04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Metabolic analysis of carotenoid dynamics and global metabolism in carotenoid mutants of Rhodospirillum rubrum using HPLC/MS methodology(2013) Bóna-Lovász, Judit; Ghosh, Robin (Prof. Dr.)In this study, Rhodospirillum rubrum, a photosynthetic, facultative anaerobic bacterium was investigated as a potentially good candidate for industrial carotenoid production. Due to its high amounts of intracellular membrane (ICM), it provides more possibilities for the storage of carotenoids than E. coli, where only the cytoplasmic membrane is available for sequestration. Since R. rubrum is an anoxygenic phototrophic bacterium, all photosynthetic operators are consequently regulated by oxygen in contrast to aerobic phototrophic microorganisms. The photosynthetic membrane (PM) and carotenoid biosynthesis can only be induced at low (pO2<0.5%) oxygen concentrations. Despite the detailed characterization of the PM there is a paucity of information regarding the biosynthesis and the assembly of the photosynthetic system. Therefore, the wild-type, ST4 (crtD-) and SLYC18 (crtC-crtD-) carotenoid mutants were studied by rapid sampled anaerobic and microaerophilic, dark fed-batch fermentations using fructose-succinate medium. The roles of the regulatory signals - O2 and ubiquinone (UQ) pool - mediating the metabolic pattern and the induction of PM biosynthesis were also investigated in this study. The metabolic pattern showed that strong oxygen and CO2 limitation resulted in extremely slow growth. High-level organic acid excretion and polyhydroxybutyrate (PHB) biosynthesis were observed, reflecting the fully reduced state of the cell. The metabolic profile, induced by the high intracellular acetate concentrations occurring in the initial phase of the growth curve, indicated that the citramalate and the ethylmalonyl pathways were active under anaerobic conditions. Succinate production, presumably via the methylmalonyl pathway, was also observed in absence of oxygen. SLYC18 was found to grow twice as fast as the wild-type under CO2- and oxygen-limited conditions. Under anaerobic conditions SLYC18 contained a two fold higher level of rhodoquinone-10 (RQ) and PHB than the wild-type. Since the high RQ level and the enhanced PHB production show a strong correlation, it is proposed here that the RQ-mediated production of NADPH occurs via a putative NADPH dehydrogenase. In order to investigate the biosynthesis of the PM, the major lipid fractions (fatty acids, phospholipids), as well as carotenoids, isoprenoid quinones and bacteriochlorophyll a (BChla) were measured simultaneously during the fermentation process. Therefore, a single-step extraction with a ternary hexane/methanol/water mixture followed by HPLC with mass spectrometric detection was developed for determination of carotenoids and other non-polar compounds present. The method is suitable for extracting large numbers of samples, which is common in systems biology studies. The procedure was able to determine 18 carotenoids, 4 isoprenoid-quinones, BChla and bacteriopheophytin a as well as four different phosphatidylglycerol species of different acyl chain compositions. The high-resolution time courses of the carotenoid biosynthesis allowed the conversion rate of each carotenogenic reaction to be studied. A strong bottleneck was observed at the reactions of CrtD. Furthermore, the second reactions of all enzymes are significantly slower than the first one. Analysis of the carotenoid dynamics indicated that several parallel carotenoid pathways can be linked at common connection points (hydroxylated carotenoids). The carotenoid biosynthesis was more strongly affected by the low oxygen concentration than the central metabolic pattern. The carotenoid mutants were the most sensitive to oxygen, resulting in growth inhibition and a 33% decrease of anaerobic carotenoid concentration in SLYC18. The carotenogenic reaction sequence was blocked in the wild-type in the presence of oxygen, which resulted in the accumulation of anhydrorhodovibrin instead of the normal major carotenoid, spirilloxanthin. The presence of oxygen clearly affected the activity of CrtD resulting in the opening of two new side-pathways: the alternative spheroidene pathway (4%) in the wild-type and ST4 and the 3,4-didehydrolycopene pathway (6%) in ST4. The existence of the 3,4-didehydrolycopene pathway, which has never been observed in R. rubrum earlier, indicated the ability of CrtI in catalyzing five desaturation steps in the presence of oxygen in the ST4 mutant. The PM biosynthesis showed a strong dependence on the changes of the [UQ]/[UQH2] ratio. Under anaerobic conditions, the metabolism is sensitive to pulses of oxygen, causing temporary inactivation the oxidative tricarboxylic acid cycle and the electron transport chain and changes in the UQ pool redox state. This oscillation pattern was analyzed by system biological methods. The systems biology analysis suggested that the isoprenoids are regulated by an exogenous signal. Considering the metabolic observations in the experiments, the regulatory signal might possibly be the [UQH2]/[UQ] ratio.Item Open Access Metabolic engineering of the photosynthetic bacterium Rhodospirillum rubrum to produce industrially interesting plant carotenoids at high level and low cost(2013) Wang, Guoshu; Ghosh, Robin (Prof. Dr.)The purple non-sulphur photosynthetic bacterium Rhodospirillum rubrum has been genetically engineered to express carotenoids, including plant-derived ones, at high level. Initially, a lycopene-producing R. rubrum strain, SLYC18, was constructed by chromosomal replacement of the late genes of the carotenoid biosynthesis, crtCD, with a kanamycin cassette. SLYC18 showed a longer lag phase than the wild-type, followed by normal growth under both photoheterotrophic and chemoheterotrophic conditions. Absorption spectroscopy and mass spectrometry of extracted carotenoids showed that SLYC18 produced lycopene almost exclusively at high levels (2 mg lycopene/g dry weight cells) under semi-aerobic, dark conditions in a high cell density medium. Using biochemical and spectroscopic analysis showed that lycopene was bound exclusively to the light-harvesting (LH) 1 complexes and reaction centers. SLYC18 exhibited also wild-type levels of LH1 complexes and intracytoplasmic membrane (ICM). In a similar strategy, the crtCD region was replaced with an Arabidopsis thaliana lycopene β-cyclase (crtL) gene flanked by a kanamycin resistance gene, to yield the β-carotene producing strain SWGK46. SWGK46 showed a higher sensitivity to oxidative stress compared to SLYC18, but still could achieve high growth rates in the exponential growth phase and yield wild-type cell densities in the stationary phase when grown both photo- and chemoheterotrophically, respectively. SWGK46 produced β-carotene at a high level of 4.4 mg/g dry weight cells in a high cell density medium. SWGK46 expressed LH1 complexes with a unique Qy near-infrared absorption maximum at 877 nm, corresponding neither to that of LH1 complexes with bound (882 nm) or no carotenoid (874 nm), respectively, indicating that β-carotene is assembled into the LH1 complexes which exist in an altered conformation. In contrast to all other carotenoid-containing strains of R. rubrum observed so far, the β-carotene content of the ICM exceeded that of the bound LH1 complex, indicating that β-carotene has also been released into the ICM phase, implying that CrtL is able to activate the endogenous carotenoid biosynthesis enzymes. Continuous growth passages of SWGK46 yield three secondary mutant phenotypes. Under dark, oxidative chemoheterotrophic conditions a brown secondary mutant (designated SWGK46B) was obtained, which exhibited strongly depressed levels of carotenoid and ICM, and a new absorption maximum at 420 nm, due to an early precursor (a protoporphyrin IX derivative) of bacteriochlorophyll biosynthesis. This phenotype is characteristic of the response of R. rubrum to extreme oxidative stress. Under photoheterotrophic conditions, two green secondary mutants (SWGK46GR and SWGK46GIRR, respectively) were observed. SWGK46GR exhibited strong carotenoid down-regulation when grown photoheterotrophically, but β-carotene production was resumed when the cultures were transferred to chemoheterotrophic conditions. The SWGK46GIRR strain showed the same phenotype under light conditions but did not resume carotenoid production when grown chemoheterotrophically in the dark. The induction of the green secondary mutants was shown to be blue light-dependent. These phenotypes have never been observed in any other R. rubrum strain so far, and probably arise from a protein-protein interaction between CrtL and the R. rubrum carotenoid biosynthesis enzymes. DNA sequence analysis indicated that the green phenotypes are due to mutation of the early genes of carotenoid biosynthesis, crtIB. Bioinformatics analysis of the A. thaliana flavoprotein CrtL indicated domains with possible sequence and structural homologies to known blue light sensors, in particular to plant flavoprotein cryptochromes, as well as to the cyanobacterial 3'-hydroxyechinenone-containing orange carotenoid protein. Possibly, the response of the lycopene β-cyclase to blue-green light may play a role in the putative interaction with the CrtIB, and thereby stimulate the production of secondary mutations.Item Open Access Isolation of alveolar plates from Coleps hirtus(2013) Lemloh, Marie-Louise; Hoos, Sina; Görtz, Hans-Dieter; Brümmer, FranzItem Open Access Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea)(2013) Lemloh, Marie-Louise; Marin, Frédéric; Herbst, Frédéric; Plasseraud, Laurent; Schweikert, Michael; Baier, Johannes; Bill, Joachim; Brümmer, Franz