Optimizing Cupriavidus necator H16 as a host for aerobic C1 conversion DOI Creative Commons
Stefano Donati, Christopher W. Johnson

Current Opinion in Biotechnology, Journal Year: 2025, Volume and Issue: 93, P. 103306 - 103306

Published: April 24, 2025

Biological systems capable of converting CO2 or CO2-derived, single-carbon (C1) compounds can be used to reduce reverse carbon emissions while establishing a circular bioeconomy provide sustainable sources the fuels, foods, and materials humanity relies on. A robust will rely upon variety microorganisms assimilating C1 them valuable products at industrial scale. While anaerobic microbes are ideal hosts for production short-chain acids alcohols, aerobic respiration well suited biosynthesis higher molecular weight products. One such organism is gram-negative soil bacterium Cupriavidus necator, which has been utilized in commercial biopolymers decades. More recently, its capability robust, growth on inspired research efforts that have advanced it toward becoming one leading bacterial C1-based biomanufacturing. This review highlights those context characteristics historically made C. necator an excellent host bioconversion processes: metabolic versatility, ability grow rapidly high cell densities, genetic amenability.

Language: Английский

Development of CRISPR-Cas9-Based Genome Editing Tools for Non-model Microorganism Erwinia persicina DOI Creative Commons
Tingfeng Cheng, Xinyan Cao,

Yuchen Wang

et al.

Synthetic and Systems Biotechnology, Journal Year: 2025, Volume and Issue: 10(2), P. 555 - 563

Published: Feb. 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.

Language: Английский

Citations

0
CnRed: Efficient, Marker-free Genome Engineering of Cupriavidus necator H16 by Adapted Lambda Red Recombineering DOI Creative Commons

Simon Arhar,

Johanna Pirchner,

Holly Stolterfoht

et al.

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

Published: Feb. 24, 2025

Due to its ability utilize carbon dioxide, native intracellular accumulation of bioplastic precursors, and a high protein content, the bacterium Cupriavidus necator offers potential solutions for social problems tackled by modern biotechnology. Yet, engineering high-performing chemolithotrophic production strains has so far been hindered lack adequate genome editing methods. In this work we present establishment lambda Red recombineering system use in H16. combination with electroporation as DNA delivery system, it enables an efficient fast gene deletion methodology utilizing either suicide plasmids or, first time, linear PCR product. The novel was validated modification three different genomic loci and, proof-of-concept, ultimately utilized stable integration Escherichia coli phytase appA into phaC1 locus. A Cre/loxP further enabled marker recycling. minimal transformation protocol robust, freedom-to-operate synthetic biology tool increasingly important bacterial host. This approach simplifies accelerates C. is expected significantly enhance future strain development efforts.

Language: Английский

Citations

0
Enzyme expression in Cupriavidus necator H16 for whole-cell biocatalysis DOI
Matteo Vajente, Maria L. Ghirardi, Sandy Schmidt

et al.

Methods in enzymology on CD-ROM/Methods in enzymology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Language: Английский

Citations

0
Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system DOI Creative Commons
Simona Della Valle, Enrico Orsi, Sjoerd C.A. Creutzburg

et al.

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

Published: March 1, 2025

Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition industrial applications. In this study, we simplified and accelerated the editing pipeline by harnessing Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR tightly control delay Cas9-based counterselection, achieving >80% efficiency at two genomic loci within 48 h after electroporation. To further increase versatility of toolbox, upgraded SIBR2.0 it regulate expression Cas12a. SIBR2.0-Cas12a could mediate gene deletion in with ~70% efficiency. Overall, streamlined necator, facilitating potential role bio-based economy.

Language: Английский

Citations

0
Cupriavidus necator as a model organism for CO2-based biotechnology DOI

Halima Aliyu Alhafiz,

Federico Di Bisceglie,

H Meier

et al.

Methods in enzymology on CD-ROM/Methods in enzymology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 1, 2025

Language: Английский

Citations

0
Optimizing Cupriavidus necator H16 as a host for aerobic C1 conversion DOI Creative Commons
Stefano Donati, Christopher W. Johnson

Current Opinion in Biotechnology, Journal Year: 2025, Volume and Issue: 93, P. 103306 - 103306

Published: April 24, 2025

Biological systems capable of converting CO2 or CO2-derived, single-carbon (C1) compounds can be used to reduce reverse carbon emissions while establishing a circular bioeconomy provide sustainable sources the fuels, foods, and materials humanity relies on. A robust will rely upon variety microorganisms assimilating C1 them valuable products at industrial scale. While anaerobic microbes are ideal hosts for production short-chain acids alcohols, aerobic respiration well suited biosynthesis higher molecular weight products. One such organism is gram-negative soil bacterium Cupriavidus necator, which has been utilized in commercial biopolymers decades. More recently, its capability robust, growth on inspired research efforts that have advanced it toward becoming one leading bacterial C1-based biomanufacturing. This review highlights those context characteristics historically made C. necator an excellent host bioconversion processes: metabolic versatility, ability grow rapidly high cell densities, genetic amenability.

Language: Английский

Citations

0