Chasing bacterial <i>chassis</i> for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

Calero, Patricia; Nikel, Pablo I.

doi:10.1111/1751-7915.13292

Cited by 207 publications

(176 citation statements)

References 283 publications

(319 reference statements)

Supporting

Mentioning

173

Contrasting

Unclassified

Order By: Relevance

“…In turn, the system also requires the use of multiple plasmids. These features are problematic for use in other bacteria, which often have a minimal number of characterized plasmids [20][21][22] . (pVeh) or a plasmid encoding constitutively expressed NHEJ (pNHEJ), and subsequently transformed in Cas9 (pCas9sp) or Cpf1 (pCpf1sp) plasmids that contained zero, one, or two gRNAs.…”

Section: Double Stranded Breaks (Dsb) In Dna Can Be Repaired By Two Mmentioning

confidence: 99%

Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

Plant¹

2019

Preprint

View full text Add to dashboard Cite

Making targeted gene deletions is essential for studying organisms, but is difficult in many prokaryotes due to the inefficiency of homologous recombination based methods. Here, I describe an easily modifiable, single-plasmid system that can be used to make rapid, sequence targeted, markerless knockouts in both a Gram-negative and a Gram-positive organism. The system is comprised of targeted DNA cleavage by Cas9 and error-prone repair by Non-Homologous End Joining (NHEJ) proteins. I confirm previous results showing that Cas9 and NHEJ can make knockouts when NHEJ is expressed before Cas9. Then, I show that Cas9 and NHEJ can be used to make knockouts when expressed simultaneously. I term the new method Potentially Organism-Agnostic Knockout (POAK) system and characterize its function in Escherichia coli and Weissella confusa. First, I develop a novel transformation 1 protocol for W. confusa. Next, I show that, as in E. coli, POAK can create knockouts in W. confusa. Characterization of knockout efficiency across galK in both E. coli and W.confusa showed that while all gRNAs are effective in E. coli, only some gRNAs are effective in W. confusa, and cut site position within a gene does not determine knockout efficiency for either organism. I examine the sequences of knockouts in both organisms and show that POAK produces similar edits in both E. coli and W. confusa. Finally, as an example of the importance of being able to make knockouts quickly, I target W. confusa sugar metabolism genes to show that two sugar importers are not necessary for metabolism of their respective sugars. Having demonstrated that simultaneous expression of Cas9 and NHEJ is sufficient for making knockouts in two minimally related bacteria, POAK represents a hopeful avenue for making knockouts in other under-utilized bacteria.

show abstract

Section: Double Stranded Breaks (Dsb) In Dna Can Be Repaired By Two Mmentioning

confidence: 99%

Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

Plant¹

2019

Preprint

View full text Add to dashboard Cite

show abstract

“…All these traits render this bacterium a robust platform for a range of industrial and environmental applications. In connection to this, the available toolbox for manipulating its genome and metabolism is still under extensive development to further enhance the applicability of P. putida as a cell platform (Calero and Nikel, 2019;Leprince et al, 2012;Mart ınez-Garc ıa and de Lorenzo, 2019).…”

Section: Introductionmentioning

confidence: 99%

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Batianis

Kozaeva

Damalas

et al. 2020

Microbial Biotechnology

Self Cite

View full text Add to dashboard Cite

Summary Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

show abstract

“…In addition to its ecological and biogeochemical relevance, the biology of Prochlorococcus is of interest because of its potential as a chassis for synthetic biology applications [41]. This cell is the simplest photosynthetic "machine" designed by nature -an attractive foundation for manipulation [41]- [44]. It has the smallest genome of any oxygenic phototroph as well as an efficient carbon concentrating mechanism [45].…”

Section: Introductionmentioning

confidence: 99%

Toward a genetic system in the marine cyanobacteriumProchlorococcus

Laurenceau

Bliem

Osburne

et al. 2019

Preprint

View full text Add to dashboard Cite

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically.Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus. To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

show abstract

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

Cited by 207 publications

References 283 publications

Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

Heterologous Cas9 and non-homologous end joining as a Potentially Organism-Agnostic Knockout (POAK) system in bacteria

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Toward a genetic system in the marine cyanobacteriumProchlorococcus

Contact Info

Product

Resources

About