System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression. Red/ET recombination perfectly complements SPL technology, since it enables easy modification of the Escherichia coli genome and can be accomplished with linear DNA (i.e., the SPL). To demonstrate the synergistic use of Red/ET and SPL for metabolic engineering applications, we replaced the native promoter of a genomic localized phosphoglucose isomerase (pgi)-lacZ reporter construct by an SPL. Using these technologies together, we were able to rapidly identify synthetic promoter sequences that resulted in activity range of 25% to 570% of the native pgi-promoter.