Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

Nature Communications, Год журнала: 2022, Номер 13(1)

Опубликована: Май 31, 2022

Abstract CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing widely-applicable engineering toolset Gram-negative bacteria. The challenge is addressed tailoring CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs streamlined protocol plasmid assembly supports multiplex editing >85% adopted to construct deconstruct complex phenotypes the soil bacterium Pseudomonas putida . Single-step an aromatic-compound production phenotype multi-step deconstruction intricate redox metabolism illustrate versatility afforded our toolbox. Hence, this approach overcomes typical limitations previous empowers programs were out reach thus far.

Язык: Английский

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Genetic engineering strategies for sustainable polyhydroxyalkanoate (PHA) production from carbon-rich wastes

Environmental Technology & Innovation, Год журнала: 2023, Номер 30, С. 103069 - 103069

Опубликована: Фев. 15, 2023

Polyhydroxyalkanoates (PHAs) are promising biopolymers for biomedical applications due to their excellent biocompatibility and biodegradability. However, the high production cost mainly resulting from pure sugar substrate limits PHA commercialization. It makes various carbon-rich wastes potential substrates production. The integration optimization of metabolic pathways can further enhance conversion PHAs. Genetic engineering strategies focusing on carbon flux energy metabolism have improved capacities targeted strains by promoting assimilation, enhancing synthesis, reducing branch metabolism. CRISPR/Cas9-based systems also served as efficient genome editing tools improve efficiency modification. modification requires fitness among strains, substrates, products. Therefore, this review outlined genetic approaches promote discussed feasibility in valorizing representative wastes. To illustrate widespread applicability support microbial cell factories with core production, involved advanced fermentation co-production engineered strains.

Язык: Английский

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Automating the design-build-test-learn cycle towards next-generation bacterial cell factories

New Biotechnology, Год журнала: 2023, Номер 74, С. 1 - 15

Опубликована: Янв. 31, 2023

Automation is playing an increasingly significant role in synthetic biology. Groundbreaking technologies, developed over the past 20 years, have enormously accelerated construction of efficient microbial cell factories. Integrating state-of-the-art tools (e.g. for genome engineering and analytical techniques) into design-build-test-learn cycle (DBTLc) will shift metabolic paradigm from almost artisanal labor towards a fully automated workflow. Here, we provide perspective on how DBTLc could be harnessed to construct next-generation bacterial factories fast, high-throughput fashion. Innovative toolsets approaches that pushed boundaries each segment are reviewed this end. We also present most recent efforts automation DBTLc, which heralds autonomous pipeline biology near future.

Язык: 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, Год журнала: 2024, Номер 52(4), С. e19 - e19

Опубликована: Янв. 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.

Язык: 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, Год журнала: 2025, Номер unknown

Опубликована: Янв. 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.

Язык: Английский

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New synthetic biology tools for metabolic control

Current Opinion in Biotechnology, Год журнала: 2022, Номер 76, С. 102724 - 102724

Опубликована: Апрель 27, 2022

In industrial bioprocesses, microbial metabolism dictates the product yields, and therefore, our capacity to control it has an enormous potential help us move towards a bio-based economy. The rapid development of multiomics data accelerated systematic understanding complex metabolic regulatory mechanisms, which allow develop tools manipulate them. last few years, machine learning-based modeling, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) derived synthetic biology tools, genetic circuits have been widely used microorganisms, gene expression, build pathways for bioproduction. This review describes latest developments control, focuses on trends challenges engineering strategies.

Язык: Английский

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Systems engineering of Escherichia coli for high-level shikimate production

Metabolic Engineering, Год журнала: 2022, Номер 75, С. 1 - 11

Опубликована: Окт. 31, 2022

Язык: Английский

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Advances in the optimization of central carbon metabolism in metabolic engineering

Microbial Cell Factories, Год журнала: 2023, Номер 22(1)

Опубликована: Апрель 21, 2023

Abstract Central carbon metabolism (CCM), including glycolysis, tricarboxylic acid cycle and the pentose phosphate pathway, is most fundamental metabolic process in activities of living organisms that maintains normal cellular growth. CCM has been widely used microbial engineering recent years due to its unique regulatory role metabolism. Using yeast Escherichia coli as representative organisms, we summarized strategies on optimization eukaryotic prokaryotic chassis, such introduction heterologous pathways key enzymes or factors, lay groundwork for future use engineering. Furthermore, bottlenecks application prospects are summarized.

Язык: Английский

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Recent Advances in CRISPR-Cas Technologies for Synthetic Biology

The Journal of Microbiology, Год журнала: 2023, Номер 61(1), С. 13 - 36

Опубликована: Янв. 1, 2023

Язык: Английский

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Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida

Metabolic Engineering, Год журнала: 2024, Номер 82, С. 157 - 170

Опубликована: Фев. 16, 2024

Sustainable aviation fuel (SAF) will significantly impact global warming in the sector, and important SAF targets are emerging. Isoprenol is a precursor for promising compound DMCO (1,4-dimethylcyclooctane) has been produced several engineered microorganisms. Recently, Pseudomonas putida gained interest as future host isoprenol bioproduction it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile P. production. We employ two computational modeling approaches (Bilevel optimization Constrained Minimal Cut Sets) to predict gene knockout optimize "IPP-bypass" pathway maximize Altogether, highest production titer was achieved at 3.5 g/L under fed-batch conditions. This combination of strain engineering on an advanced biofuels vital significance enabling process that use renewable streams.

Язык: Английский

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