RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage‐encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double‐strand DNA (dsDNA), or single‐strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non‐model bacteria, both Gram‐negative and ‐positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non‐model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non‐model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter‐selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet‐based tools for oligo selection from genome sequences are also described, as is the use of oligo‐mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision‐making paths for strategically applying annealase‐dependent or oligo‐mediated recombination in non‐model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species

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“…Ec d (Wu, Li, et al, 2017) λ Red Gam, Beta, and Exo (Khandelwal et al, 2018) Gram-positive Actinobacteria b Corynebacterium glutamicum…”

Section: Recetmentioning

confidence: 99%

Recombineering in Non‐Model Bacteria

Corts¹,

Thomason

Costantino

et al. 2022

Current Protocols

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“…More recently, new methods used to engineer bacterial chromosomal genes have been implemented in Z. mobilis. Wu et al (2017) demonstrated the use of Enterobacteriophage RecT system to perform direct genes knockout. Using this method, only 60 bp homology regions were required for integration.…”

Section: Genetic Engineering Tools Available For Z Mobilismentioning

confidence: 99%

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds

Braga

Gomes

Rainha

et al. 2021

Bioresour. Bioprocess.

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Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved. Graphical Abstract

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“…In some cases, the expression of λ red recombineering genes was proven to be toxic, as shown for the enterobacteriaceae Pantoea ananatis ( Katashkina et al, 2009 ), requiring the use of mutant strains to express λ red genes ( Katashkina et al, 2009 ). Adaptation of Rac recombineering was proven successful in case of Zymomonas mobilis ( Wu et al, 2017 ), Acinetobacter baumannii ( Tucker et al, 2019 ), Corynebacterium glutamicum ( Huang et al, 2017 ) and Pseudomonas putida ( Choi and Lee, 2020 ) among others.…”

Section: λ Red -Based Recombineering In Other Bactmentioning

confidence: 99%

Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics

2020

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Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering-or recombinationmediated genetic engineering-represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ∼40-50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double-and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls.

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RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Cited by 12 publications

References 27 publications

Recombineering in Non‐Model Bacteria

Recombineering in Non‐Model Bacteria

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds

Bacterial Genetic Engineering by Means of Recombineering for Reverse Genetics

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