Cell-free styrene biosynthesis at high titers

Grubbe, William S.; Rasor, Blake J.; Krüger, Antje; Jewett, Michael C.; Karim, Ashty S.

doi:10.1101/2020.03.05.979302

“…CFPS decreases the time from DNA to soluble protein and can be used to synthesize functional catalytic enzymes (Karim and Jewett, 2016). The hybrid approach of cell-free protein synthesis metabolic engineering (CFPS-ME) has been successfully adapted to prototype production of polyhydroxyalkanoate (Kelwick et al, 2018), 1,4-butanediol (Wu et al, 2015;Wu et al, 2017), and styrene (Grubbe et al, 2020). Yet, even with the rapid ability to synthesize and test enzymes in vitro, these examples have typically utilized only a small set of enzyme homologs in their optimization strategies.…”

Section: Introductionmentioning

confidence: 99%

In vitro prototyping of limonene biosynthesis using cell-free protein synthesis

Dudley

¹

,

Karim

²

,

Nash

³

et al. 2020

Metabolic Engineering

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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..

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“…First, our rapid, cell-free framework facilitates the study of large number of pathway combinations. Already, cell-free prototyping has proved useful for optimizing several enzymatic pathways including isoprenoids (Dudley et al, 2016), fatty acids (Liu et al, 2010), farnesene (Zhu et al, 2014), phenylalanine (Ding et al, 2016), nonoxidative glycolysis (Bogorad et al, 2013) , polyhydroxyalkanoates (Kelwick et al, 2018), 1,4butanediol (Wu et al, 2015;Wu et al, 2017), styrene (Grubbe et al, 2020), and 3 hydroxybutyrate/n-butanol . Here, we used CFPS to express and study numerous enzyme homologs.…”

Section: Discussionmentioning

confidence: 99%

“…CFPS decreases the time from DNA to soluble protein and can be used to synthesize functional catalytic enzymes (Karim and Jewett, 2016). The hybrid approach of cell-free protein synthesis metabolic engineering (CFPS-ME) has been successfully adapted to prototype production of polyhydroxyalkanoate (Kelwick et al, 2018), 1,4-butanediol (Wu et al, 2015;Wu et al, 2017), and styrene (Grubbe et al, 2020). Yet, even with the rapid ability to synthesize and test enzymes in vitro, these examples have typically utilized only a small set of enzyme homologs in their optimization strategies.…”

Section: Introductionmentioning

confidence: 99%

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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“…16 Cell-free biosynthesis of styrene has also been shown, surpassing the highest published in vivo titer without processing steps by more than an order of magnitude. 24 Toxic pretreated biomass hydrolysates can even be used as feedstocks. 25 Taken together, these results set the stage for new sustainable manufacturing practices, including agile domestic bio-readiness capabilities, that provide economically viable alternatives to fossil fuel-derived chemicals.…”

Section: Where the Technology Can Gomentioning

confidence: 99%