Metabolic Profiling of <i>Escherichia coli</i>-Based Cell-Free Expression Systems for Process Optimization

Miguez, April M.; McNerney, Monica P.; Styczynski, Mark P.

doi:10.1021/acs.iecr.9b03565

Cited by 33 publications

(51 citation statements)

References 31 publications

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“…In cases where increased yield is needed, there are some general approaches to further improve yield that we have yet to apply to this strain, such as: increasing the surface area to volume ratio, [56][57][58] implementing machine learning to improve reaction composition, 59 and conducting metabolomic studies to identify and optimize key metabolites. 60 However, even with these improvements, the SHuffle strain may continue to express less protein than other strains that are exogenously supplemented with T7RNAP and DsbC simply due to exhausted machinery. Yet, the facile, 'one-pot' approach presented in this work can still make a significant impact at the prototyping phase where a large amount of extract is needed to test many sequence candidates.…”

Section: Resultsmentioning

confidence: 99%

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

Dopp

Reuel

2019

Preprint

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In vitro expression of proteins from E. coli extract is a useful method for prototyping and production of cytotoxic or unnatural products. However, proteins that have multiple disulfide bonds require custom extract that to date requires careful addition of exogenous, purified isomerase enzymes. Many important proteins such as enzymes, cytokines, and monoclonal antibodies require disulfide bonds for stabilization and/or proper activity. Currently the only solution to expressing these products is to purchase commercial kits (such as PUREfrex® (GeneFrontier) or PURExpress® (New England BioLabs)) or to grow up the KGK10 strain and supplement with purified enzymes (T7 RNA polymerase and disulfide bond isomerase C). This multistep process can limit the accessibility of such extract to some groups that wish to rapidly prototype proteins with disulfide bonds. In this work, we present a "one-pot" solution that does not require addition of supplemental enzymes. This is done using a commercially available SHuffle® T7 Express lysY strain of E. coli that can express both the needed T7 polymerase and DsbC isomerase enzymes. We find optimal growth conditions for our 1 L cultures in 2.5 L shake flasks (5.6 and 3.9 hr for harvest and induction respectively) using a luciferase (from Gaussia princeps) that contains 5 disulfide bonds as our reporter protein. The method presented here uses a continuous pass homogenizer and pilot scale lyophilizer to produce large volumes of extract; also, optimal reconstitution ratios of the dried extract are found. To show the broad applicability of the extract, three other enzymes containing ≥ 3 disulfide bonds (hevamine, endochitinase A, and periplasmic AppA) were also expressed from minimal genetic templates that had undergone rolling circle amplification.

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Section: Resultsmentioning

confidence: 99%

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

Dopp

Reuel

2019

Preprint

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“…While extremely useful as a standard across labs due to ease of measurement, the approach hides potentially large and complex underlying differences between methods [ 35 ], ignores the fraction of non-functional protein produced [ 151 ], and could poorly predict performance for some applications. The recent application of proteomics [ 112 , [152] , [153] , [154] ] and metabolomics [ 155 ] tools are important advances to better understand the underlying differences between extracts and CFE systems more generally.…”

Section: Discussionmentioning

confidence: 99%

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

Cole¹,

Miklos²,

Chiao³

et al. 2020

Synthetic and Systems Biotechnology

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Cell-free systems that mimic essential cell functions, such as gene expression, have dramatically expanded in recent years, both in terms of applications and widespread adoption. Here we provide a review of cell-extract methods, with a specific focus on prokaryotic systems. Firstly, we describe the diversity of Escherichia coli genetic strains available and their corresponding utility. We then trace the history of cell-extract methodology over the past 20 years, showing key improvements that lower the entry level for new researchers. Next, we survey the rise of new prokaryotic cell-free systems, with associated methods, and the opportunities provided. Finally, we use this historical perspective to comment on the role of methodology improvements and highlight where further improvements may be possible.

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“…First, it is the precursor to pantothenic acid (vitamin B 5 ) and thus to Coenzyme A, an essential cofactor for many key metabolic pathways including the TCA cycle, fatty acid biosynthesis, and acetyl-CoA production 24,25 . Second, and perhaps even more noteworthy, we have previously shown that supplementing β -alanine to a CFE reaction increases protein expression 21 , indicating that its levels are (either directly or indirectly) important to CFE.…”

Section: Levels Of Multiple Key Metabolic Pathways Evolve Over the Course Of A Cfe Reactionmentioning

confidence: 96%

Metabolic Dynamics inEscherichia coli-based Cell-Free Systems

Miguez

Zhang

Piorino

et al. 2021

Preprint

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The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential to push the field even further. However, CFE systems still face some outstanding challenges, including endogenous metabolic activity that is poorly understood yet has a significant impact on CFE productivity. Here, we use metabolomics to characterize the temporal metabolic changes in CFE systems and their constituent components, including significant metabolic activity in central carbon and amino acid metabolism. We find that while changing the reaction starting state via lysate pre-incubation impacts protein production, it has a comparatively small impact on metabolic state. We also demonstrate that changes to lysate preparation have a larger effect on protein yield and temporal metabolic profiles, though general metabolic trends are conserved. Finally, while we improve protein production through targeted supplementation of metabolic enzymes, we show that the endogenous metabolic activity is fairly resilient to these enzymatic perturbations. Overall, this work highlights the robust nature of CFE reaction metabolism as well as the importance of understanding the complex interdependence of metabolites and proteins in CFE systems to guide optimization efforts.

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Metabolic Profiling of Escherichia coli-Based Cell-Free Expression Systems for Process Optimization

Cited by 33 publications

References 31 publications

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

Simple, Functional, Inexpensive Cell Extract forin vitroPrototyping of Proteins with Disulfide Bonds

Methodologies for preparation of prokaryotic extracts for cell-free expression systems

Metabolic Dynamics inEscherichia coli-based Cell-Free Systems

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