Vol 53, No 7 (2017)

Quantitatively characterizing the intracellular carbon flux distribution provides useful information for both fundamental and applied investigations into the cellular metabolism at the system level, such as the roles of different metabolic pathways and individual reactions, metabolic state characterization, metabolic differences between the strains, and clues regarding strategies for producer-strain improvement. A variety of methods have been developed to characterize the metabolic state of the cell by determining its intracellular flux distribution, and together, they are called metabolic flux analysis (MFA) or fluxomics. These methods, in addition to other X-omics technologies (i.e., genomics, transcriptomics, proteomics, and metabolomics) constitute a recent arsenal of the system biology estimation approaches. One of the most well-developed approaches for intracellular carbon flux estimation in vivo in (quasi) steady-state conditions is ¹³C-MFA, which uses substrates that are labeled with a heavy carbon (¹³C). Applying ¹³C-MFA requires the coordination of experts in biochemistry, applied mathematics and nuclear magnetic resonance (NMR) or mass spectrometry. Therefore, the authors have prepared a three-part review highlighting the different but equally important aspects of ¹³C-MFA. In the first part, which is presented below, the focus is on the basic principles of ¹³C-MFA, such as stoichiometric model development, labeling experiments and experimental data extraction. The principles of the labeling experiments modeling and quantitative carbon flux estimation and statistics are discussed in the second part. The final part reviews recent achievements in fundamental and applied investigations of bacterial metabolism achieved using ¹³C-MFA.

We isolated seven algologically and five bacteriologically pure cultures of toxin-producing cyanobacteria from Turgen gorge (Kazakhstan), Karlovy Vary (Czech Republic), and Shar-Nuur Lake, Bayan Ulgiiregion (Mongolia) springs. According to the Daphnia magna test, Desertifilum sp. and Nostoc sp. strains were the most toxic in the test of isolated strains (complete death of all test organisms was detected after 48 h). These strains possessed the highest inhibitory effect on proliferation of the HeLa cancer cell line. The Anabaena sp. 35 and Nostoc sp. 4 strains were also high toxic. Model strains Synechocystis PCC 6803 and Synechococcus elongates PCC 7942, as well as the strain isolated in the present work, Synechococcus sp. 55, were less toxic. Mass spectrometry made it possible to assign cyanobacterial toxins to cyclic depsipeptides. Two cyclic depsipeptides, micropeptin T and oscillapeptin, were detected in Desertifilum sp. extracts. Cryptophycin and small amounts of cyclic depsipeptide micropeptin SD were detected in Nostoc sp. extract.

Two previously obtained, full-size, fully human antibodies that reversibly bind the active form of an enzyme belonging to the subtype EC 3.2.1, which is used for substitutive enzymatic therapy in lysosomal storage diseases, have been characterized by surface plasmon resonance and biolayer interferometry methods. It was shown under normal physiological conditions that the designed antibodies specifically bound with the antigen (KD ~ 10^–8 M) and rapidly dissociated at neutral pH in 60% ethylene glycol while leaving the enzymatic activity unchanged. Dan ue to their properties, the developed antibodies can be used in industry as affinity ligand in the isolation of therapeutic substances of the enzyme.