Promoters are the most important tools to control and regulate the gene expression in synthetic biology and metabolic engineering. The expression of target genes in Escherichia coli is usually controlled by the high-strength inducible promoter with the result that the abnormally high transcription of these genes creates excessive metabolic load on the host, which decreases product formation. The constitutive expression systems are capable of avoiding these defects. In this study, to enrich the application of constitutive promoters in metabolic engineering, four promoters from the glycolytic pathway of E. coli were cloned and characterized using the enhanced green fluorescent protein as reporter. Among these promoters, P-gapA was determined as the strongest one, the strength of which was about 8.92% of that of the widely used inducible promoter P-T7. This promoter was used to control the expression of heterologous xylose reductase in E. coli for xylitol synthesis so as to verify its function in pathway engineering. The maximum xylitol titer (40.6 g.L-1) produced by engineered E. coli under the control of the constitutive promoter P-gapA was obviously higher than that under the control of the inducible promoter PT7, indicating the feasibility and superiority of promoter PgapA in the metabolic engineering of E. coli. (C) 2014 The Chemical Industry and Engineering Society of China, and Chemical Industry Press. All rights reserved.