Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism Yun Chen a , Laurent Daviet b , Michel Schalk b , Verena Siewers a , Jens Nielsen a,n a Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden b Firmenich SA, Corporate R&D Division, CH-1211 Geneva 8, Switzerland article info Article history: Received 29 June 2012 Received in revised form 1 October 2012 Accepted 5 November 2012 Available online 17 November 2012 Keywords: Acetyl-CoA Cell factory Saccharomyces cerevisiae a-Santalene abstract Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of a-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products. & 2012 Elsevier Inc. All rights reserved. 1. Introduction Saccharomyces cerevisiae is the most widely used cell factory employed in the production of food and beverage products, bioethanol, vaccines, therapeutic proteins and nutraceuticals (Nevoigt, 2008). With its long term use for large-scale bioethanol production this organism is well implemented in the fermenta- tion industry. Furthermore, S. cerevisiae is often the organism of choice when a new process has to be developed due to its robustness towards harsh environmental conditions, the ease of genetic manipulation, and the extensive available knowledge about its physiology and biochemistry (Nielsen and Jewett, 2008). There is therefore much interest to develop S. cerevisiae as a general cell factory platform. Such a platform cell factory can preferentially efficiently convert the raw material, today typically glucose/fructose derived from starch or sucrose, but in the future also pentoses derived from lignocellulose (Garcia Sanchez et al., 2010; Ha et al., 2011), into so-called precursor metabolites that are then further converted into the product of interest (Nielsen and Jewett, 2008). One of these key metabolites is acetyl-CoA that is used as precursor for the production of a wide range of industrially very interesting products including isoprenoids (mainly used as flavours and fragrances, biodiesels, antimala- rial and anticancer drugs, antibiotics, rubber, dietary supple- ments, food ingredients and vitamins), polyketides (antibiotics, anticancer drugs and immunosuppressors), lipids (such as dietary supplements, pharmaceuticals and biodiesels), polyhydroxyalk- anoates and 1-butanol (Fig. 1). These products are produced by pathways that usually drain acetyl-CoA from the cytosol. In connection with the reconstruc- tion of synthetic pathways it is generally desirable to position these pathways in this compartment as this will minimize issues related to product secretion. Therefore, the supply of sufficient amounts of the precursor acetyl-CoA in the cytoplasm is crucial. As illustrated in Fig. 1, acetyl-CoA is, however, produced and used in several different compartments, i.e., the cytosol, mitochondria and the peroxisomes, and in S. cerevisiae this metabolite cannot be transported directly between the different compartments without the carnitine/acetyl-carnitine shuttle or glyoxylate cycle. Even though S. cerevisiae holds all components of the carnitine trans- port system, it is not capable of de novo synthesis of carnitine (van Roermund et al., 1999) and in industrial fermentations, it would be too expensive to add this component to the medium. Thus, the carnitine shuttle is excluded as a possible route to channel acetyl-CoA between different compartments. In yeast, acetyl-CoA in the cytosol is produced from acetate that is derived from acetaldehyde, which is formed by de- carboxylation of pyruvate. Acetaldehyde can also be converted to ethanol by alcohol dehydrogenase, and during growth on glucose the majority of the glycolytic flux is directed towards ethanol due to the so-called Crabtree effect in yeast (Van Hoek et al., 1998; Vemuri et al., 2007). Besides the main alcohol dehydrogenase (Adh1p) there are several alcohol dehydrogenases that can catalyze the conversion of acetaldehyde to ethanol Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/ymben Metabolic Engineering 1096-7176/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymben.2012.11.002 n Corresponding author. Fax: þ46 31 772 3801. E-mail address: nielsenj@chalmers.se (J. Nielsen). Metabolic Engineering 15 (2013) 48–54