(712c) Combinatorial Metabolic Engineering with Genomic Libraries Constructed from Degenerate Oligonucleotide-Primed PCR with Thermodynamically Designed Primers

Authors: 
Freedman, B. G., Virginia Tech
Senger, R. S., Virginia Tech

A genomic library consists of a heterogeneous mixture of DNA fragments derived from one or multiple organisms.  Combinatorial metabolic engineering through the cloning and enrichment of a genomic library to confer resistance to an applied stress has proven successful in several applications.  Genomic library production and cloning is an essential and non-trivial component of enrichment studies designed to discover new functionalities of genomic fragments. The process of creating a genomic library often involves collecting large amounts of genomic DNA, fractionating by physical force or enzymatic digestion, end-polishing, and cloning.  This method requires a substantial amount of trial-and-error to guarantee adequate genomic coverage, and cloning efficiencies are often low. Using degenerate oligonucleotide-primed PCR (DOP-PCR) amplification protocols, we have developed new methodology that can generate microbial genomic libraries of gene-sized fragments (1-10 kb) and incorporate at least 95% of a host genome from less than 1 ng of starting material. Additionally, this method has proven highly reproducible by other researchers, and entire libraries can be packaged reliably into an expression vector in only 60 hours. Genomic distributions of the amplified material from three laboratory strains were calculated using massively parallel high-throughput Illumina® sequencing technologies.  Primers were designed to contain both degenerate (NNNNNN) and non-degenerate sequences, and thermodynamic calculations with NUPACK software enabled optimization of primer sequences to yield both large genomic fragments and high genome coverage. By considering the thermodynamics of oligonucleotide folding, we have further optimized this method to reduce amplification biases. The utility of the DOP-PCR generated genomic libraries was demonstrated through enrichment in Escherichia coli K12 to increase butanol tolerance and in Clostridium cellulolyticum to identify genes enabling increased utilization of multiple cellulosic feed stocks.  The methods related to thermodynamic DOP-PCR primer design and library cloning will be discussed along with results of the combinatorial metabolic engineering studies.