AI-driven high-throughput droplet screening of cell-free gene expression

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: March 19, 2025

Cell-free gene expression (CFE) systems enable transcription and translation using crude cellular extracts, offering a versatile platform for synthetic biology by eliminating the need to maintain living cells. However, Such are constrained cumbersome composition, high costs, limited yields due numerous additional components required biocatalytic efficiency. Here, we introduce DropAI, droplet-based, AI-driven screening strategy designed optimize CFE with throughput economic DropAI employs microfluidics generate picoliter reactors utilizes fluorescent color-coding system address screen massive chemical combinations. The in-droplet is complemented in silico optimization, where experimental results train machine-learning model estimate contribution of predict high-yield By applying significantly simplified composition an Escherichia coli-based system, achieving fourfold reduction unit cost expressed superfolder green protein (sfGFP). This optimized formulation was further validated across 12 different proteins. Notably, established E. coli successfully adapted Bacillus subtilis-based through transfer learning, leading doubled yield prediction. Beyond CFE, offers high-throughput scalable solution combinatorial optimization biochemical systems. often authors propose droplet-AI combined approach perform efficient CFE. work led improved cost-effectiveness.

Language: Английский

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Cell-free synthetic biology for natural product biosynthesis and discovery

Chemical Society Reviews, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Cell free synthetic biology enables a broad range of approaches relevant to the generation, biosynthetic characterisation, bioengineering, activity profiling, and production complex natural products.

Language: Английский

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In Vitro Transcription–Translation in an Artificial Biomolecular Condensate

ACS Synthetic Biology, Journal Year: 2023, Volume and Issue: 12(7), P. 2004 - 2014

Published: June 21, 2023

Biomolecular condensates are a promising platform for synthetic cell formation and constitute potential missing link between the chemical cellular stage of origins life. However, it has proven challenging to integrate complex reaction networks into biomolecular condensates, such as cell-free in vitro transcription–translation (IVTT) system. Integrating IVTT successfully is one precondition condensation-based formation. Moreover, would provide proof concept that principle compatible with central dogma, hallmarks Here, we have systemically investigated compatibility eight different (bio)molecular incorporation. Of these candidates, found green fluorescent protein-labeled, intrinsically disordered cationic protein (GFP-K72) single-stranded DNA (ssDNA) can form up μM expression. This shows indeed networks, confirming their use platforms hinting at possible role origin

Language: Английский

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Advancing synthetic biology through cell-free protein synthesis

Computational and Structural Biotechnology Journal, Journal Year: 2023, Volume and Issue: 21, P. 2899 - 2908

Published: Jan. 1, 2023

The rapid development of synthetic biology has enabled the production compounds with revolutionary improvements in biotechnology. DNA manipulation tools have expedited engineering cellular systems for this purpose. Nonetheless, inherent constraints persist, imposing an upper limit on mass and energy conversion efficiencies. Cell-free protein synthesis (CFPS) demonstrated its potential to overcome these been instrumental further advancement biology. Via removal cell membranes redundant parts cells, CFPS provided flexibility directly dissecting manipulating Central Dogma feedback. This mini-review summarizes recent achievements technique application a wide range projects, such as minimal assembly, metabolic engineering, recombinant therapeutics, well biosensor vitro diagnostics. In addition, current challenges future perspectives developing generalized cell-free are outlined.

Language: Английский

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Synthetic Biology of Natural Products Engineering: Recent Advances Across the Discover–Design–Build–Test–Learn Cycle

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(9), P. 2684 - 2692

Published: Aug. 20, 2024

Advances in genome engineering and associated technologies have reinvigorated natural products research. Here we highlight the latest developments field across discover–design–build–test–learn cycle of bioengineering, from recent progress computational tools for AI-supported mining, enzyme pathway engineering, compound identification to novel host systems new techniques improving production levels, place these trends context responsible research innovation, emphasizing importance anticipatory analysis at early stages process development.

Language: Английский

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Cell-Free Gene Expression: Methods and Applications

Chemical Reviews, Journal Year: 2024, Volume and Issue: 125(1), P. 91 - 149

Published: Dec. 19, 2024

Cell-free gene expression (CFE) systems empower synthetic biologists to build biological molecules and processes outside of living intact cells. The foundational principle is that precise, complex biomolecular transformations can be conducted in purified enzyme or crude cell lysate systems. This concept circumvents mechanisms have evolved facilitate species survival, bypasses limitations on molecular transport across the wall, provides a significant departure from traditional, cell-based rely microscopic cellular "reactors." In addition, cell-free are inherently distributable through freeze-drying, which allows simple distribution before rehydration at point-of-use. Furthermore, as nonliving, they provide built-in safeguards for biocontainment without constraints attendant genetically modified organisms. These features led increase development use CFE over past two decades. Here, we discuss recent advances highlight how transforming efforts cells, control genetic networks, manufacture biobased products.

Language: Английский

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Single-Walled Carbon Nanotube Probes for Protease Characterization Directly in Cell-Free Expression Reactions

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 14, 2025

Proteins can be rapidly prototyped with cell-free expression (CFE) but in most cases there is a lack of probes or assays to measure their function directly the cell lysate, thereby limiting throughput these screens. Increased needed build standardized, sequence data sets feed machine learning guided protein optimization. Herein, we describe use fluorescent single-walled carbon nanotubes (SWCNT) as effective for measuring protease activity lysate. Substrate proteins were conjugated carboxymethyl cellulose-wrapped SWCNT, yielding stable and sensitive detection limit 6.4 ng/mL bacterial from Streptomyces griseus. These successfully measured subtilisin unpurified CFE reactions, surpassing commercial assays. Furthermore, they enabled continuous monitoring during synthesis both purified lysate-based systems without compromising expression. Surface passivation techniques, such pre-incubation lysate supplement components, reduced initial signal loss improved probe stability complex environment. modular used, described, high-throughput screening optimization proteases and, change substrate, wider range other hydrolases.

Language: Английский

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Laboratory evolution in Novosphingobium aromaticivorans enables rapid catabolism of a model lignin-derived aromatic dimer

Applied and Environmental Microbiology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 23, 2025

ABSTRACT Lignin contains a variety of interunit linkages, leading to range potential decomposition products that can be used as carbon and energy sources by microbes. β-O-4 linkages are the most common in native lignin, associated catabolic pathways have been well characterized. However, fate mono-aromatic intermediates result from dimer cleavage has not fully elucidated. Here, we experimental evolution identify mutant strains Novosphingobium aromaticivorans with improved catabolism model aromatic containing linkage, guaiacylglycerol-β-guaiacyl ether (GGE). We identified several parallel causal mutations, including single nucleotide polymorphism promoter an uncharacterized gene roughly doubled growth yield GGE. characterized enzyme demonstrated it oxidizes intermediate GGE catabolism, β-hydroxypropiovanillone, vanilloyl acetaldehyde. Identification this its key role furthers our understanding for lignin-derived compounds. IMPORTANCE degradation is step both cycling nature biomass conversion fuels chemicals. Bacteria catabolize compounds, but complexity lignin means full mineralization requires numerous often results slow growth. Using evolution, monomer, β-hydroxypropiovanillone. A significantly increased bacterial either β-hydroxypropiovanillone or related dimer. This work expands repertoire known genes demonstrates compounds may due misregulation under laboratory conditions rather than inherent challenges.

Language: Английский

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Cell-Free Systems to Mimic and Expand Metabolism

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 29, 2025

Cell-free synthetic biology incorporates purified components and/or crude cell extracts to carry out metabolic and genetic programs. While protein synthesis has historically been the primary focus, more metabolism researchers are now turning toward cell-free systems either prototype pathways for cellular implementation or design new-to-nature reaction networks that incorporate environmentally relevant substrates new energy sources. The ability design, build, test enzyme combinations in vitro accelerated efforts understand bottlenecks engineer high-yielding pathways. However, only a small fraction of possibilities explored systems, from model organisms remain most common starting points. Expanding scope include organisms, alternative pathways, non-natural chemistries will enhance our bio-based chemical conversions.

Language: Английский

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Pitfalls of transcriptomics and selection of the most appropriate transcriptomic technique

Elsevier eBooks, Journal Year: 2025, Volume and Issue: unknown, P. 15 - 34

Published: Jan. 1, 2025

Language: Английский

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