse reactions, but the inhibition was not very significant, and was disregarded here.…”

Section: Experimental Partmentioning

confidence: 99%

“…This cascade reaction was investigated previously using purified enzymes 18,42 and cell-free extracts (CFEs). 18,42 In this paper, the aim was to simplify the system by avoiding the need for enzyme isolation and purification. Another objective was to investigate the performance of the reaction catalyzed by CFE compared with that using LWCB.…”

Section: Introductionmentioning

confidence: 99%

“…In this paper, we tested the hypothesis on an in vitro approach by evaluating the kinetics of LWCB and comparing the results with the previously reported ones with CFE. 18,42 Furthermore, the model developed for the reaction with CFE can be used to determine the ratio of transaminase and aldolase activities for coexpression in LWCB necessary to achieve good process metrics. For a nonbiological multienzymatic synthesis, the kinetic modeling of the individual reaction steps is crucial to evaluate the best enzymes and the optimal reactor configuration.…”

Section: Introductionmentioning

confidence: 99%

“…Details of the procedure, 42 as well as the analytical method, are described in the literature. 18 2.3. Activity of Transaminase and Aldolase.…”

Section: Introductionmentioning

confidence: 99%

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Cascade Synthesis of l-Homoserine Catalyzed by Lyophilized Whole Cells Containing Transaminase and Aldolase Activities: The Mathematical Modeling Approach

Katulić

Sudar

Hernández

et al. 2021

Ind. Eng. Chem. Res.

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Aldolase and transaminase coexpressed in Escherichia coli cells and lyophilized (i.e., lyophilized whole-cell biocatalyst (LWCB)) were used as biocatalysts for the one-pot cascade synthesis of L-homoserine with substrate cycling. The kinetic analysis of enzymes within lyophilized cells was performed to evaluate the behavior of the system. The best result among the performed fed-batch reactor experiments achieved was 640.8 mM (76.3 g L −1 ) of L-homoserine with a volume productivity of 2.6 g L −1 h −1 . This is comparable with the results of the same cascade synthesis using cell-free extracts (CFEs) and significantly better than the reports in the literature applying fermentation technology. The approach applied here can serve as guidance for the design of microbial cells with an optimal ratio of expressed enzymes that act as biocatalysts in the cascade, resulting in lower biocatalyst cost, no need for the addition of expensive coenzymes, and enhanced enzyme stability as compared with cell-free extracts.

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Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

Madsen

Woodley

2023

ChemCatChem

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In vitro transaminase catalyzed asymmetric synthesis is not a new phenomenon in the biocatalytic toolbox. Our group published a review on process consideration for these reactions in 2011, in which process metrics and the unfavorable reaction equilibrium was deduced to be the main bottlenecks at the time. Now, a decade later, this has been solved by different process methods presented in this review, with the development of enzymatic cascades one of the main solutions. Today, the new bottleneck is the downstream processing, due to the complexity of transaminase reactions, in which a ketone and an amine as substrates and a new ketone and a new amine as products. How to recover the produced amine from the resulting product mixture is considered and will be the key challenge for scale‐up of such systems in the near future.

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Cascade enzymatic synthesis of l-homoserine – mathematical modelling as a tool for process optimisation and design

Abstract: Mathematical modelling enabled cascade reaction optimisation; 100% increase in product concentration and 18% increase in volume productivity compared to previous work.

Cited by 15 publications

References 31 publications

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

Getting the Most Out of Enzyme Cascades: Strategies to Optimize In Vitro Multi-Enzymatic Reactions

Cascade Synthesis of l-Homoserine Catalyzed by Lyophilized Whole Cells Containing Transaminase and Aldolase Activities: The Mathematical Modeling Approach

Considerations for the Scale‐up of in vitro Transaminase‐Catalyzed Asymmetric Synthesis of Chiral Amines

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