ABSTRACT
Central to the concept of metabolic engineering is the modication of a microorganism’s genome to help overproduce a target metabolite, inhibit the formation of an undesirable byproduct, and/or to improve the tness of the microorganism for industrial applications. In the early years of metabolic engineering, genetic modication was carried out randomly by chemical or transposon mutagenesis, followed by screening or selecting for a desired phenotype. With the onset of recombinant DNA technology, the addition, deletion, or modication of specic genes or operons began to be carried out in a systematic manner. Since the early 1990s, researchers employing Saccharomyces cerevisiae have enjoyed a distinct advantage in chromosomal engineering strategies, since the high rate of homologous recombination in yeast had aorded the means to generate gene deletions via transformation of a PCR product [1-3]. Alterations to bacterial genomes, especially with non-E. coli bacteria, were not as easily performed. Bacterial gene replacements typically involved transformation of a nonreplicating plasmid containing a deleted or modied gene, followed by a low-frequency plasmid integration event into the chromosome. Selection or screening for resolution events was followed by phenotypic screens and/or Southern gels to identify gene replacement candidates.
