BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING 2,3-Butanediol production from starch by engineered Klebsiella pneumoniae G31-A Flora Tsvetanova & Penka Petrova & Kaloyan Petrov Received: 5 November 2013 /Accepted: 14 November 2013 /Published online: 10 December 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract 2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renew- able feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying α-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simulta- neous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3- BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process. Keywords 2,3-Butanediol . Starch . Klebsiella pneumoniae . Amylase . Bacillus licheniformis Introduction 2,3-Butanediol (2,3-BD) is a bivalent alcohol with extensive industrial uses. The interest in its production increased remark- ably in the last decade because of its application as a liquid fuel, along with its common usage in manufacture of antifreezes, printing inks, perfumes, moistening, and softening agents. 2,3- BD is also a chiral compound, reagent in a number of asym- metric chemical syntheses (Xiu and Zeng 2008; Zeng and Sabra 2011). It is platform chemical for valuable derivatives such as methyl ethyl ketone and 1,3-butadiene (Syu 2001). The microbial production of 2,3-BD is an attractive alter- native to the more costly chemical synthesis. Several bacterial species, belonging to Klebsiella , Enterobacter , Serratia , Paenibacillus polymyxa , Bacillus licheniformis , Bacillus subtilis , and Bacillus amyloliquefaciens are able to secrete 2,3-BD (Ji et al. 2011; Jurchescu et al. 2013). Among them, the species Klebsiella pneumoniae is known as the best for industrial use because of its more complete fermentation, broad substrate spectrum, and cultural adaptability (Garg and Jain 1995; Ma et al. 2009). It is capable of degrading a variety of hexoses, pentoses, and disaccharides and produces mainly meso-2,3-BD via mixed-acid pathway that yields several liq- uid by-products: lactic, succinic, and acetic acid and ethanol (Syu 2001). This is the reason that, so far, a number of engineered Klebsiella strains were manipulated with the pur- pose to redistribute the metabolic flux predominantly to 2,3- BD synthesis (Kumar et al. 2012; Kim et al. 2013; Ji et al. 2010, 2013), and at the same time, the attempts to expand the substrate utilization properties of 2,3-BD-producing strains are very limited (Zheng et al. 2008). In bacterial metabolism, the monosaccharides are first con- verted to pyruvate via a combination of Embden–Mayerhof and pentose phosphate pathways. Then, two molecules of pyruvate are converted to one molecule of α-acetolactate, subsequently reduced to acetoin. The acetoin is reduced to 2,3-BD by a reversible reaction (Ji et al. 2011). Among all Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-5418-4) contains supplementary material, which is available to authorized users. F. Tsvetanova : K. Petrov (*) Institute of Chemical Engineering, Bulgarian Academy of Sciences, 103, Acad. G. Bonchev str., 1113 Sofia, Bulgaria e-mail: kaloian04@yahoo.com P. Petrova Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bonchev str., 1113 Sofia, Bulgaria Appl Microbiol Biotechnol (2014) 98:2441–2451 DOI 10.1007/s00253-013-5418-4