A synthetic biochemistry platform for cell free production of monoterpenes from glucose

Korman, Tyler P.; Opgenorth, Paul H.; Bowie, James U.

doi:10.1038/ncomms15526

Cited by 190 publications

(162 citation statements)

References 48 publications

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“…Our investigation demonstrates that cell‐free biosynthesis using the IUP is a promising route for the production of high‐value complex cyclic diterpenoids like taxadiene. The IUP takes advantage of a low‐cost feedstock, isoprenol, and uses a relatively small number of enzymes (five for diterpenes and four for mono‐ or sesquiterpenoids) compared with other cell‐free systems for isoprenoid production (Korman et al, ). When compared with in vivo systems, cell‐free production can sustain higher flux as there is no competition for precursors to support biological functions meaning theoretically all of the substrates could be converted into the product.…”

Section: Resultsmentioning

confidence: 99%

“…To use a low‐cost feedstock like glucose, glycolysis enzymes must also be included along with terpene pathway enzymes to precisely balance cofactor generation and utilization steps. This was demonstrated for the production of monoterpenes, which required 27 enzymes to sustainably synthesize monoterpenes from glucose (Korman, Opgenorth, & Bowie, ). While they were able to achieve high titers (>11 g/L over 7 days) of pinene, sabinene, and limonene, and mitigate the toxicity effects which limit monoterpene production in vivo, the high cost for the production, purification, and maintenance of so many enzymes would be prohibitive at scale.…”

Section: Introductionmentioning

confidence: 99%

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Cell free biosynthesis of isoprenoids from isopentenol

Ward

Chatzivasileiou

Stephanopoulos

2019

Biotech & Bioengineering

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Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far. K E Y W O R D Sbiocatalysis, enzyme kinetics, isoprenoids, metabolic control analysis, taxadiene

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cell free biosynthesis of isoprenoids from isopentenol

Ward

Chatzivasileiou

Stephanopoulos

2019

Biotech & Bioengineering

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“…Over the past several years, cell‐free chemical biosynthesis using microbial cells with engineered pathways has emerged as a promising alternative for the biomanufacturing industry . This new approach averts the tedious and challenging tasks to engineer and optimize synthetic pathways in live cells, and conquers the slow mass transfer controlled by both cell membranes and cellular processes .…”

Section: Introductionmentioning

confidence: 99%

Hierarchical Micro‐ and Mesoporous Zn‐Based Metal–Organic Frameworks Templated by Hydrogels: Their Use for Enzyme Immobilization and Catalysis of Knoevenagel Reaction

Cheng

Švec

et al. 2019

Small

121

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Encapsulation of enzymes in metal–organic frameworks (MOFs) is often obstructed by the small size of the orifices typical of most reported MOFs, which prevent the passage of larger‐size enzymes. Here, the preparation of hierarchical micro‐ and mesoporous Zn‐based MOFs via the templated emulsification method using hydrogels as a template is presented. Zinc‐based hydrogels featuring a 3D interconnecting network are first produced via the formation of hydrogen bonds between melamine and salicylic acid in which zinc ions are well distributed. Further coordination with organic linkers followed by the removal of the hydrogel template produces hierarchical Zn‐based MOFs containing both micropores and mesopores. These new MOFs are used for the encapsulation of glucose oxidase and horseradish peroxidase to prove the concept. The immobilized enzymes exhibit a remarkably enhanced increased operational stability and enzymatic activity with a kcat/km value of 85.68 mm s–1. This value is 7.7‐fold higher compared to that found for the free enzymes in solution, and 2.7‐fold higher than enzymes adsorbed on conventional microporous MOFs. The much higher catalytic activity of the mesoporous conjugate for Knoevenagel reactions is demonstrated, since the large pores enable easier access to the active sites, and compared with that observed for catalysis using microporous MOFs.

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“…We also demonstrated pathway modularity by swapping out the isoprenoid synthetase to produce pinene and bisabolene. Our results suggest that previous cell-free isoprenoid systems, which have so far, to our knowledge reported fewer than 20 enzyme and reaction combinations per study (Chen et al, 2013;Dirkmann et al, 2018;Korman et al, 2017;Korman et al, 2014;Rodriguez and Leyh, 2014;Zhu et al, 2014), could benefit from screening more enzyme variants. iPROBE provides the ability to test dozens of enzyme homologs in hundreds of combinations without needing to reengineer a cell or re-assemble DNA.…”

Section: Introductionmentioning

confidence: 66%

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Dudley¹,

Karim²,

Nash³

et al. 2020

Preprint

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Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design. Dudley, et al. -Cell-free prototyping of limonene biosynthesis 3 TOC Figure Highlights • Applied the iPROBE framework to build the nine-enzyme pathway to produce limonene • Assessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance • Iteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions • Extended pathway to biofuel precursors pinene and bisabolene Dudley, et al. -Cell-free prototyping of limonene biosynthesis 4

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A synthetic biochemistry platform for cell free production of monoterpenes from glucose

Cited by 190 publications

References 48 publications

Cell free biosynthesis of isoprenoids from isopentenol

Cell free biosynthesis of isoprenoids from isopentenol

Hierarchical Micro‐ and Mesoporous Zn‐Based Metal–Organic Frameworks Templated by Hydrogels: Their Use for Enzyme Immobilization and Catalysis of Knoevenagel Reaction

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

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