BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose Tony Sanny & Marina Arnaldos & Stephanie A. Kunkel & Krishna R. Pagilla & Benjamin C. Stark Received: 9 June 2010 / Revised: 30 July 2010 / Accepted: 1 August 2010 / Published online: 18 August 2010 # Springer-Verlag 2010 Abstract Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was de- pendent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol. Keywords Bioethanol . Glucose . Lignocellulose . Vitreoscilla hemoglobin . Xylose Introduction The hemoglobin from the bacterium Vitreoscilla (VHb) is the first discovered and one of the best characterized of the microbial hemoglobins. In its native host, as well as in heterologous hosts transformed with the VHb gene (vgb), a major function appears to be to bind oxygen, particularly under microaerobic conditions, and deliver it to the terminal oxidase of the respiratory chain, thus allowing aerobic growth when oxygen is limiting (Webster 1987; Ramandeep et al. 2001; Park et al. 2002). The expression of VHb in heterologous hosts may also enhance activity of oxygenases in some cases (Lin et al. 2003), be involved in NO detoxification (Kaur et al. 2002), and may influence the activity of bacterial transcription factors (Anand et al. 2010). The expression of VHb in Eschercihia coli appears to affect the expression of hundreds of genes, some positively and some negatively (Roos et al. 2004), and so its overall effects on heterologous hosts may be complex. In any event, engineering of a variety of bacteria with vgb has been shown to improve their growth and ability to degrade aromatic compounds and produce various valuable metabolites. Engineering with vgb and expression of VHb Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2817-7) contains supplementary material, which is available to authorized users. T. Sanny : S. A. Kunkel : B. C. Stark Biology Division, Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, IL, USA M. Arnaldos : K. R. Pagilla Department of Civil, Architectural, and Environmental Engineering, Illinois Institute of Technology, Chicago, IL, USA B. C. Stark (*) BCPS/Biology, Illinois Institute of Technology, Chicago, IL 60616, USA e-mail: starkb@iit.edu Appl Microbiol Biotechnol (2010) 88:1103–1112 DOI 10.1007/s00253-010-2817-7