1,721,021 research outputs found
Metabolic engineering of Pseudomonas putida for the production of aromatics from glucose
No full textBildung, Isolierung und Nachweis der mikrobiell gebildeten Speicherstoffe Polyphosphat und Polyhydroxybuttersäure bei ausgewählten Mikroorganismen
No full textThe present paper is aimed at investigating to what extent the production of the microbial reserve substances polyphosphate and polyhydroxybutyrate can be enhanced and whether they can be isolated. In addition, the possibility of utilising these substances beyond their biological function in the field of environmentally friendly products and technologies is addressed. Owing to the comprehensive method development and the examination of both reserve substances, the present paper consists of three parts, namely methods of detecting the polymer compounds polyphosphate and polyhydroxybutyrate, the microbial production of polyphosphate and the microbial production of polyhydroxybutyrate. Laboratory-scale test series were performed to produce the reserve substances polyphosphate and polyhydroxybutyrate through selective enrichment of microorganisms. The methods for the isolation as well as the qualitative and quantitative detection of the reserve substances were developed and/or established. The produced polyphosphates were detected qualitatively and quantitatively by means of microscopy, fluorescence photometry and gel electrophoresis. The polyphosphates were quantified after extraction by measurement as orthophosphate. Due to a lack of uniform standards, however, the detection methods involve methodological difficulties regarding the determination of the individual chain lengths of the polyphosphates and their quantification, which is why the results should be seen with a critical eye and the detection methods require optimisation. Nevertheless, a simultaneous look at the process flow of all employed detection methods reveals consistent results, which is why the combination of the employed methods can be regarded as purposeful. The produced polyhydroxybutyrate was isolated from the cell and detected qualitatively and quantitatively. The employed detection methods included microscopy, enzyme-mediated assay and gas chromatography. The detection methods for polyhydroxybutyrate yielded satisfactory results, given the availability of suitable standard substances. The phosphate uptake and phosphate accumulation was examined on the basis of the microorganisms in sewage sludge, giving special regard to the fungus species Cunninghamella elegans. It was found that phosphate is accumulated in the cell predominantly as polyphosphate through the activity of microorganisms. At unfavourable culture conditions, such as a change in the pH value, directly or indirectly induced precipitation reactions, such as the chemical precipitation of struvite (magnesium ammonium phosphate) occurred additionally. The carbon/nitrogen/phosphorus ratio in the employed medium was taken as a basis for the assessment of the test results. Compared to the microorganisms in the sewage sludge, the fungus species Cunninghamella elegans was found to be capable of removing from the medium and accumulating in the cell as polyphosphate the tenfold amount of phosphate. Cunninghamella elegans was able to fix a maximum of 10% phosphate in the biomass, whereas the sewage sludge biocoenosis only fixed 3% of phosphate at maximum. Cunninghamella elegans produced a promising biomass yield and accumulated large amounts of phosphate within a short period of time. However, the technical utilisation of polyphosphate production is impracticable due to the necessity of separating the polyphosphates from the biomass, which is not possible without hydrolysis. The microbial production and accumulation of polyhydroxybutyrate was examined on the basis of the bacterial species Paracoccus denitrificans and Cupriavidus necator. Despite varying culture conditions and very high carbon consumption, it was not possible to achieve a high yield of biomass and polyhydroxybutyrate, which is why the method requires optimisation. The highest yield of 3-hydroxybutyrate with Paracoccus denitrificans and Cupriavidus necator amounted to 50% of the dry weight at maximum. In view of the test results achieved at laboratory scale, a transfer to industrial scale should be considered and investigated. The economical utilisation of the reserve substances requires a high cell density of microorganisms as well as a high yield. The tested microorganisms were therefore selected individually for each reserve substance. The further processing of industrial wastewater appears to be a starting point for reducing the production costs of polymers. As part of the tests, industrial process water was added to the employed media as a source of phosphate. Despite some progress in the research into reserve substances, the isolation of the produced reserve substances from microorganisms and their quantification is still difficult, since losses may occur in the course of hydrolysis.At present, the production and extraction of the microbial reserve substances polyphosphate and polyhydroxybutyrate is not yet economical and the method’s technical feasibility is limited
Rational engineering of the methylerythritol 4-phosphate (MEP) pathway for terpenoid production through metabolic control analysis
No full textCompartment engineering to increase metabolic flux through the mevalonate pathway in S. cerevisiae
No full textMetabolic flux analysis for biosynthesis of volatile terpenoids in model Lamiaceae plants
Get PDFEssentielle Öle von Laminaceen haben eine wirtschaftliche Bedeutung auf Grund ihres hohen Gehaltes an Terpenen die in den Trichomen produziert werden. Wenig verstanden wird bislang die Regulation der Biosynthese dieser Terpene. Diese Regulation wurde in der vorliegenden Studie mittels Metabolischer Flussanalyse (MFA) in Pfefferminze und Oregano untersucht. Die Flussanalyse ergab einen Beitrag des Mevalonat Biosynthese Weges zur Monoterpensynthese und einen Beitrag des “Nicht-Mevalonat” Weges zur Sequiterpensythese. Diese Interaktion der Stoffwechselwege zur Synthese von Mono und Sequiterpenen konnte quantifiziert werden. Gleichzeitig wurde die Wichtigkeit des oxidativen Pentosephosphatweges über die Bereitstellung von Reduktionsäquivalenten und RuBisCo zur effizienten CO2 Refixierung nachgewiesen. Diese Studie stellt einen Beitrag zum “metabolic engeneering” dieser Stoffwechselwege dar, da es “bottlenecks” in diesen Synthesewegen aufzeigt.Essential oil of Lamiaceae plants has commercial value due to the enrichment of volatile terpenes in trichomes. However, overall metabolic regulation for the production of these volatile compounds is still not fully understood. The present study was to investigate the metabolism towards terpene production in trichomes which was achieved by performing metabolic flux analysis (MFA) in peppermint and oregano. The flux analysis provided evidence for the contribution of the alternate mevalonate route to monoterpene production and non-mevalonate route to sesquiterpene production, and quantified the cross-talk between non-mevalonate and mevalonate route. In addition, MFA supported a prominent role for the oxidative pentose phosphate pathway in providing reductants for terpene biosynthesis and RuBisCO in refixing CO2 thereby contributing to the carbon use efficiency in peppermint trichomes. This study can advance the metabolic engineering of this cell by uncovering the metabolic bottlenecks in the precursor pathways for further increasing the productivity of these high value compounds
Multiscale modeling of drug-induced toxicity in humans
No full textDrug toxicity poses a crucial problem in drug development and particularly in clinical care and in publichealth. Although in vitro experiments can obtain valuable information of the mechanisms underlyingdrug-induced toxicity, there is still a clear lack of approaches translating such in vitro findings into apatient situation. In this thesis, a novel translational approach is presented and is successfully applied inthree different studies to investigate the onset of adverse drug events at patient level. Here, multiscalemodeling enables the coupling of in vitro concentration-response relationships, at the cellular level, withdrug concentration-time profiles, at the organism level, to predict cellular responses following drug administrationof different doses in vivo. The application of the developed translational approach allowedstudying drug-induced toxicity in humans (i) for acute and chronic administration of azathioprine in aproof-of-concept study, (ii) for fifteen hepatotoxic drugs in a comparative manner focusing on drug administrationof therapeutic and toxic doses, and (iii) for a drug combination therapy of acetaminophenand caffeine. The presented translational approach may lead to useful knowledge for clinical applicationgained from in vitro experiments thereby contributing to the ongoing discussion of the predictive valueof preclinical research. Overall, the results shown here provide novel insights into drug-induced toxicitywithin a patient context and, thus, may improve patient safety in drug development
Reaction and process-optimization of modular synthetic enzyme cascades towards diols and hydroxy ketones meeting industrial demands
No full textHydroxy ketones and diols are chiral building blocks of interest in several active pharmaceutical ingredients. While classic chemical synthesis is able to provide high product concentrations, it oftentimes lacks the required regio- and stereoselectivity and encompasses ecologically problematic conditions or waste. In both regards, enzymes could provide a valuable synthesis alternative. Especially carboligases and NADPH dependent alcohol dehydrogenases would allow a synthesis of these valuable building blocks from inexpensive bulk chemicals. Unfortunately, these enzymes operate preferentially under diluted aqueous conditions, which are unsuitable to provide economically sustainable product concentrations. Hence, this thesis aims to explore methods of process intensification to lift the enzymatic synthesis of hydroxy ketones and diols to industrially relevant levels that are economically feasible. As such, the three subjects (i) NADPH regeneration, (ii) unconventional reaction media, and (iii) process design are intensively studied. In NADPH regeneration two highly atom efficient substrate coupled methods are explored: coproduct recycling and smart cosubstrates. While in the first method recycles coproduct into the main synthesis to gain the final product, the other method generates a value added coproduct, which makes both methods profitable regeneration methods. Alternatively, also whole cell NADPH regeneration from citrate was explored, which revealed promising cost reduction features besides easy applicability. A second aim is the increase of substrate concentrations by the employment of an unconventional hydrophobic reaction environment. Here, a microaqueous reaction system (MARS) proves to be beneficial in increasing substrate concentrations to 500 mM beyond their solubility limit in aqueous conditions. The importance of buffer amounts in this system to ensure catalytic activity as well as the impact of green solvent selection on the system are reflected. Beyond the synthesis application and setup characteristics of MARS for artificial enzyme cascades, it also facilitates product isolation, which makes it a highly interesting reaction environment. As third optimization step, batch and continuous process designs are evaluated to enhance space time yields. Here, in both systems the issue of possible substrate toxicity, which may limit yields, is circumvent by employing and optimizing technical methods. This allowed space time yields of up to 165 g L 1 d-1 in batch and 7296 g L 1 d-1 in continuous application. These three methods are combined, applied and compared in their profitability to fulfil industrial benchmarks. Notably, all these methods are well suited to lift the multi-step biocatalytic synthesis of diols and hydroxy ketones into industrial scale. A catalyst cost analysis reveals enzyme stability and, thereby, the catalyst selection as key factor for a profitable enzymatic process layout. In addition to economic profitability also ecologic sustainability of enzymatic syntheses is assessed. This identifies an application of MARS as indeed highly environmentally sustainable with E factors ranking between 12 to 45
Accelerated Development of Phototrophic Bioprocesses: A Conceptual Framework
No full textPhototrophic systems regained interest as feedstocks for bio-economy but their industrial exploitation mostly fails for economic reasons until today. Due to lacking high throughput photobioreactors and supporting methodologies, especially early stage screening suffers from low efficiency. In this context, a framework for accelerated phototrophic process development was designed. Focusing lipid production by Chlorella vulgaris as a microalgal model process, the full spectrum from strain maintenance, via cultivation and analytics to experimental design was addressed while parallelized cultivation was focused as a key technology.Contrary to well-established serial subculturing, strain maintenance was done by cryopreservation of glucose-adapted cells. Thus, an easy-to-use protocol was set up and optimized. According to specially developed growth pattern analysis, post-thawing viabilities of 63 ± 2 % were achieved and phototrophic pre-cultivation lead to highly reproducible adaptation to light.Enabling elevated throughput cultivation, a 48-well microtiter plate-based micro-photobiore-actor was designed and growth was precisely and non-invasively monitored via scattered light. Strictly controlled conditions permitted a high comparability along the wells of a plate (± 5%) while small scale and fast mixing ensured excellent light supply of the cultures. The system was shown to offer good scalability to established laboratory scale photobioreactors.To handle samples from microscale cultivation, a dye-based assay was set up and assay conditions were optimized towards efficient and precise quantification of Chlorella vulgaris’ intracellular lipid accumulation. Assay automation resulted in minimal hands-on-times while errors due to fluctuating performance of manual operators could be excluded.These technologies were merged into a framework for the accelerated development of phototrophic processes while Kriging-assisted experimental design was used to take full advantage of the improved experimental capacity. Within as little as four experimental rounds, the volumetric productivity of the model lipid production process was approximately tripled solely via medium optimization and synergistic multi-parameter interaction could be revealed.Though this framework was suitable to accelerate phototrophic process development, it may only be seen as an initial blueprint. Future improvement of the micro-photobioreactor and intensified robotic integration will enable more complex processes and thus extend the range of application from screening tasks to the acquisition of kinetic data concerning metabolism of phototrophic microorganisms or even simulation of dynamic environmental conditions
What can we do with smut? Organic acid production from glycerol with Ustilaginaceae
No full textThe rapidly growing need for energy and fuel combined with an increased awareness of the detrimental influence of fossil resources has evoked the investigation of renewable alternatives, such as biodiesel. The production process of biodiesel, however, results in a huge waste stream of crude glycerol, reducing the economic and ecological advantage.The overall aim of this thesis was to establish a production process for organic acids from biodiesel derived glycerol with Ustilaginaceae. The biodiversity within this family is well known and in a screening of 126 Ustilaginaceae, two promising candidates for organic acid production were found - Ustilago trichophora for malic acid production and U. vetiveriae for itaconic acid production. Glycerol uptake and growth rate of both strains were improved by adaptive laboratory evolution. Selection of the best growing single colony for each strain and medium and process optimization drastically improved the production values.The itaconic acid titer of U. vetiveriae TZ1 was increased to 35 g L 1 produced at a production rate of 0.09 g L 1 h 1. Simultaneously about 60 g L 1 malic acid were formed. In first metabolic engineering approaches overexpressing the mitochondrial transporter Mtt1 and the regulator of the itaconic acid gene cluster Ria1, both from U. maydis, the production values could be shifted in favor of itaconic acid increasing it by 1.5 and 2.0 fold, respectively. Simultaneously, the malic acid titer was reduced by 96 % and 61 %, respectively.For U. trichophora TZ1 the malic acid titer was improved to nearly 200 g L 1 produced within 264 h reaching a maximal production rate of 1.53 g L 1 h 1. Since the knowledge on this obscure U. trichophora was scarce, potential native target genes for metabolic engineering were identified after de novo genome sequencing. To enable the improvement of malic acid production with U. trichophora TZ1 by metabolic engineering, existing tools, such as antibiotic markers and promoters, were investigated and adapted to be suitable for creation of overexpression mutants. Using these tools, overexpression mutants for two different malate dehydrogenases (Mdh1 and Mdh2), pyruvate-carboxylase (Pyc) and two different malic acid transporters (Ssu1 and Ssu2) were generated. While overexpression of Pyc did not improve malic acid production, transformants overexpressing Mdh1 and Mdh2 and malic acid transporters Ssu1 and Ssu2 showed an up to 38 % increased malic acid production rate and an up to 54 % increased yield in shake flasks compared to U. trichophora TZ1. In bioreactor cultivations with the mutant overexpressing Ssu2, an increased production rate could not be observed. Due to a drastically lowered optical density, however, this strain had a 29 % higher specific production rate. Additionally, the product yield was improved by 1.4 fold.These results clearly strengthen the applicability of Ustilaginaceae as industrially valuable production organisms. By this valorization of biodiesel derived crude glycerol, the overall biodiesel bio refinery concept is improved on an economic as well as ecological level
Engineering of Scale-Down Bioreactor Setup: Deciphering Metabolic Phenotype of Corynebacterium glutamicum under Simulated Bioreactor Inhomogeneity
No full textPerformance losses during the scale-up from laboratory into production scale and the associated increase of production costs imperil the competitiveness of sustainable biotechnological bulk products. Since the formation of environmental gradients in the large-scale was identified as the major obstacle for process scalability, scale-up simulation in the laboratory scale became one of the most important prediction tools. This work hooks up with the so-called scale-down strategy by providing a flexible state of the art two compartment scale down device, which is designed for easy process parallelization. The system is composed of two connected and fully controlled stirred tank reactors (STR) implemented in a parallel cultivation platform.To validate the STR-STR application for Corynebacterium glutamicum processes, it was compared to an already established scale-down device consisting of a plug-flow reactor (PFR) connected to a STR. Apart from differing side-product levels, very similar results were observed for the metabolic phenotype and bioprocess performance when sole oxygen perturbations were simulated, although the differing setups provide opposed back mixing profiles.A standardized scale-down workflow for the systematic separation and recombination of critical scale-up parameters was established, including state of the art technology for intracellular metabolite, protein and transcript analysis. Within a comprehensive study of 13 different combinations of oxygen, substrate and pH fluctuations, the workflow was used to investigate the scalability of a C. glutamicum based 1,5-diaminopentane production process. Thereby C. glutamicum provided an outstanding level of robustness to oxygen and substrate inhomogeneities. Using omics based methods, it could be shown, that the flexible rearrangement of the central metabolism is the key element to overcome shortage in NAD+ recycling. Furthermore, formed L-lactate served as reversible and flexible external buffer for carbon and redox equivalents. Only acidic and alkaline pH fluctuation of one pH unit compromised C. glutamicum process performance. Especially, acidic pH levels inhibited regulation of the fermentative key pathway and by that the adaption to short term oxygen limitations. This reduced the biomass and product formation in a significant manner. Consequently, pH was identified to be one of the most sensitive parameter for C. glutamicum processes scale-up. Nevertheless, this systematic investigation of intracellular adaption mechanisms provided a comprehensive metabolic characterization of an adaptive and robust microbial strain for large-scale production
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