A lysate proteome engineering strategy for enhancing cell-free metabolite production

Garcia, David; Dinglasan, Jaime Lorenzo N.; Shrestha, Him K.; Abraham, Paul E.; Hettich, Robert L.; Doktycz, Mitchel J.

doi:10.1016/j.mec.2021.e00162

Cited by 18 publications

(31 citation statements)

References 54 publications

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“…The optimization of CFE systems has incorporated several engineered strains for increased protein synthesis yields 27 , 57 – 59 and for specialized applications, such as producing proteins with disulfide bonds 60 or noncanonical amino acids 61 , 62 . In contrast, nearly all cell-free, crude extract-based metabolite biosynthesis to date has relied on strains with wildtype metabolism expressing pathway enzymes 36 , 38 , 47 with few examples of strain or extract modifications to increase in vitro product titers 63 , 64 . Therefore, we sought to test the central hypothesis of this work with the system described above in place.…”

Section: Resultsmentioning

confidence: 99%

“…This reflects previous efforts to engineer strains for CFE to increase protein yields 27 , 57 , 58 and enable specialized applications 60 – 62 through gene knockouts and/or complementation, but the focus here was on reshaping metabolic flux for small molecule synthesis rather than translation. To our knowledge, this is the first report using multiplexed dCas9 modulation to enhance cell-free biosynthesis, using multiple gRNAs that can be implemented and adjusted more rapidly than knocking out enzymes in vivo 63 or selectively removing them in vitro 64 . Our approach has several key features.…”

Section: Discussionmentioning

confidence: 99%

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An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Rasor

Brown

et al. 2021

Nat Commun

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Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Rasor

Brown

et al. 2021

Nat Commun

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“…To quantify the lysate-based cell-free synthesis of common fermentation products from glucose, lysates derived from strains grown in 2xYPTG media were fed 100 µM glucose as a primary carbon source 8 Molar concentrations for all target analytes were calculated from their respective standard curves. Glucose was consumed within the first 3 h of the reaction and mainly fermented to lactate (Figure 2A,B).…”

Section: Representative Resultsmentioning

confidence: 99%

“…In CFME applications, this mode of detection has been most commonly used with HPLC columns that separate compounds based on a combination of size exclusion and ligand exchange mechanisms, or ion-moderated partition chromatography 5 , 6 , 8 , 13 . This particular technique is used to quickly quantify the consumption of sugar substrates like glucose as well as the formation of fermentation products like succinate, lactate, formate, acetate, and ethanol in lysate-based CFME reactions 8 . Recording the concentration changes of these compounds via HPLC has been useful for both elucidating the potential of crude cell extracts to pool central metabolic precursors and understanding how pathway flux is redirected through fermentative pathways during complex metabolic conversions from glucose in lysates 6 , 8 , 14 .…”

Section: Introductionmentioning

confidence: 99%

“…Seminal CFME studies in E. coli cell extracts confirm that fermentation compounds accumulate as end-products of glucose catabolism and also occur as unwanted by-products in lysates that overexpress exogenous enzymes 6 , 15 . It is suggested that fermentative metabolism plays a necessary role in regenerating redox equivalents of cofactors (i.e., NAD(P)H and ATP) to sustain glycolytic reactions 8 . Hence, an HPLC-based optical detection method designed to separate fermentation products is a useful and commonly applied tool when executing various lysate-based CFME tasks.…”

Section: Introductionmentioning

confidence: 99%

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Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Dinglasan

Reeves

Hettich

et al. 2021

JoVE

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Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell's survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13 C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/ MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

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Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping

Rasor

Vögeli

Jewett

et al. 2022

Methods in Molecular Biology

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A lysate proteome engineering strategy for enhancing cell-free metabolite production

Cited by 18 publications

References 54 publications

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping

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