BACKGROUND: 2,3-Butanediol (2,3-BD) is a valuable chemical that can be biosynthesized from many kinds of substrates. For commercial biological production of 2,3-BD, it is desirable to use cheap substrate without pretreatment, such as starch. However, there have been few reports on the production of 2,3-BD directly from starch. RESULTS: In this work, gene malS coding for alpha-amylase (EC 3.2.1.1) precursor was inserted into plasmid pUC18K, and secretory over-expression of at-amylase was achieved by engineered Klebsiella pneumoniae. The extracellular recombinant amylase accelerated the hydrolyzation of starch, and one-step production of 2,3-BD from starch was carried out by engineered K. pneumoniae. A 2,3-BD concentration of 3.8 g L-1 and yield of 0.19 g 2,3-BD g(-1) starch were obtained after 24 h fermentation. CONCLUSION: The one-step production of 2,3-BD from starch was achieved by secretory over-expression of amylase in K. pneumoniae. This would simplify the process and reduce the production cost considerably by enabling use of starch with minimal pretreatment. (c) 2008 Society of Chemical Industry.