Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase Stefan de Kok, Duygu Yilmaz, Erwin Suir, Jack T. Pronk, Jean-Marc Daran, Antonius J.A. van Maris n Department of Biotechnology, Delft University of Technology and Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands article info Article history: Received 11 March 2011 Received in revised form 16 May 2011 Accepted 1 June 2011 Available online 14 June 2011 Keywords: Metabolic engineering Synthetic biology Yeast Energetics b-Phosphoglucomutase Maltase abstract Increasing free-energy conservation from the conversion of substrate into product is crucial for further development of many biotechnological processes. In theory, replacing the hydrolysis of disaccharides by a phosphorolytic cleavage reaction provides an opportunity to increase the ATP yield on the disaccharide. To test this concept, we first deleted the native maltose metabolism genes in Saccharomyces cerevisiae. The knockout strain showed no maltose-transport activity and a very low residual maltase activity (0.03 mmol mg protein 1 min 1 ). Expression of a maltose phosphorylase gene from Lactobacillus sanfranciscensis and the MAL11 maltose-transporter gene resulted in relatively slow growth (m aerobic 0.09 70.03 h 1 ). Co-expression of Lactococcus lactis b-phosphoglucomutase accelerated maltose utilization via this route (m aerobic 0.21 70.01 h 1 , m anaerobic 0.10 70.00 h 1 ). Replacing maltose hydrolysis with phosphorolysis increased the anaerobic biomass yield on maltose in anaerobic maltose- limited chemostat cultures by 26%, thus demonstrating the potential of phosphorolysis to improve the free-energy conservation of disaccharide metabolism in industrial microorganisms. & 2011 Elsevier Inc. All rights reserved. 1. Introduction Showcases such as the biotechnological production of 1,3-propa- nediol with Escherichia coli (Nakamura and Whited, 2003) and the anti-malarial precursor artemisinic acid with Saccharomyces cerevisiae (Ro et al., 2006) demonstrate the maturation of metabolic engineer- ing. Introduction and optimization of heterologous enzymes and pathways through metabolic modeling, synthetic biology and high throughput screening allow production of a wide range of biological molecules (Dietrich et al., 2010; Na et al., 2010). The development of efficient microorganisms that closely approximate maximum theore- tical product yields requires cellular homeostasis of the redox cofactors (e.g. NAD(P)(H)) and free energy (e.g. in the form of ATP) for growth, cellular maintenance and/or product formation (Boender et al., 2009; Nasution et al., 2008; Sharma et al., 2007). Aerobic respiration enables redox cofactor regeneration for product pathways that would otherwise result in a surplus of NAD(P)H (Grewal and Kalra, 1995; Kimura, 2003) and can also provide the cells with ATP via oxidative phosphorylation. However, aeration of industrial scale fermentations is expensive due to the cost of stirring, air compression and cooling. In addition, aeration results in dissimilation of part of the substrate to CO 2 , thereby decreasing the product yield. Therefore, where possible, it would be beneficial to produce commodity chemicals through redox-neutral pathways, as is the case for alcohol, lactic acid and many metabolic engineering targets, which allows industrial production under anaerobic conditions. In the conversion of glucose via a classical Embden-Meyerhof glycolytic pathway, substrate-level phosphorylation results in the net formation of 2 ATP for each molecule of glucose converted. A challenging situation arises for products of interest whose forma- tion from glucose does not result in a net formation of ATP when produced through redox-neutral routes under anaerobic conditions. In many such cases, the ATP formed in glycolysis by substrate-level phosphorylation may subsequently be used for carboxylation reac- tions (Zelle et al., 2010; Zhang et al., 2009), product export (van Maris et al., 2004b) or the formation of acyl-CoA esters (Singh et al., 2010; van Maris et al., 2004a). Production of lactic acid by metabo- lically engineered S. cerevisiae is an illustrative example. The conversion of glucose to 2 molecules of lactic acid yields 2 ATP. However, in S. cerevisiae export of lactic acid is hypothesized to require 1 ATP per lactic acid. This results in a ‘zero-ATP pathway’ from glucose to extracellular lactic acid. This phenomenon presents an intrinsic limitation for efficient production of lactic acid under anaerobic conditions and at low pH (van Maris et al., 2004b) since, without a net formation of ATP, cells cannot grow or fulfill the free- energy requirements for cellular maintenance. Increasing free-energy (ATP) conservation from the conversion of substrate into product is of major importance for such ‘zero ATP pathways’. This study will explore the possibilities to increase Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ymben Metabolic Engineering 1096-7176/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2011.06.001 n Corresponding author. Fax: þ31 15 278 2355. E-mail address: A.J.A.vanMaris@TUDelft.nl (A.J.A. van Maris). Metabolic Engineering 13 (2011) 518–526