(251e) Multiplex Recombineering: Progress toward the Multigenic Insertion of Regulatory Elements and Potential Applications In Metabolic Engineering

Microbes can be used as biocatalysts for the production of commodity chemicals or fuels. However, industrial conditions can stress these organisms resulting in reduced production. Engineering stress tolerance in these strains can be difficult because many of the genes involved are unknown. We are currently identifying individual and/or contiguous genes involved in a stress tolerance phenotype by creating genome wide libraries where genes are present on a plasmid in increased copy or genes are ?knocked out? by an interruption on the chromosome. Microarray analysis is used to identify mutants that propagate in the presence of stress and evaluate their relative fitness. These strategies have been successfully applied in our lab to identify E. coli genes involved in tolerance to acetate, succinate, 3-hydroxypropionate, napthol, ethanol, aspartate antimetabolites, and biofilm formation. However, these strategies do not identify associations of multiple, non-contiguous genes that are involved with stress tolerance. It is known that multiple perturbations in gene expression can result in greater stress tolerance. Methods are available to produce libraries of mutants where many genes are up regulated and down regulated within an individual but the identification of those genes remains difficult. We have designed and are constructing pools of synthetic DNA containing barcode tags, regulatory elements and gene homology regions that allow precise insertion upstream of almost every gene in E. coli. The barcode tags allow genome wide identification of genes containing synthetic regulatory elements in a complex population of mutants on a universal microarray. Synthetic regulatory elements that increase or decrease expression, respectively, include the PLtetO-1 promoter fused to a ribosome binding site or an ?inert? sequence that replaces the native ribosome binding site and start codon. The pool of synthetic DNA is then transformed into E. coli and chromosomal insertion is catalyzed by the bacteriophage lambda-Red proteins, termed ?Recombineering?. Multiplex DNA synthesis and multiplex recombineering are currently being optimized. Multiplex recombineering is a potential new way to generate a library of mutants with genome wide multigenic changes in expression. Individuals in the libraries can be selected for growth in stress conditions and genes involved in tolerance identified on a barcode tag microarray. This method has the potential to create libraries where genes are up regulated and/or down regulated and mutants are identified relatively easily. Additionally, genetic diversity may be tuned by the simultaneous or sequential insertion of regulatory elements for multiple genes.