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( Christoph J. Bolten ),( Hartwig Schroder ),( Jeroen Dickschat ),( Christoph Wittmann ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.8
In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the △metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.
Dynamic Respiratory Measurements of Corynebacterium glutamicum using Membrane Mass Spectrometry
Wittmann, Christoph,Yang, Tae Hoon,Kochems, Irene,Heinzle, Elmar 한국미생물 · 생명공학회 2001 Journal of microbiology and biotechnology Vol.11 No.1
The present work presents a novel approach for the dynamic quantification of respiration rates on a small scale by using lysine-producing Corynebacterium glutamicum ATCC 21253. Cells sampled from batch cultures at different times were incubated in a 12-m1 scale bioreactor equipped with a membrane mass spectrometer. Under dynamic conditions, gas exchange across the gas-liquid phase, specific respiration rates, and RQ values were precisely measured. For this purpose, suitable mass balances were formulated. The transport coefficients for 0_2 and C0_2, crucial for calculating the respiration activity, were determined as k_La_O2=9.18h^-1 and k_la_co2=5.10h^-1 at 400rpm. The application of the proposed method to batch cultures of C. glutamicum ATCC 21253 revealed the maximum specific respiration rates of q_O2=8.4mmol g^-1h^-1 and q_C02=8.7mmol g^-1 h^-1 in the middle of the exponential growth phase after 5h of cultivation. When the cells changed from growth to lysine production due to the depletion of the essential amino acids threonine, methionine, and leucine, q_O2 and q_co2 decreased significantly and RQ increased. The respiration data exhibited an excellent agreement with previous cultivations of the strain [13]. This confirms the potential of the developed approach to realistically reflect the metabolic activities of cells at their point of sampling. The short-term influence of added threonine, methionine, and leucine was highest during the shift from growth to lysine production, where q_O2 and q_co2 increased by 50% within one minute after the pulse addition of these compounds. Non-growing, yet lysine-producing cells taken from the end of the batch cultivation revealed no metabolic stimulation with the addition of threonine, methionine, and leucine.
( Christoph J. Bolten ),( Elmar Heinzle ),( Rolf Muller ),( Christoph Wittmann ) 한국미생물 · 생명공학회 2009 Journal of microbiology and biotechnology Vol.19 No.1
In the present work, the metabolic network of primary metabolism of the slow-growing myxobacterium Sorangium cellulosum was reconstructed from the annotated genome sequence of the type strain So ce56. During growth on glucose as the carbon source and asparagine as the nitrogen source, So ce56 showed a very low growth rate of 0.23 d-1, equivalent to a doubling time of 3 days. Based on a complete stoichiometric and isotopomer model of the central metabolism, 13C metabolic flux analysis was carried out for growth with glucose as carbon and asparagine as nitrogen sources. Normalized to the uptake flux for glucose (100%), cells recruited glycolysis (51%) and the pentose phosphate pathway (48%) as major catabolic pathways. The Entner-Doudoroff pathway and glyoxylate shunt were not active. A high flux through the TCA cycle (118%) enabled a strong formation of ATP, but cells revealed a rather low yield for biomass. Inspection of fluxes linked to energy metabolism revealed that S. cellulosum utilized only 10% of the ATP formed for growth, whereas 90% is required for maintenance. This explains the apparent discrepancy between the relatively low biomass yield and the high flux through the energy-delivering TCA cycle. The total flux of NADPH supply (216%) was higher than the demand for anabolism (156%), indicating additional reactions for balancing of NADPH. The cells further exhibited a highly active metabolic cycle, interconverting C3 and C4 metabolites of glycolysis and the TCA cycle. The present work provides the first insight into fluxes of the primary metabolism of myxobacteria, especially for future investigation on the supply of cofactors, building blocks, and energy in myxobacteria, producing natural compounds of biotechnological interest.
Evaluation of a New Workplace Protection Factor―Measuring Method for Filtering Facepiece Respirator
Sun, Chenchen,Thelen, Christoph,Sanz, Iris Sancho,Wittmann, Andreas Occupational Safety and Health Research Institute 2020 Safety and health at work Vol.11 No.1
Background: This study aims to assess whether the TSI PortaCount (Model 8020) is a measuring instrument comparable with the flame photometer. This would provide an indication for the suitability of the PortaCount for determining the workplace protection factor for particulate filtering facepiece respirators. Methods: The PortaCount (with and without the N95-Companion<sup>TM</sup>) was compared with a stationary flame photometer from Moores (Wallisdown) Ltd (Type 1100), which is a measuring instrument used in the procedure for determining the total inward leakage of the particulate filtering facepiece respirator in the European Standard. Penetration levels of sodium chloride aerosol through sample respirators of two brands (A and B) were determined by the two measuring systems under laboratory conditions. For each brand, thirty-six measurements were conducted. The samples were split into groups according to their protection level, conditioning before testing, and aerosol concentration. The relationship between the gauged data from two measuring systems was determined. In addition, the particle size distribution inside the respirator and outside the respirator was documented. Linear regression analysis was used to calculate the association between the PortaCount (with and without the N95-Companion<sup>TM</sup>) and the flame photometer. Results: A linear relationship was found between the raw data scaled with the PortaCount (without N95-Companion<sup>TM</sup>) and the data detected by the flame photometer (R<sup>2</sup> = 0.9704) under all test conditions. The distribution of particle size was found to be the same inside and outside the respirator in almost all cases. Conclusion: Based on the obtained data, the PortaCount may be applicable for the determination of workplace protection factor.