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Microorganisms
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Current Prokaryotic Genome Engineering
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Current Prokaryotic Genome Engineering

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (30 June 2023) | Viewed by 9876

Special Issue Editors

Dr. Tamás Fehér

E-Mail Website
Guest Editor
Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, Hungary
Interests: bacterial and phage genome editing; mutagenesis; transposable elements; metabolic engineering; synthetic biology
Dr. Antonia P. Sagona

E-Mail Website
Guest Editor
School of Life Sciences, University of Warwick, Gibbet Hill Campus, Coventry CV4 7AL, UK
Interests: human cell biology; phage therapy; synthetic and molecular biology; phage–host–human cells interactions; molecular microbiology; microscopy

Special Issue Information

Dear Colleagues,

The invention of DNA sequencing and DNA synthesis more than four decades ago radically transformed molecular biology, including microbial genetics. These not only gave rise to an unprecedented increase in the resolution of genetic analysis but also opened up the possibility of precise genome modification. During the elapsed time, one could witness the emergence of numerous genome editing techniques applying plasmids, linear DNA, transposable elements, phage recombinases and integrases, CRISPR-Cas, de novo genome synthesis, and others. Today, the appearance of novel genome engineering strategies, as well as new combinations of existing systems, warrant the constant development of this field. The areas of application are just as exciting: fundamental biology, virulence, antimicrobial development, vaccines, phage therapy, food biotechnology, metabolic engineering, biosensors, and genetic circuits are just a few examples underlining the potential of this topic.

The aim of this Special Issue is to provide a snapshot of current prokaryotic genome editing. Descriptions of new techniques or applications of existing methods for new purposes or on novel microbial strains are all welcome. Since both the toolbox and the investigated biological questions link bacterial and bacteriophage gene editing, we take these two topics as one whole. As Guest Editors of the Special Issue, we invite you to submit research articles, short communications, and review articles concerning the modification of eubacterial, archaeal, or bacteriophage genomes.

Dr. Tamás Fehér
Dr. Antonia Sagona
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Microorganisms is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • gene editing
  • genome engineering
  • genome reduction
  • modified bacteriophage
  • engineered prokaryotic cells
  • synthetic genome

Published Papers (3 papers)

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Research

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16 pages, 1996 KiB  
Article
Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System
by Luísa D. F. Santos, Laëtitia Caraty-Philippe, Emmanuelle Darbon and Jean-Luc Pernodet
Microorganisms 2022, 10(4), 828; https://doi.org/10.3390/microorganisms10040828 - 16 Apr 2022
Cited by 3 | Viewed by 2135
Abstract
Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report [...] Read more.
Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis. Full article
(This article belongs to the Special Issue Current Prokaryotic Genome Engineering)
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31 pages, 3611 KiB  
Article
inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes
by Ranti Dev Shukla, Ágnes Zvara, Ákos Avramucz, Alona Yu. Biketova, Akos Nyerges, László G. Puskás and Tamás Fehér
Microorganisms 2022, 10(2), 236; https://doi.org/10.3390/microorganisms10020236 - 21 Jan 2022
Viewed by 2636
Abstract
Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered [...] Read more.
Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered a viable alternative; however, to date, it has been of limited use due to its tedious nature and often being limited to a single copy. We introduce here a strategy that uses bacterial insertion sequences, which are the simplest autonomous transposable elements to insert and amplify genetic cargo into a bacterial chromosome. Transgene insertion can take place either as transposition or homologous recombination, and copy number amplification is achieved using controlled copy-paste transposition. We display the successful use of IS1 and IS3 for this purpose in Escherichia coli cells using various selection markers. We demonstrate the insertion of selectable genes, an unselectable gene and a five-gene operon in up to two copies in a single step. We continue with the amplification of the inserted cassette to double-digit copy numbers within two rounds of transposase induction and selection. Finally, we analyze the stability of the cloned genetic constructs in the lack of selection and find it to be superior to all investigated plasmid-based systems. Due to the ubiquitous nature of transposable elements, we believe that with proper design, this strategy can be adapted to numerous other bacterial species. Full article
(This article belongs to the Special Issue Current Prokaryotic Genome Engineering)
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Review

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27 pages, 2564 KiB  
Review
Past, Present, and Future of Genome Modification in Escherichia coli
by Hirotada Mori, Masakazu Kataoka and Xi Yang
Microorganisms 2022, 10(9), 1835; https://doi.org/10.3390/microorganisms10091835 - 14 Sep 2022
Cited by 2 | Viewed by 3429
Abstract
Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the [...] Read more.
Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design. Full article
(This article belongs to the Special Issue Current Prokaryotic Genome Engineering)
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