Genome-scale promoter engineering by coselection MAGE

Abstract

Multiplex Automated Genome Engineering (MAGE) employs short oligonucleotides to scarlessly modify genomes. However, insertions of >10 bases are still inefficient, but can be improved substantially by selection of highly modified chromosomes. Here, we describe Co-Selection MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

“…9 To improve the insertion efficiency of short oligonucleotides (>10 bases), Wang et al proposed a co-selection strategy and combinatorially inserted multiple T7 promoters simultaneously into 12 genomic operons, enabling the rapid optimization of the biosynthesis of aromatic amino acid derivatives. 19 Moreover, by applying over 110 MAGE cycles, they simultaneously inserted hexa-histidine sequences into 38 essential genes that encode the complete translation machinery and realized its in vitro co-purification. 20 Although MAGE-related techniques are efficient and easy to perform, their application is restricted by the development of high-throughput methodologies to screen mutants with desired phenotypes.…”

Recent advances and versatility of MAGE towards industrial applications

“…Whilst Wang et al (2012a) have used MAGE to scarlessly modify genomes, insertions of more than 10 bases remains inefficient. Insertion limits could be improved significantly by the selection of highly modified chromosomes, through the newly developed technique of 'coselection' MAGE (CoS-MAGE), used recently to optimize the biosynthesis of aromatic amino acid derivatives by the combinatorial insertion of multiple T7 promoters simultaneously into 12 genomic operons.…”

What If Extinction Is Not Forever?

“…The most likely would be improvements in genetic engineering, such as the targeted replacement of large stretches of genomic DNA ( 6).…”