ORIGINAL PAPER Increasing reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05 Andrew Iverson • Erin Garza • Jinfang Zhao • Yongze Wang • Xiao Zhao • Jinhua Wang • Ryan Manow • Shengde Zhou Received: 8 November 2012 / Accepted: 7 February 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehy- drogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose ) 2 acetyl-CoA ? 4 NADH ) 2 ethanol) in the engineered strain, Escherichia coli SZ420 (DfrdBC DldhA DackA DfocA-pflB DpdhR:: pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (Y RPG = 4) under anaerobic con- ditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual Y RPG value of *3.6, with xylitol as the major fermentation product and acetate as the by-product. Keywords E. coli Á Catabolism Á NADH output Á Reducing power Á Xylitol Á Xylose Introduction Cellulosic biomass has the potential to become a resource for microbial fermentative production of renewable fuels and valuable green chemicals (Aristidou and Penttila 2000). Many of these chemicals are reduced products when compared to the oxidation states of biomass-derived sug- ars, such as glucose and xylose. The production of these reduced products from cellulosic biomass will most likely utilize anaerobic fermentation by commercially proven microorganisms such as Escherichia coli. One of the challenges of anaerobic fermentative pro- duction of reduced products is the limitation of reducing power output (NADH) of sugar catabolism. During microbial anaerobic fermentation, NAD and NADH play a central role as electron carriers for enzyme catalyzed metabolic reactions. NAD serves as an electron acceptor in substrate catabolism, while NADH provides the reducing power for the reduction of metabolic intermediates that yield redox neutral and/or reduced products. A balance of NAD reduction and NADH oxidation is needed for con- tinuous fermentation (De Graef et al. 1999). However, most microorganisms, if not all, have a fixed number of NADH molecules that can be formed using a given carbon source. For example, two NADH can be normally formed A. Iverson Á J. Zhao Á Y. Wang Á X. Zhao Á J. Wang (&) Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, College of Bioengineering, Hubei University of Technology, Wuhan 430068, People’s Republic of China e-mail: wangjinhua@mail.hbut.edu.cn A. Iverson Á E. Garza Á R. Manow Á S. Zhou (&) Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA e-mail: szhou@niu.edu 123 World J Microbiol Biotechnol DOI 10.1007/s11274-013-1285-5