Directed Combinatorial Mutagenesis of Escherichia coli for Complex Phenotype Engineering

Engineering of strains for industrial production requires the targeted improvement of multiple complex traits ranging from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for generating targeted genomic modifications at high efficiency along with high throughput phenotypic screening and growth strategies to rapidly engineer multiple traits in Escherichia coli. Using iCREATE, we engineered rapid glucose and xylose co-consumption, tolerance to hydrolysate inhibitors, and 3-hydroxypropionate (3HP) production in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened targeting ~40,000 mutations across 30 genes; including global and high level regulators that regulate the global gene expression, transcription factors that play important roles in genome level transcription, and enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants conferring increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. The best producing quadruple mutant strain BGHPht was tested under high furfural and high acetate hydrolysate fermentation, demonstrating a 6.3 and 7-fold increase in productivity relative to the parent strain, respectively. This technology enables a single researcher to iteratively generate hundreds of thousands of designer variants and to map each of these variants to a selected phenotype using the designed barcode.