Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper‐butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable ...resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro‐organism. In this study, the effects of Type I restriction‐modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7‐fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 103 CFU/ml‐donor, which was 15.3‐fold higher than the wild‐type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end‐products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR‐Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5–25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro‐organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.
The conjugation efficiency of Clostridium tyrobutyricum was improved by 15.3‐fold after the deletion of type I restriction‐modification system and native plasmid in this strain; the result provides valuable guidance for establishing efficient DNA transformation methods in non‐model microorganisms. Thanks to the increased conjugation efficiency, the CRISPR‐Cas9/Cpf1 systems were successfully implemented for genome editing in the mutant, expanding the genetic toolbox for unlocking the potential of this organism as a chassis for valuable biofuel and biochemical production.
Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of ...Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In bacteria, foreign nucleic acids are silenced by clustered, regularly interspaced, short palindromic repeats (CRISPR)--CRISPR-associated (Cas) systems. Bacterial type II CRISPR systems have been ...adapted to create guide RNAs that direct site-specific DNA cleavage by the Cas9 endonuclease in cultured cells. Here we show that the CRISPR-Cas system functions in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies similar to those obtained using zinc finger nucleases and transcription activator-like effector nucleases.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We employ the CRISPR-Cas system of Streptococcus pyogenes as programmable RNA-guided endonucleases (RGENs) to cleave DNA in a targeted manner for genome editing in human cells. We show that complexes ...of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33%.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive abilities and diverse metabolic activities, and are therefore widely utilized in bioconversion, biosynthesis and ...bioremediation. However, due to the high GC-content of the genome (~70%), together with low transformation and recombination efficiency, the efficient genome editing of Rhodococcus remains challenging. In this study, we report for the first time the successful establishment of a CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the restriction-modification system, the transformation efficiency of R. ruber was enhanced by 89-fold, making it feasible to obtain enough colonies for screening of mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61, the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated triple-plasmid recombineering system was developed with high efficiency of gene deletion, insertion and mutation. Finally, this new genome editing method was successfully applied to engineer R. ruber for the bio-production of acrylamide. By deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained with reduced byproduct formation and enhanced catalytic stability. Compared with the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells could be recycled from 1 batch to 4 batches.
•Bypassing RM system of Rhodococcus enhanced the transformation efficiency by 89-fold.•Introducing bacteriophage recombinases enabled dsDNA recombineering of Rhodococcus.•A CRISPR/Cas9 method was developed for Rhodococcus genome editing for the first time.•The engineered Rhodococcus could achieve 50% acrylamide production for 4 batches.
Genetic intractability presents a fundamental barrier to the manipulation of bacteria, hindering advancements in microbiological research. Group A
Streptococcus
(GAS), a lethal human pathogen ...currently associated with an unprecedented surge of infections worldwide, exhibits poor genetic tractability attributed to the activity of a conserved type 1 restriction modification system (RMS). RMS detect and cleave specific target sequences in foreign DNA that are protected in host DNA by sequence-specific methylation. Overcoming this “restriction barrier” thus presents a major technical challenge. Here, we demonstrate for the first time that different RMS variants expressed by GAS give rise to genotype-specific and methylome-dependent variation in transformation efficiency. Furthermore, we show that the magnitude of impact of methylation on transformation efficiency elicited by RMS variant TRD
AG
, encoded by all sequenced strains of the dominant and upsurge-associated
emm
1 genotype, is 100-fold greater than for all other TRD tested and is responsible for the poor transformation efficiency associated with this lineage. In dissecting the underlying mechanism, we developed an improved GAS transformation protocol, whereby the restriction barrier is overcome by the addition of the phage anti-restriction protein Ocr. This protocol is highly effective for TRD
AG
strains including clinical isolates representing all
emm
1 lineages and will expedite critical research interrogating the genetics of
emm
1 GAS, negating the need to work in an RMS-negative background. These findings provide a striking example of the impact of RMS target sequence variation on bacterial transformation and the importance of defining lineage-specific mechanisms of genetic recalcitrance.
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•The genome editing system for wild-type P. aeruginosa KT1115 were established.•Nitric oxide, not nitrate, is crucial for RLs production.•A two-stage fermentation strategy were ...developed to improve RLs productivity.•The RLs productivity increased by 55% compared to wild-type strain KT1115.
Nitrate plays a crucial role in the high-efficient fermentation production of rhamnolipids (RLs). However, the underlying mechanism remains unclear. Firstly, by knocking out the restriction endonuclease PaeKI and utilizatiing the endogenous CRISPR-Cas-mediated single-plasmid recombineering system, a genome editing system for P. aeruginosa KT1115 has been established. Secondly, an engineered strain KT1115ΔpaeKIΔnirS was obtained with a 87% of reduction in nitric oxide (NO) accumulation and a 93% of reduction in RLs production, revealing the crucial role of NO signaling molecule produced from nitrate metabolism in RLs production. Finally, by combining metabolic engineering of the nitrate metabolism pathway with nitrogen feeding, a new two-stage fermentation process was developed. The fermentation production period was reduced from 168 h to 120 h while achieving a high yield of 0.8 g/g, and the average productivity increased by 55%. In all, this study provides a novel insights in the RLs biosynthesis and fermentation control strategy.
Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically ...important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type–like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.
Considered a "Generally Recognized As Safe" (GRAS) bacterium, the plant growth-promoting rhizobacterium
has been widely applied in: agriculture, medicine, industry, and environmental remediation.
...species not only accelerate plant growth and degrade toxic substances in wastewater and soil but also produce industrially-relevant enzymes and antimicrobial peptides. Due to a lack of genetic manipulation tools and methods, exploitation of the bioresources of naturally isolated
species has long been limited. Genetic manipulation tools and methods continue to improve in
, such as shuttle plasmids, promoters, and genetic tools of CRISPR. Furthermore, genetic transformation systems develop gradually, including: penicillin-mediated transformation, electroporation, and magnesium amino acid-mediated transformation. As genetic manipulation methods of homologous recombination and CRISPR-mediated editing system have developed gradually,
has come to be regarded as a promising microbial chassis for biomanufacturing, expanding its application scope, such as: industrial enzymes, bioremediation and bioadsorption, surfactants, and antibacterial agents. In this review, we describe the applications of
bioproducts, and then discuss recent advances and future challenges in the development of genetic manipulation systems in this genus. This work highlights the potential of
as a new microbial chassis for mining bioresources.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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