Automated in vivo enzyme engineering accelerates biocatalyst optimization

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: April 24, 2024

Achieving cost-competitive bio-based processes requires development of stable and selective biocatalysts. Their realization through in vitro enzyme characterization engineering is mostly low throughput labor-intensive. Therefore, strategies for increasing while diminishing manual labor are gaining momentum, such as vivo screening evolution campaigns. Computational tools like machine learning further support efforts by widening the explorable design space. Here, we propose an integrated solution to challenges whereby ML-guided, automated workflows (including library generation, implementation hypermutation systems, adapted laboratory evolution, growth-coupled selection) could be realized accelerate pipelines towards superior

Language: Английский

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The pAblo·pCasso self-curing vector toolset for unconstrained cytidine and adenine base-editing in Gram-negative bacteria

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(4), P. e19 - e19

Published: Jan. 5, 2024

Abstract A synthetic biology toolkit, exploiting clustered regularly interspaced short palindromic repeats (CRISPR) and modified CRISPR-associated protein (Cas) base-editors, was developed for genome engineering in Gram-negative bacteria. Both a cytidine base-editor (CBE) an adenine (ABE) have been optimized precise single-nucleotide modification of plasmid targets. CBE comprises deaminase conjugated to Cas9 nickase from Streptococcus pyogenes (SpnCas9), resulting C→T (or G→A) substitutions. Conversely, ABE consists fused SpnCas9 A→G T→C) editing. Several nucleotide substitutions were achieved using these plasmid-borne base-editing systems novel protospacer adjacent motif (PAM)-relaxed (SpRY) variant. Base-editing validated Pseudomonas putida other bacteria by inserting premature STOP codons into target genes, thereby inactivating both fluorescent proteins metabolic (antibiotic-resistance) functions. The functional knockouts obtained via reverted the wild-type genotype ABE. Additionally, series induction-responsive vectors facilitate curing platform single cultivation step, simplifying complex strain programs without relying on homologous recombination yielding plasmid-free, bacterial cells.

Language: Английский

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CIFR (Clone–Integrate–Flip-out–Repeat): a toolset for iterative genome and pathway engineering of Gram-negative bacteria

Metabolic Engineering, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Advanced genome engineering enables precise and customizable modifications of bacterial species, toolsets that exhibit broad-host compatibility are particularly valued owing to their portability. Tn5 transposon vectors have been widely used establish random integrations desired DNA sequences into genomes. However, the iteration procedure remains challenging because limited availability reusability selection markers. We addressed this challenge with CIFR, a mini-Tn5 integration system tailored for iterative engineering. The pCIFR incorporate attP attB sites flanking an antibiotic resistance marker select insertion. Subsequent removal determinants is facilitated by Bxb1 integrase paired user-friendly counter-selection marker, both encoded in auxiliary plasmids. CIFR delivers engineered strains harboring stable insertions free any cassette, allowing tool. was validated Pseudomonas putida, Escherichia coli, Cupriavidus necator, underscoring its portability across diverse industrially relevant hosts. toolbox calibrated through combinatorial chromoprotein genes P. generating displaying color palette. also introduced carotenoid biosynthesis pathway putida two-step process, showcasing potential tool balancing. broad utility expands toolkit metabolic engineering, construction complex phenotypes while opening new possibilities genetic manipulations.

Language: Английский

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DuBA.flow─A Low-Cost, Long-Read Amplicon Sequencing Workflow for the Validation of Synthetic DNA Constructs

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(2), P. 457 - 465

Published: Jan. 31, 2024

Modern biological science, especially synthetic biology, relies heavily on the construction of DNA elements, often in form plasmids. Plasmids are used for a variety applications, including expression proteins subsequent purification, heterologous pathways production valuable compounds, and study functions mechanisms. For all critical step after plasmid is its sequence validation. The traditional method determination Sanger sequencing, which limited to approximately 1000 bp per reaction. Here, we present highly scalable in-house rapid validation amplified sequences using long-read Nanopore sequencing. We developed two-step amplicon transposase strategies provide maximum flexibility dual barcode also an automated analysis pipeline quickly reliably analyze sequencing results easy-to-interpret each sample. user-friendly DuBA.flow start-to-finish widely applicable. Furthermore, show that construct can be performed by barcoded colony PCR thus accelerating research.

Language: Английский

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A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation

Cell Reports, Journal Year: 2024, Volume and Issue: 43(4), P. 113979 - 113979

Published: March 22, 2024

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited the stationary growth phase under nutrient-limited conditions, requiring customized strategies costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach reroute carbon flux remove regulatory constraints using synthetic biology. We construct collection of Pseudomonas putida-overproducing strains at expense lignin-related compounds growth-coupling approaches. PHA was successfully achieved during phase, resulting in up 46% PHA/cell dry weight while maintaining balanced carbon-to-nitrogen ratio. Our additionally validated an upcycling scenario enzymatically hydrolyzed polyethylene terephthalate feedstock. These findings potential revolutionize address global plastic crisis overcoming complexities traditional

Language: Английский

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Leveraging Engineered Pseudomonas putida Minicells for Bioconversion of Organic Acids into Short-Chain Methyl Ketones

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

Published: Jan. 3, 2025

Methyl ketones, key building blocks widely used in diverse industrial applications, largely depend on oil-derived chemical methods for their production. Here, we investigated biobased production alternatives short-chain adapting the solvent-tolerant soil bacterium Pseudomonas putida as a host ketone biosynthesis either by whole-cell biocatalysis or using engineered minicells, chromosome-free bacterial vesicles. Organic acids (acetate, propanoate and butanoate) were selected main carbon substrate to drive of acetone, butanone 2-pentanone. Pathway optimization identified efficient enzyme variants from Clostridium acetobutylicum Escherichia coli, tested with both constitutive inducible expression cognate genes. By implementing these optimized pathways P. which can be prepared through simple three-step purification protocol, feedstock was converted into target methyl ketones. These results highlight value combining morphology pathway engineering noncanonical hosts establish alternative bioprocesses toxic chemicals that are difficult produce conventional approaches.

Language: Английский

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Synthetic biology strategies for sustainable bioplastic production by yeasts

The Journal of Microbiology, Journal Year: 2025, Volume and Issue: 63(3), P. e2501022 - e2501022

Published: March 28, 2025

Language: Английский

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Evaluating the Performance of Automated Machine Learning (AutoML) Tools for Heart Disease Diagnosis and Prediction

AI, Journal Year: 2023, Volume and Issue: 4(4), P. 1036 - 1058

Published: Dec. 1, 2023

Globally, over 17 million people annually die from cardiovascular diseases, with heart disease being the leading cause of mortality in United States. The ever-increasing volume data related to opens up possibilities for employing machine learning (ML) techniques diagnosing and predicting conditions. While applying ML demands a certain level computer science expertise—often barrier healthcare professionals—automated (AutoML) tools significantly lower this barrier. They enable users construct most effective models without in-depth technical knowledge. Despite their potential, there has been lack research comparing performance different AutoML on data. Addressing gap, our study evaluates three tools—PyCaret, AutoGluon, AutoKeras—against datasets (Cleveland, Hungarian, combined dataset). To evaluate efficacy against conventional methodologies, we crafted ten using standard practices exploratory analysis (EDA), cleansing, feature engineering, others, utilizing sklearn library. Our toolkit included an array models—logistic regression, support vector machines, decision trees, random forest, various ensemble models. Employing 5-fold cross-validation, these traditionally developed demonstrated accuracy rates spanning 55% 60%. This is markedly inferior that tools, indicating latter’s superior capability generating predictive Among AutoGluon emerged as tool, consistently achieving between 78% 86% across datasets. PyCaret’s varied, 65% 83%, dependency nature dataset. AutoKeras showed fluctuation performance, accuracies ranging 54% 83%. findings suggest can simplify generation robust potentially surpass those through traditional methodologies. However, must also consider limitations explore strategies overcome them. successful deployment high-performance designed via could revolutionize treatment prevention globally, impacting patient care.

Language: Английский

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A versatile microbial platform as a tunable whole-cell chemical sensor

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: Sept. 27, 2024

Language: Английский

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AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(7), P. 2227 - 2237

Published: July 8, 2024

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering microbes will not be sufficient meet ever-increasing demand for novel producer strains. Here we describe AutoBioTech platform, a fully automated laboratory system with 14 devices perform operations strain construction without human interaction. Using modular workflows, this platform enables transformations Escherichia coli plasmids assembled via cloning. A CRISPR/Cas9 toolbox compatible existing cloning frameworks allows and flexible genome editing E. coli. In addition, workflows have been established transformation Gram-positive model organism Corynebacterium glutamicum by conjugation electroporation, latter proving more robust technique. Overall, excels at versatility due modularity seamless transitions between modules. This accelerate Gram-negative bacteria.

Language: Английский

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