Synthetic Biology for Cell-Free Biosynthesis: Fundamentals of Designing Novel In Vitro Multi-Enzyme Reaction Networks

Morgado, Gaspar; Gerngross, Daniel; Roberts, Tania Michelle; Panke, Sven

doi:10.1007/10_2016_13

Cited by 32 publications

(26 citation statements)

References 157 publications

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“…The results of the prototyping were subsequently validated with in vivo assays. Cell-free metabolic engineering is thus a promising tool to identify the best combination of enzymes that work together [103,104]. Combining CFPS with cell-free metabolic engineering simplifies the manipulation of metabolic pathways and avoids the need for time-intensive engineering of organisms [105,106].…”

Section: Applications Of Cell-free Protein Synthesismentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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Cell-free protein synthesis (CFPS) has become an established tool for rapid protein synthesis in order to accelerate the discovery of new enzymes and the development of proteins with improved characteristics. Over the past years, progress in CFPS system preparation has been made towards simplification, and many applications have been developed with regard to tailor-made solutions for specific purposes. In this review, various preparation methods of CFPS systems are compared and the significance of individual supplements is assessed. The recent applications of CFPS are summarized and the potential for biocatalyst development discussed. One of the central features is the high-throughput synthesis of protein variants, which enables sophisticated approaches for rapid prototyping of enzymes. These applications demonstrate the contribution of CFPS to enhance enzyme functionalities and the complementation to in vivo protein synthesis. However, there are different issues to be addressed, such as the low predictability of CFPS performance and transferability to in vivo protein synthesis. Nevertheless, the usage of CFPS for high-throughput enzyme screening has been proven to be an efficient method to discover novel biocatalysts and improved enzyme variants.

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Section: Applications Of Cell-free Protein Synthesismentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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“…To circumvent potential problems caused by cellular complexity 28 , in-vitro enzyme cascades have been used as an alternative to whole-cell biocatalysis. Compared with chemical approaches, enzyme cascades enable the production under moderate, environmentally friendly conditions and, most importantly, provide stereo-selectivity for product formation 29,30 . However, although much simpler than intact cells, one-pot enzyme cascades function similar to metabolic pathways and are subject to regulatory properties (e.g., inhibition by pathway intermediates, (co-)substrates and products) and need quantitative tools for analysis.…”

mentioning

confidence: 99%

“…To identify the rate-limiting step(s) and to optimize enzyme cascades, currently mainly combinatorial approaches are used with non-biased combinations of cascade elements and/or reaction conditions (e.g., buffer composition, pH and temperature) [31][32][33] (for reviews, see ref. 30 ). Rational approaches involving computation and modelling have so far rarely been included [34][35][36][37] and the results for computational pathway design are often hampered by missing data for enzyme properties and incomplete information of systems behaviour.…”

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confidence: 99%

A combined experimental and modelling approach for the Weimberg pathway optimisation

et al. 2020

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The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD + recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cellfree extracts.

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“…This is especially appealing for product formation from cheap and more easily available substrates and might also be considered as an alternative for KDPG synthesis. Nevertheless, enzyme cascades are more complex to understand and to optimize them is much more complicated and time consuming (Morgado et al, 2018) than single enzyme systems as reported here.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

Krevet

Shen

Bohnen

et al. 2020

Front. Bioeng. Biotechnol.

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The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a V max of 61.6 U mg −1 and a K M of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.

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Synthetic Biology for Cell-Free Biosynthesis: Fundamentals of Designing Novel In Vitro Multi-Enzyme Reaction Networks

Cited by 32 publications

References 157 publications

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

A combined experimental and modelling approach for the Weimberg pathway optimisation

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

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