Expression and Functional Analysis of the Compact Thermophilic Anoxybacillus flavithermus Cas9 Nuclease DOI Open Access

Anastasiya Matveeva,

Alexander Ryabchenko,

Viktoria Petrova

et al.

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(23), P. 17121 - 17121

Published: Dec. 4, 2023

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools, as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology medicine. The subclass of thermophilic is actively due to the advances sequencing allowing meticulous examination various microorganisms' genomes search systems. most prominent effectors known date are GeoCas9, ThermoCas9, IgnaviCas9, AceCas9, others. These characterized by a varying temperature range activity stringent PAM preferences; thus, further diversification naturally occurring presents an intriguing task. This study focuses generating construct express compact nuclease (AnoCas9) microorganism Anoxybacillus flavithermus displaying 37-60 °C preference 5'-NNNNCDAA-3' vitro. Here, we highlight close relation AnoCas9 GeoCas9 family effectors. AnoCas9, beyond broadening repertoire nucleases, suggests areas requiring presence thermostable CRISPR/Cas systems vitro, such libraries' enrichment, allele-specific isothermal PCR,

Language: Английский

Biosynthesis of plant neuroactive alkaloids treating Alzheimer’s disease DOI Creative Commons

Quanyu Yin,

Zhen Zhu, Mengquan Yang

et al.

Frontiers in Pharmacology, Journal Year: 2025, Volume and Issue: 16

Published: Feb. 5, 2025

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder characterized by cognitive decline and memory impairment. With increasing global prevalence, the need for effective therapeutic interventions critical. Among currently approved treatments, acetylcholinesterase inhibitors (AChEIs) like huperzine A galantamine stand out due to their neuroprotective roles. These plant-derived alkaloids have demonstrated significant efficacy in alleviating symptoms acetylcholine levels brain.While numerous other plant exhibit varying degrees of neuroactive properties, remain only marketed as specific treatments AD diseases. For example, such berberine (from Berberis species) rhynchophylline Uncaria rhynchophylla) shown potential targeting amyloid-beta (Aβ) aggregation, oxidative stress, tau hyperphosphorylation. Similarly, harmine has ability inhibit hyperphosphorylation through dual inhibition glycogen synthase kinase-3 beta (GSK-3β) specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). However, these metabolites not yet advanced clinical applications (Ng et al., 2015;Rezaul Islam 2024).The elucidation biosynthetic pathways marks advancement understanding biochemistry specialized metabolism. It advances our but also provides opportunities sustainable scalable production synthetic biology approaches. By leveraging this approach, researchers can reconstruct natural products microbial or systems, facilitating efficient reducing reliance on native sources valuable compounds (Liu 2023;Zhang 2023;Bai 2024a;Teng 2024). This opinion highlights implications discoveries future research application treatment.Huperzine A, derived from Huperzia serrata (Lycopodiaceae), well-known AChEI that been widely used traditional Chinese medicine (Ma Gang, 2004;Yang 2017;Wang 2020;Zhang The pathway provided crucial insights into formation Lycopodium uncovered enzymes with novel functions (Li 2022;Ushimaru Abe, 2023;Cheng Recent studies identified three neofunctionalized α-carbonic anhydrase-like (CAL) responsible key Mannich-like condensations form core carbon-carbon bonds alkaloids, steps construction polycyclic skeletons.Through transcriptome analysis enzyme characterization, Nett al. CAL-1 CAL-2, which promote annulation reactions (Nett 2023;Liu 2024a;Zamar proceeds stereospecific modifications scaffold tailoring, involving additional Fe(II)-dependent dioxygenases, introduce oxidation final bioactive (Figure 1) 2021;Nett 2023;Ushimaru 2023). findings shed light complex evolution species, suggesting evolved metabolite defense mechanism. breakthrough deepens opens door bioengineering chassis, resources (Zhang 2022;Gao 2024b;Golubova 2024).Galantamine, an alkaloid plants Amaryllidaceae family, particularly daffodils (Narcissus spp.), another treatment (Prvulovic 2010). Similar was recently elucidated, providing invaluable its (Kilgore 2014;Li 2018;Li 2019;Hu 2021;Mehta discovery began identifying precursor, 4'-O-methylnorbelladine (4OMN), followed coupling catalyzed cytochrome P450 NtCYP96T6. facilitates para-ortho (p-o') necessary produce skeleton. Subsequent methylation reduction steps, NtNMT1 NtAKR1 respectively, complete biosynthesis 2) (Mehta profound metabolic engineering. sourced primarily populations daffodils, biosynthesize it engineered systems holds promise Additionally, galantamine's helps understand how generate chemical diversity simple precursors, foundation engineering related value.The underscores complexity elegance Both share common feature acting inhibitors, though evolutionary origins differ significantly. families, distinct pressures, developed highly allow synthesize intricate structures. While represents advancement, several challenges remain.First, vivo functional roles are fully understood. speculated they serve against herbivores, regulatory mechanisms governing elusive (Chavez Further ecological could provide important medicinal plants, pathways, interactions environment (Szypuła Pietrosiuk, 2024).Second, scalability producing heterologous remains challenge. transient expression N.benthamiana proof-of-concept biosynthesis, translating industrial-scale will require optimization gene expression, precursor supply, enzymatic activity plant-based platforms 2024b;Yang Optimizing enhancing activity, achieving high-yield critical bottlenecks.Microbial platforms, Saccharomyces cerevisiae Pichia pastoris, offer promising avenues large-scale ease genetic manipulation 2023;Yang On hand, chassis Nicotiana benthamiana unique advantages, including environments compartmentalized cells conducive 2023;Golubova 2024;Liu 2024b). Advances CRISPR-based genome editing, multi-gene assembly, flux pivotal overcoming current limitations (Liao 2023;Xie 2023;Teng tools, create paving way accessible therapeutics disease.Finally, discovering new species should be overlooked. likely represent fraction produce. Systematic exploration yield AChE diseases.The moment neuropharmacology. deepen metabolism practical treatments. As population ages burden diseases grows, plantderived continue play essential role treatment. lies intersection biology, engineering, sciences, innovative solutions neurological disorders.MY: Conceptualization, Writing-original draft. MY, QY, ZZ: Writing-review editing.

Language: Английский

Citations

1
Recent advances in genome mining and synthetic biology for discovery and biosynthesis of natural products DOI

Mingpeng Wang,

Lei Chen, Zhaojie Zhang

et al.

Critical Reviews in Biotechnology, Journal Year: 2024, Volume and Issue: unknown, P. 1 - 21

Published: Aug. 12, 2024

Natural products have long served as critical raw materials in chemical and pharmaceutical manufacturing, primarily which can provide superior scaffolds or intermediates for drug discovery development. Over the last century, natural contributed to more than a third of therapeutic production. However, traditional methods producing drugs from become less efficient expensive over past few decades. The combined utilization genome mining synthetic biology based on sequencing, bioinformatics tools, big data analytics, genetic engineering, metabolic systems promises counter this trend. Here, we reviewed recent (2020–2023) examples used resolve challenges production products, such variety, poor efficiency, low yield. Additionally, emerging design principles, building strategies its application prospects NPs synthesis also been discussed.

Language: Английский

Citations

6
Advancing microbial production through artificial intelligence-aided biology DOI
Xinyu Gong, Jianli Zhang, Qi Gan

et al.

Biotechnology Advances, Journal Year: 2024, Volume and Issue: 74, P. 108399 - 108399

Published: June 24, 2024

Language: Английский

Citations

4
Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling DOI Creative Commons
Jason Fontana,

David Sparkman-Yager,

Ian Faulkner

et al.

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

Published: July 27, 2024

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming expression, but poor predictability of guide RNA folding can disrupt control. Here, we correlate efficacy modified RNAs (scRNAs) CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA rate into the active structure (rS = 0.8). This also enables forward design scRNAs, allowing us system three synthetic CRISPRa promoters that orthogonally activate (>35-fold) chosen outputs. Through combinatorial tuning, profile three-dimensional space expressing two different biosynthetic pathways, demonstrating variable production pteridine and human milk oligosaccharide products. approach aids optimization metabolic may accelerate routine effective regulation bacterial hosts. Guide affects functionality systems. authors report gRNA together creation orthogonal demonstrate application coli.

Language: Английский

Citations

4
Bibliometric Analysis of Probiotic Bacillus in Food Science: Evolution of Research Trends and Systematic Evaluation DOI
Lijun Zhao, Shijie Liu,

Miaoyun Li

et al.

Probiotics and Antimicrobial Proteins, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 23, 2025

Language: Английский

Citations

0
Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit DOI
Xingzhong Cao, Xiaojuan Wang, Ruirui Chen

et al.

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

Published: March 4, 2025

Bacillus subtilis is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology metabolic engineering technologies of B. have lagged behind most widely used Saccharomyces cerevisiae Escherichia coli. CRISPR (an acronym clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, programmable DNA cleavage, which revolutionized manner genome editing. In 2016, technology was first introduced into been intensely upgraded since then. this Review, we discuss recently developed key additions to toolkit design in with gene editing, transcriptional regulation, enzyme modulation. Second, advances efficient biochemicals proteins are discussed. Finally, conclude perspectives on challenges opportunities CRISPR-based biotechnology subtilis, wishing that can be comparable traditional microorganisms such E. coli S. someday soon.

Language: Английский

Citations

0
Inducible regulating homologous recombination enables precise genome editing in Pichia pastoris without perturbing cellular fitness DOI
Fan Bai, Peng Cai, Lun Yao

et al.

Trends in biotechnology, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Language: Английский

Citations

0
The structural biology of type III CRISPR-Cas systems DOI
Xuzichao Li, Jie Han,

Jie Yang

et al.

Journal of Structural Biology, Journal Year: 2024, Volume and Issue: 216(1), P. 108070 - 108070

Published: Feb. 22, 2024

Language: Английский

Citations

3
Yarrowia lipolytica: A promising microbial platform for sustainable squalene production DOI
Hany Elsharawy,

Moath Refat

Biocatalysis and Agricultural Biotechnology, Journal Year: 2024, Volume and Issue: 57, P. 103130 - 103130

Published: March 24, 2024

Language: Английский

Citations

3
Shortening electron transfer distance to enhance chemicals and electric energy production in Escherichia coli DOI
Guangjie Liang, Yuan Liu,

Ziyun Gu

et al.

Chemical Engineering Journal, Journal Year: 2024, Volume and Issue: 497, P. 154932 - 154932

Published: Aug. 17, 2024

Language: Английский

Citations

3