1 3 J Ind Microbiol Biotechnol (2014) 41:913–930 DOI 10.1007/s10295-014-1439-y BIOCATALYSIS Study of metabolic network of Cupriavidus necator DSM 545 growing on glycerol by applying elementary flux modes and yield space analysis Markan Lopar · Ivna Vrana Špoljaric ´ · Nikolina Cepanec · Martin Koller · Gerhart Braunegg · Predrag Horvat Received: 18 December 2013 / Accepted: 13 March 2014 / Published online: 9 April 2014 © Society for Industrial Microbiology and Biotechnology 2014 can be further increased if NAD/NADH or FAD/FADH 2 were assumed to contribute in the reaction of GLY-3-P DH. As a major outcome, it was demonstrated that experimen- tally determined yields for biomass and PHB with respect to glycerol fit well to the values obtained in silico when the Entner–Doudoroff pathway (ED) dominates over the glyco- lytic pathway; this is also the case if the Embden–Meyer- hof–Parnas pathway dominates over the ED. Keywords Cupriavidus necator · Elementary flux modes · Glycerol · PHB · Yield space analysis Introduction Polyhydroxyalkanoates (PHAs) serve as carbon- and energy- storage compounds in microorganisms and genetically modified plants [5]. They are natural, biodegradable macro- molecules with material traits similar to those of synthetic petrochemically derived plastics and are accessible by the biotechnological conversion of renewable sources [17]. The downside of such processes for industrial biopolymer manu- facturing is still the product price. The use of different inex- pensive waste materials for PHA biosynthesis is a promising strategy for reducing production costs and, at the same time, handling industrial waste-disposal problems [16]. Among all described PHAs, the homopolyester poly-3-hydroxybutyrate (PHB) is the most common representative in nature and, since decades, the most profoundly investigated. Biodiesel is a biofuel produced through transesterifica- tion of vegetable oils or animal fats [15]. Glycerol emerg- ing from biodiesel production (principal by-product of the process; mass ratio of biodiesel to glycerol is 9:1) at increasing quantities [14, 15], constitutes a potential carbon substrate for PHA production. Abstract A metabolic network consisting of 48 reac- tions was established to describe intracellular processes during growth and poly-3-hydroxybutyrate (PHB) produc- tion for Cupriavidus necator DSM 545. Glycerol acted as the sole carbon source during exponential, steady- state cultivation conditions. Elementary flux modes were obtained by the program Metatool and analyzed by using yield space analysis. Four sets of elementary modes were obtained, depending on whether the pair NAD/NADH or FAD/FADH 2 contributes to the reaction of glycerol-3-phos- phate dehydrogenase (GLY-3-P DH), and whether 6-phos- phogluconate dehydrogenase (6-PG DH) is present or not. Established metabolic network and the related system of equations provide multiple solutions for the simultaneous synthesis of PHB and biomass; this number of solutions Electronic supplementary material The online version of this article (doi:10.1007/s10295-014-1439-y) contains supplementary material, which is available to authorized users. M. Lopar · I. V. Špoljaric ´ · N. Cepanec · P. Horvat Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6/IV, 10000 Zagreb, Croatia M. Koller Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010, Graz, Austria M. Koller · G. Braunegg ARENA, Arbeitsgemeinschaft für Ressourcenschonende and Nachhaltige Technologien, Graz University of Technology, Inffeldgasse 23, 8010 Graz, Austria M. Koller (*) Institute of Chemistry, University of Graz, Stremayrgasse 16/IV, 8010 Graz, Austria e-mail: martin.koller@uni-graz.at Downloaded from https://academic.oup.com/jimb/article/41/6/913/5995257 by guest on 13 July 2022