Strategy for directing combinatorial genome engineering in Escherichia coli

Abstract

We describe a directed genome-engineering approach that combines genome-wide methods for mapping genes to traits [Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LBA, Gill RT (2010) Nat Biotechnol 28:856–862] with strategies for rapidly creating combinatorial ribosomal binding site (RBS) mutation libraries containing billions of targeted modifications [Wang HH, et al. (2009) Nature 460:894–898]. This approach should prove broadly applicable to various efforts focused on improving production of fuels, chemicals, and pharmaceuticals, among other products. We used barcoded promoter mutation libraries to map the effect of increased or decreased expression of nearly every gene in Escherichia coli onto growth in several model environments (cellulosic hydrolysate, low pH, and high acetate). Based on these data, we created and evaluated RBS mutant libraries (containing greater than 100,000,000 targeted mutations), targeting the genes identified to most affect growth. On laboratory timescales, we successfully identified a broad range of mutations (>25 growth-enhancing mutations confirmed), which improved growth rate 10–200% for several different conditions. Although successful, our efforts to identify superior combinations of growth-enhancing genes emphasized the importance of epistatic interactions among the targeted genes (synergistic, antagonistic) for taking full advantage of this approach to directed genome engineering.

Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals

“…There is no established strategy to predict the epistatic interactions of target genes, and searching for the optimal combination of beneficial genetic traits is challenging (28). A general model is included to illustrate interactions among the four genetic traits for furfural tolerance (Fig.…”

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

“…MAGE uses recombineering (6) to simultaneously incorporate multiple single-strand DNA (ssDNA) oligonucleotides (oligos), and thereby rapidly creates desired allele combinations and combinatorial genomic libraries. From accelerated optimization of biosynthetic pathways (5,(7)(8)(9) to the construction of a so-called "genomically recoded organism" (10)(11)(12), MAGE has allowed genome-engineering endeavors of unparalleled complexity in Escherichia coli. Functionality of ssDNA-mediated recombineering has been described in various other species, including lactic acid bacteria (13), Corynebacterium glutamicum (14), and Bacillus subtilis (15).…”

White biotechnology: State of the art strategies for the development of biocatalysts for biorefining

“…More recently, alternative methods for the creation of synthetic promoter libraries containing different 5' UTR elements reporter genes, such as GFP or RFP, were developed for E. coli (Mutalik et al , 2013a, Mutalik et al , 2013b, Sandoval et al , 2012, Stanton et al , 2014 and…”