Engineering extracellular electron transfer pathways of electroactive microorganisms by synthetic biology for energy and chemicals production

Chemical Society Reviews, Journal Year: 2023, Volume and Issue: 53(3), P. 1375 - 1446

Published: Dec. 20, 2023

The excessive consumption of fossil fuels causes massive emission CO

Language: Английский

---

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

New Biotechnology, Journal Year: 2023, Volume and Issue: 74, P. 1 - 15

Published: Jan. 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.

Language: Английский

---

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

---

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

---

A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9

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

Published: Feb. 1, 2025

CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, window, and off-target effects. Here, a series iteratively improved cytosine editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an range covering 38 nucleotides upstream downstream PAM, without which 6.3 times larger than that traditional CBEs. Additionally, lower achieved when incorporating high-fidelity mutations at R61A Q964A in FrCas9n, while maintaining high efficiency. final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene facilitating mechanistic studies, functional exploration, protein evolution microbes. Cytosine still face several limitations practical microbial genome engineering. authors develop high-efficiency, unbiased, wide-window, low based on protein, demonstrating effective various microorganisms.

Language: Английский

---

Emergent CRISPR–Cas-based technologies for engineering non-model bacteria

Current Opinion in Microbiology, Journal Year: 2023, Volume and Issue: 75, P. 102353 - 102353

Published: July 4, 2023

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated proteins (Cas) technologies brought a transformative change in the way bacterial genomes are edited, and plethora of studies contributed to developing multiple tools based on these approaches. Prokaryotic biotechnology benefited from implementation such genome engineering strategies, with an increasing number non-model species becoming genetically tractable. In this review, we summarize recent trends microbes using CRISPR–Cas technologies, discussing their potential supporting cell factory design towards biotechnological applications. These efforts include, among other examples, modifications as well tunable transcriptional regulation (both positive negative). Moreover, examine how toolkits for organisms enabled exploitation emergent processes (e.g. native synthetic assimilation on-carbon substrates). Finally, discuss our slant future domesticating light most advances ever-expanding field.

Language: Английский

---

Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration

Biotechnology Advances, Journal Year: 2023, Volume and Issue: 63, P. 108102 - 108102

Published: Jan. 18, 2023

Enzymes need to be efficient, robust, and highly specific for their effective use in commercial bioproduction. These properties can introduced using various enzyme engineering techniques, with random mutagenesis directed evolution (DE) often being chosen when there is a lack of structural information -or mechanistic understanding- the enzyme. The screening or selection step DE limiting part this process, since it must ideally (ultra)-high throughput, specifically target catalytic activity have an accurately quantifiable metric said activity. Growth-coupling strategies involve coupling desired cellular metabolism therefore growth, where growth (rate) becomes output metric. Redox cofactors (NAD+/NADH NADP+/NADPH) recently been identified as promising molecules coupling, owing essentiality ubiquitous nature. cofactor oxidation reduction disrupted through metabolic culturing conditions, rendering cell inviable unless 'rescue' reaction complements imposed deficiency. Using principle, variants displaying improved rates selected increased rate cell. In recent years, several E. coli strains developed that are deficient NAD+/NADH NADP+/NADPH pairs, non-canonical redox pairs NMN+/NMNH NCD+/NCDH, which provides researchers versatile toolbox platforms. A range dependent enzymes engineered variety these strains, demonstrating power growth-coupling technique engineering. This review aims summarize involved creating auxotrophic reduced oxidized state cofactors, resulting successes them Perspectives on unique features potential future applications also presented.

Language: Английский

---

Urban green infrastructure and its role in sustainable cities: A comprehensive review

World Journal of Advanced Research and Reviews, Journal Year: 2024, Volume and Issue: 21(2), P. 928 - 936

Published: Feb. 17, 2024

Urban Green Infrastructure (UGI) plays a pivotal role in shaping sustainable cities by integrating natural elements into the urban landscape. This comprehensive review explores multifaceted contributions of UGI towards enhancing environmental quality, social well-being, and economic resilience settings. As continue to grapple with challenges such as climate change, air water pollution, heat island effect, emerges key solution for fostering sustainability. The delves various components UGI, encompassing green spaces, forests, roofs, permeable surfaces. It examines how these collectively contribute mitigating issues absorbing carbon dioxide, reducing pollutants, attenuating impacts extreme weather events. Additionally, serves biodiversity hotspot, supporting diverse flora fauna within boundaries. Beyond its benefits, significantly influences dynamics well-being. presence spaces fosters recreational opportunities, promotes mental health, strengthens community cohesion. Accessible well-designed infrastructure contributes equitable distribution addressing justice concerns areas. Economically, proves be an asset property values, energy consumption through temperature regulation, local businesses. successful case studies that have effectively implemented achieve development, drawing insights best practices potential challenges. underscores integral creating resilient cities. By environmental, social, dimensions, holistic approach aligns evolving needs populations imperatives future. Understanding intricacies implementation provides foundation policymakers, planners, researchers collaboratively work greener, healthier, more environments.

Language: Английский

---

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

---

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

---