Site-Directed Mutagenesis Using Oligonucleotide-Based Recombineering

Gerlach, Roman G.; Blank, Kathrin; Wille, Thorsten

doi:10.5772/46241

Cited by 3 publications

(2 citation statements)

References 77 publications

(105 reference statements)

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“…The technology referred to as “ en passant mutagenesis ” has been shown to be effective in inducing large-scale changes in viral genomes without scarring [ 76 ] and for removing F-derivative sequences from the viral genome during reconstruction in cells [ 77 ]. However, the accumulation of point mutations or deletions within the I-SceI recognition site during the second round of homologous recombination [ 78 , 79 ], as well as the self-ligation of the I-SceI enzyme cleavage site, can lead to high background noise [ 79 , 80 ], negatively impacting screening efficiency (after the second round of recombination, the screening efficiency drops from 95% to 1–15%) [ 81 ].…”

Section: Screening Methods Of Herpesvirus Mutantsmentioning

confidence: 99%

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research

Hao

Tang

et al. 2023

Microorganisms

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Herpesviruses are major pathogens that infect humans and animals. Manipulating the large genome is critical for exploring the function of specific genes and studying the pathogenesis of herpesviruses and developing novel anti-viral vaccines and therapeutics. Bacterial artificial chromosome (BAC) technology significantly advanced the capacity of herpesviruses researchers to manipulate the virus genomes. In the past years, advancements in BAC-based genome manipulating and screening strategies of recombinant BACs have been achieved, which has promoted the study of the herpes virus. This review summarizes the advances in BAC-based gene editing technology and selection strategies. The merits and drawbacks of BAC-based herpesvirus genome editing methods and the application of BAC-based genome manipulation in viral research are also discussed. This review provides references relevant for researchers in selecting gene editing methods in herpes virus research. Despite the achievements in the genome manipulation of the herpes viruses, the efficiency of BAC-based genome manipulation is still not satisfactory. This review also highlights the need for developing more efficient genome-manipulating methods for herpes viruses.

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Section: Screening Methods Of Herpesvirus Mutantsmentioning

confidence: 99%

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research

Hao

Tang

et al. 2023

Microorganisms

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“…These methods are based on two successive rounds of recombination, similar to other counterselection systems. The approaches can be categorized into two types (18). The first type requires integration of a duplicated sequence with the I-SceI recognition site sequences in a first round of recombination.…”

mentioning

confidence: 99%

High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Yang

Sun

Huang

et al. 2014

Appl Environ Microbiol

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Steps Towards Deciphering the Post-Polyketide Synthase Tailoring Steps in the Phoslactomycin Biosynthesis Pathway

Marimo

2000

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Phoslactomycins (PLMs) are a group of natural products belonging to a polyketides class.These polyketides are synthesized by sequential reaction catalyzed by a collection of enzymes activities called polyketide synthases. A polyketide is a large class of diverse compounds that are characterized by more than two carbonyl groups connected by single intervening carbon atoms. In other words, a polyketide is a polymer whose monomer is a ketide. The PLMs are also known as phosphazomycins or phospholines. These compounds were isolated based on antifungal and antitumor activities. This array of promising biological activities has stimulated research into the field of PLMs for treatment of various diseases such as aspergillosis. A significant success has been reported in understanding and manipulating PLM biosynthesis. However, its post-polyketide biosynthetic mechanism remains to be elucidated. In this study, we established steps needed to pave the way for the elucidation of the post-polyketide synthase tailoring steps in the phoslactomycin biosynthetic pathway. Various, biological activities of polyketide natural products are often linked with specific structural motifs, biosynthetically introduced after construction of the polyketide core. Therefore, investigation of such "post-polyketide synthase (PKS)" modifications is important, and the accumulated knowledge on these processes can be applied for combinatorial biosynthesis to generate new polyketide derivatives with enhanced biological activity.In this study, the enzymes and genes responsible for the modification of the phoslactomycin moiety have been investigated to verify their functions and to study how ii they are coordinated to achieve the desired phoslactomycin. The proposed modification steps in the PLM biosynthesis pathway involves, PlmT4 a cytochrome P450 monooxygenase, PlmT5, a kinase, and PlmT8 an oxidoreductase. These enzymes were successfully cloned, overexpressed, and purified from an overexpression vector. Mutant strains for two genes plmT4 and plmT8 were either constructed or studied. The function of PlmT4 tailoring enzyme was characterized, by gene disruption and an in vitro enzyme activity assay. The isolation of PLM 1 an intermediate analog from plmT4 mutant strain and the observation of a malonylated PLMs, suggests that the malonyl side chain is introduced during polyketide chain formation These results, will pave the way to delineate the intermediary steps between the PLM PKS product(s) that is released from the PLM PKS and the formation of the final phoslactomycin.

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Site-Directed Mutagenesis Using Oligonucleotide-Based Recombineering

Cited by 3 publications

References 77 publications

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research

Bacterial-Artificial-Chromosome-Based Genome Editing Methods and the Applications in Herpesvirus Research

High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Steps Towards Deciphering the Post-Polyketide Synthase Tailoring Steps in the Phoslactomycin Biosynthesis Pathway

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