Опубликована: Янв. 1, 2025
Язык: Английский
Nature Communications, Год журнала: 2024, Номер 15(1)
Опубликована: Апрель 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
Язык: Английский
Процитировано
34
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
Процитировано
21
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
Процитировано
3
Biotechnology Advances, Год журнала: 2023, Номер 68, С. 108239 - 108239
Опубликована: Авг. 22, 2023
Synthetic biology transforms the way we perceive biological systems. Emerging technologies in this field affect many disciplines of science and engineering. Traditionally, synthetic approaches were commonly aimed at developing cost-effective microbial cell factories to produce chemicals from renewable sources. Based on this, immediate beneficial impact environment came reducing our oil dependency. However, is starting play a more direct role environmental protection. Toxic released by industries agriculture endanger environment, disrupting ecosystem balance biodiversity loss. This review highlights that can help protection providing remediation systems capable sensing responding specific pollutants. Remediation strategies based genetically engineered microbes plants are discussed. Further, an overview computational facilitate design application tools presented.
Язык: Английский
Процитировано
30
Metabolic Engineering, Год журнала: 2024, Номер 82, С. 238 - 249
Опубликована: Фев. 23, 2024
Язык: Английский
Процитировано
16
Synthetic and Systems Biotechnology, Год журнала: 2025, Номер 10(2), С. 555 - 563
Опубликована: Фев. 19, 2025
Erwinia persicina is a bacterium that has been known to produce secondary metabolites, such as andrimid, pink pigment, and exopolysaccharides, infect more than twenty plant species. However, traditional gene manipulation methods have hindered by the inefficient of suicide plasmid-mediated genome editing. In this study, we describe successful application CRISPR-Cas9 system in E. persicina. Efficient editing was achieved substituting native gRNA promoter with J23119 single-plasmid (pRed_Cas9_ΔpoxB) optimizing design. The use double gRNAs led deletion 42 kb genomic fragment, incorporation sacB screening marker facilitated iterative knockouts. Additionally, 22 plasmid containing self-resistance conjugally transferred into persicina, resulting insertion 6.4 fragment 100 % efficiency. Furthermore, demonstrated expression shinorine, an anti-UV compound, within chassis. This study establishes promising chassis for synthetic biology provides model gene-editing systems non-model microorganisms.
Язык: Английский
Процитировано
2
Biotechnology Advances, Год журнала: 2025, Номер unknown, С. 108522 - 108522
Опубликована: Янв. 1, 2025
Язык: Английский
Процитировано
1
Nature Communications, Год журнала: 2023, Номер 14(1)
Опубликована: Окт. 21, 2023
Abstract A true circular carbon economy must upgrade waste greenhouse gases. C1-based biomanufacturing is an attractive solution, in which one (C1) molecules (e.g. CO 2 , formate, methanol, etc.) are converted by microbial cell factories into value-added goods (i.e. food, feed, and chemicals). To render cost-competitive, we adapt metabolism to perform chemical conversions at high rates yields. this end, the biotechnology community has undertaken two (seemingly opposing) paths: optimizing natural C1-trophic microorganisms versus engineering synthetic C1-assimilation de novo model microorganisms. Here, pose how these approaches can instead create synergies for strengthening competitiveness of as a whole.
Язык: Английский
Процитировано
20
EMBO Reports, Год журнала: 2024, Номер 25(4), С. 1694 - 1699
Опубликована: Фев. 12, 2024
Язык: Английский
Процитировано
8
Water Conservation Science and Engineering, Год журнала: 2024, Номер 9(2)
Опубликована: Июль 2, 2024
Язык: Английский
Процитировано
8