Recent advances and versatility of MAGE towards industrial applications

Abstract

The genome engineering toolkit has expanded significantly in recent years, allowing us to study the functions of genes in cellular networks and assist in overproduction of proteins, drugs, chemicals and biofuels. Multiplex automated genome engineering (MAGE) has been recently developed and gained more scientific interest towards strain engineering. MAGE is a simple, rapid and efficient tool for manipulating genes simultaneously in multiple loci, assigning genetic codes and integrating nonnatural amino acids. MAGE can be further expanded towards the engineering of fast, robust and over-producing strains for chemicals, drugs and biofuels at industrial scales.

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

“…First, genes encoding for the orthogonal AcFRS/tRNA CUA Opt system [75] and four amber codon-containing TolC membrane channel for colicin ColE1 translocation were integrated into the intergenic region of the bacterial chromosome. Resulting organisms were used for the generation of aa-RSs library by diversification by multiplex automated genome engineering (MAGE) [84] using single-stranded DNA (ssDNA) pool designed for the mutagenesis of selected amino acids. Obtained diversified cell pool was subjected for the negative selection in the presence of colicin Col E1 to eliminate not orthogonal aa-RS; the remaining orthogonal AcFRS library went through the positive selection in the presence of NAA to isolate variants with improved enzyme efficiency.…”