Targeted mutagenesis: A sniper-like diversity generator in microbial engineering

Abstract

Mutations, serving as the raw materials of evolution, have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions. Global and targeted mutagenesis are two main methods of obtaining various mutations, distinguished by the range of action they can cover. While the former one stresses the mining of novel genetic loci within the whole genomic background, targeted mutagenesis performs in a more straightforward manner, bringing evolutionary escape and error catastrophe under control. In this review, we classify the existing techniques of targeted mutagenesis into two categories in terms of whether the diversity is generated in vitro or in vivo, and briefly introduce the mechanisms and applications of them separately. The inherent connections and development trends of the two classes are also discussed to provide an insight into the next generation evolution research.

High-throughput microbioreactor provides a capable tool for early stage bioprocess development

“…In recent years, both the development of host strains and the identification/design of promising protein candidates have accelerated significantly with steady progress in the fields of molecular biology, genetic engineering, synthetic biology, protein engineering, and bioinformatics. These advances encompass high-throughput (HTP) methodologies, such as HTP proteomics and protein crystallization, microarray technologies, RNA sequencing, large-scale genome sequencing, and gene editing technologies [1][2][3][4][5][6] . The construction of combinatorial libraries was strongly supported by growing structural and genome sequence databases 7 .…”

Phage Therapy: What Have We Learned?

“…Additionally, they should be ready for the introduction or exchange of genomic modules (e.g., an appropriate restriction-modification system or phage receptors determining gene cassettes), enabling these strains to serve as microbial cell factories for the propagation of selected therapeutic phages. Methodologies enabling the abolishment of mobile genetic elements and other genome fragments using genome shuffling, recombineering, oligo-mediated allelic replacement, or genome editing using CRISPR/Cas-assisted selection of desired clones have been developed for model bacteria, even on a genome-wide scale [ 201 , 202 , 203 , 204 , 205 , 206 , 207 , 208 , 209 ]. The repertoire of genetic engineering tools that extend the ability of genomic manipulations to bacteria other than E. coli using the newest strategies has been constantly increasing, providing means to edit genomes belonging to genera represented by the most problematic bacterial pathogens, including potential phage propagation strains [ 210 , 211 , 212 , 213 , 214 , 215 , 216 , 217 , 218 ].…”

Directed evolution to improve protein folding in vivo

“…Classic random mutagenesis techniques such as chemical and physical mutagenesis, and error prone PCR, transposon insertion mutagenesis, gene shuffling as well as more recently developed technologies for targeted mutagenesis including Multiplex Automated Genome Engineering (MAGE) facilitate the introduction of genetic changes in vitro and in vivo . The diverse methods are described in several excellent reviews and articles and will not be discussed further here [ 21 – 24 ].…”