Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-S.42] Kinetic modeling of biochemical systems for optimization of biotechnological production Kirill Peskov 1,∗ , Nail Demin 1,2
Gizzatkulov 1 , Galina
Lebedeva 3 , Oleg
Institute for Systems Biology SPb, Russian Federation A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russian Federation 3 Edinburgh University, United Kingdom Keywords: Strain improvement; Pyrimidine biosynthesis; Penicillin acylase; Kinetic modeling 2
Kinetic modeling of biochemical systems is a systems biology method which allows taking into model account different types of experimental information (both in vitro and in vivo). Predictions of kinetic models have wide aplication range and could be used for the development of the strain improvement programmes and instruments for optimization of biotechnological production. Here, we describe two examples of such approach. At the ﬁrst example a program of gene modiﬁcations of E. coli strain which produces one of the components of thymidine biosynthesis has been developed. For this aim kinetic model of pyrimidine biosynthesis has been developed and veriﬁed against experimental data and 9 predictions has been suggested indicating what and how gene modiﬁcations should be implemented in existing industrial strain. Practical realization of this program has increased the yield of this necessary component on an industrial scale on 23%. In the framework of the second study on the basis of kinetic model of penicillin acylase and technological features of biotechnological synthesis of 6-aminopenicillan acid we have developed software (PACS) and implemented it in technological processes of pharmaceutical company GlaxoSmithKline. In this software all requirements of biotechnologists who deal with control of 6aminopenicillan acid production in industrial conditions have been completely realized. Software algorithm allows both to regulate reaction conditions and to give to the biotechnologist the optimal strategy of components adding. That allows achievement of the maximal efﬁciency of the biotechnological process. Implementation of the software has led to the enzyme savings amounted about 10% and to the considerable increase of the relative yield of product. It is important that obtained results have not only completely repaid research efforts but also led to the signiﬁcant proﬁt. doi:10.1016/j.jbiotec.2010.09.849 [P-S.43] Nonlinear Enzyme-catalytic Kinetics of Glycerol Dissimilation to 1,3-Propanediol by Klebsiella pneumoniae
catalytic kinetics on reductive and oxidative pathway, the inhibition of 3-hydroxypropionaldehyde (3-HPA) to glycerol dehydratase (GDHt), 1,3-PD oxydoreductase (PDOR) and glycerol dehydrogenase (GDH), and transport of byproduct such as acetate acid and succinic acid across cell membrane were all taken into consideration. Moreover, the enzyme-catalytic kinetics of GDH, pyruvate kinase (PK), pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH) were also investigated. Comparisons between simulated and experimental results indicated that the model could be used to describe the continuous fermentation under steady states reasonably. The intracellular concentrations of intermediates of oxidative pathway such as dihydroxyacetone, dihydroxyacetone phosphate, phosphoenolpyruvate, pyruvate, succinic acid, lactic acid, 2,3-butanediol (2,3-BD), format, ethanol, and acetate acid, could also be predicted for continuous cultivations. The simulation results disclosed that the transport of succinic acid and acetate acid across cell membrane was passive diffusion complied with Fick diffusion law. Furthermore, the inhibition of 3-HPA to GDH was weaker than that to GDHt and PDOR. This model would give new insights into the metabolic and genetic regulation of dha regulon of glycerol metabolism. doi:10.1016/j.jbiotec.2010.09.850 [P-S.44] Development of energizing cell-free protein synthesis system from E. coli extracts HC Kim ∗ , KH Lee, YC Kwon, SC Kang, DM Kim Chungnam National University, Republic of Korea Keywords: cell-free protein synthesis; ATP regeneration; continuous cell-free protein synthesis; glycolytic pathway Despite the promising potential of cell-free protein synthesis, the high cost of the required reagents has plagued its practical application. In particular, most of the reagent cost is attributed to the expenses for conventional energy sources such as phosphoenol pyruvate (PEP) and creatine phosphate (CP). In this study, for the preparative expression of recombinant proteins at a reduced energy cost, we attempted to use the glycolytic intermediates to drive the cell-free protein synthesis in a continuous-exchange cell-free protein synthesis (CECF) reaction. Among the glycolytic intermediates examined, use of fructose 1,6-bisphosphate (FBP) gave the highest yield of protein synthesis, producing approximately 7 mg/ml of a chloramphenicol acetyl transferase (CAT) protein. In this work, we have examined a variety of compounds that can derive the regeneration of ATP during cell-free protein synthesis. Our results demonstrate the possibility of using cell-free protein synthesis as a realistic alternative to the conventional in vivo expression technologies.
Yaqin Sun ∗ , Hu Teng, Zhilong Xiu School of Life Science and Biotechnology, Dalian University of Technology, China Keywords: Glycerol dissimilation; 1,3-Propanediol; Enzymecatalytic kinetics; 3-hydroxypropionaldehyde Glycerol can be converted to 1,3-propanediol(1,3-PD) by Klebsiella pneumoniae under anaerobic and aerobic conditions. The main metabolic pathway of glycerol fermentation include oxidative and reductive pathway. In this study, the twenty-three dimensional nonlinear dynamical system was presented to describe the continuous culture of glycerol metabolism in K. pneumoniae, in which the enzyme-