A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology

Casini, Arturo; Chang, Frank; Eluere, Raissa; King, Andrew; Young, Eric M.; Dudley, Quentin M.; Karim, Ashty S.; Pratt, Katelin; Bristol, Cassandra; Forget, Anthony L.; Ghodasara, Amar; Warden-Rothman, Robert; Gan, Rui; Cristofaro, Alexander; Borujeni, Amin Espah; Ryu, Min-Hyung; Li, Jian; Kwon, Yong Chan; Wang, He; Tatsis, Evangelos C.; Rodríguez-López, Carlos; O’Connor, Sarah E.; Medema, Marnix H.; Fischbach, Michael A.; Jewett, Michael C.; Voigt, Christopher A.; Gordon, D. Benjamin

doi:10.1021/jacs.7b13292

Cited by 126 publications

(109 citation statements)

References 160 publications

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“…Heterologous production of bioproducts has been demonstrated for a very large number of 58 compounds and in a wide variety of microbial hosts 1,2 . Yet, even the most well-designed 59 heterologous pathway requires considerable additional work to reach titers, rates and yields 60 (TRY) necessary for the adoption of these systems by industry 3,4 .…”

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confidence: 99%

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Banerjee

Eng

et al. 2020

Preprint

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33Achieving high titer rates and yields (TRY) remains a bottleneck in the production of 34 heterologous products through microbial systems, requiring elaborate engineering and many 35 iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of 36 the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the 37 inherent trade-off between cellular use of carbon towards growth vs. target metabolite 38 production. We hypothesized that being able to strongly couple product formation with growth 39 may lead to improvements across both metrics. In this study, we use elementary mode analysis 40 to predict metabolic reactions that could be targeted to couple the production of indigoidine, a 41 sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We 42 then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 43 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting 44 engineered P. putida strain could achieve high TRY. The engineered pairing of product 45 formation with carbon use also shifted production from stationary to exponential phase and the 46 high TRY phenotype was maintained across scale. In one design cycle, we constructed an 47 engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g 48 indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in 49 exponential phase. These desirable phenotypes were maintained from batch to fed-batch 50 cultivation mode, and from 100ml shake flasks to 250 mL ambr® and 2 L bioreactors. 51 52 53 54 55 56

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confidence: 99%

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Banerjee

Eng

et al. 2020

Preprint

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“…Crude lysates are becoming increasingly popular for prototyping metabolism because lysates contain endogenous metabolism, alternate substrates, and cofactors (Miguez et al, 2019;Schuh et al, 2019). Additionally, when provided with an energy source, amino acids, NTPs, and excess cofactors, crude lysates contain the translational machinery for cell-free protein synthesis (CFPS) which enables rapid production of proteins (Carlson et al, 2012;Casini et al, 2018;Chen et al, 2020;Des Soye et al, 2019;Huang et al, 2018;Jaroentomeechai et al, 2018;Jewett et al, 2008;Kightlinger et al, 2019;Lin et al, 2020;Silverman et al, 2019;Stark et al, 2019;Stark et al, 2018). CFPS decreases the time from DNA to soluble protein and can be used to synthesize functional catalytic enzymes (Karim and Jewett, 2016).…”

Section: Introductionmentioning

confidence: 99%

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

Dudley¹,

Karim²,

Nash³

et al. 2020

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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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“…This was successful in generating (+)‐DHCD from waste orange peel; however, limonene cytotoxicity impacted on the upper level of feedstock concentrations that could be applied . To overcome this cytotoxicity, another study designed a cell‐free system for ( R )‐carvone production from glucose . However, due to time constraints only limonene was efficiently produced.…”

Section: Introductionmentioning

confidence: 99%

From Bugs to Bioplastics: Total (+)‐Dihydrocarvide Biosynthesis by Engineered Escherichia coli

et al. 2019

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The monoterpenoid lactone derivative (+)‐dihydrocarvide ((+)‐DHCD) can be polymerised to form shape‐memory polymers. Synthetic biology routes from simple, inexpensive carbon sources are an attractive, alternative route over chemical synthesis from (R)‐carvone. We have demonstrated a proof‐of‐principle in vivo approach for the complete biosynthesis of (+)‐DHCD from glucose in Escherichia coli (6.6 mg L−1). The pathway is based on the Mentha spicata route to (R)‐carvone, with the addition of an ′ene′‐reductase and Baeyer–Villiger cyclohexanone monooxygenase. Co‐expression with a limonene synthesis pathway enzyme enables complete biocatalytic production within one microbial chassis. (+)‐DHCD was successfully produced by screening multiple homologues of the pathway genes, combined with expression optimisation by selective promoter and/or ribosomal binding‐site screening. This study demonstrates the potential application of synthetic biology approaches in the development of truly sustainable and renewable bioplastic monomers.

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A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology

Cited by 126 publications

References 160 publications

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

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

From Bugs to Bioplastics: Total (+)‐Dihydrocarvide Biosynthesis by Engineered Escherichia coli

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