A directed evolution approach to engineering oxygen resistant Fe-Fe hydrogenases

Diese Arbeit stellt einen Ansatz vor eine künstliche Selektion für FeFe-Hydrogenasen in E. Coli zu entwerfen, und damit große Bibliotheken mutierter Hydrogenasen auszusortieren, mit dem Ziel Mutanten mit einer erhöhten Halbwertszeit in aerober Umgebung zu isolieren. Die biologische Herstellung von Wasserstoff durch Kopplung von Photosynthese und Hydrogenasen ist ein vielversprechender Ansatz, der allerdings Sauerstofftolerante Hydrogenasen voraus setzt. Für einen Ansatz mittels gerichteter Evolution müssen große Bibliotheken zufällig mutierter Varianten eines Enzyms hergestellt werden und von diesen dann solche auszuwählen, die eine erhöhte Funktionalität in dem erwünschten Parameter aufweisen – im Fall diese Projektes eine erhöhte Aktivität von Hydrogenasen in erhöhten Sauerstoffpartialdrücken. Gesetzt den Fall, dass eine Selektion zum herausfiltern erwünschter Mutationen angewendet werden kann ist gerichtete Evolution die leistungsfähigste Methode für die Entwicklung neuer Proteine. Zu diesem Zweck wurde im Silver -Lab ein E. Coli Stamm entwickelt, dessen Wachstum auf eine aktive Hydrogenase für die Herstellung von Cystein und Methionin angewiesen ist. Eine künstliche Elektronentransportkette transferiert Elektronen von der Hydrogenase zu einer transgenen Sulfit-Reduktase, wodurch Sulfit reduziert und somit verwendet werden kann und das Wachstum unter selektiven Bedingungen (fehlender reduzierter Schwefel) stark erhöht wird. Ein Protokoll zur Herstellung großer Mutanten-Bibliotheken von verschiedenen Hydrogenasen mittels fehlerhafter-PCR und deren aussortieren in dem synthetischen Selektionsstamm bei verschiedenen Sauerstofflevels wurde entwickelt. Weiters wurden verschiedene knock-out-Stämme um die Selektivität des Sortierungsprozesses zu erhöhen erzeugt sowie alternative Ansätze die Selektivität zu erhöhen getestet. Nach der Charakterisierung möglicher Treffer aus den Wachstumsuntersuchungen wurden die Aktivitäten der Mutanten mit in vivo und in vitro Ansätzen gemessen. Außerdem wurde ein Protokoll zur biochemischen Aufreinigung der Hydrogenasen angewendet. Mehrere Mutanten wurden aus den Wachstumsuntersuchungen isoliert. Die meisten wiesen eine stark verminderte Aktivität im Vergleich zum Wildtyp auf. Auch ihre Halbwertszeiten in O2-atmosphere waren nicht erhöht. Das legt den Schluss nahe, dass das Ziel das Wachstum von E. Coli streng an Hydrogenaseaktivität zu koppeln nicht geglückt ist und wir daher nicht in der Lage waren, O2-resistente Hydrogenasen zu isolieren. Dennoch stellt das Projekt, als ein erster Ansatz einer Selektion zur Entwicklung verbesserter Hydrogenasen und einer der ersten Versuche Hydrogenasen weiterzuentwickeln eine sehr innovative Methode dar und unsere Ergebnisse gewähren wertvolle Einsichten in die Herausforderungen und Hindernisse die es zu meistern gilt um letztendlich erfolgreich Hydrogenasen – und biologische Systeme im Allgemeinen – weiterzuentwickeln.This thesis presents an approach to design an artificial selection for hydrogenase function in E. coli, and use it to screen large mutant libraries if Fe-Fe hydrogenases, with the aim of isolating mutants with an increased half-life in aerobic environments. The biological production of hydrogen by the direct connection of photosynthesis to hydrogenases is a promising option for renewable energy but requires oxygen-tolerant hydrogenases. A directed evolution approach entails making large libraries of randomly mutated versions of an enzyme and then selecting variants that have an increased functionality for the desired parameter – in our case hydrogenase activity in increased O2 partial pressures. Directed evolution is the most powerful tool available to protein engineering, provided a system to select for beneficial mutations can be applied. To that end an E. coli strain has been developed at the Silver lab that is dependent upon an active hydrogenase for cysteine and methionine synthesis. An artificial electron transport pathway shuttles electrons from the hydrogenase to a transgenic Sulfite-reductase, allowing the reduction and incorporation of sulfite, greatly improving growth on selective media (lacking reduced sulfur). A protocol to generate large plasmid libraries of mutated hydrogenases of different origins using error-prone PCR and screen them in the artificial selection strain at different O2-levels was developed. Also knock-out strains to increase the selectivity of the screening process were generated and alternative approaches to improve the selectability of hydrogenases in our system as well as to increase the range of selectable oxygen levels were developed. Characterization of putative hits by growth assays was followed by measuring activities of hydrogenases using both in vitro and in vivo assays in order to characterize promising mutants. A protocol for the anaerobic purification of hydrogenases was also applied. Several mutants could be isolated from the growth assays, most of which were active but showed reduced activity compared to wild type. Their half-lives in O2 were not improved either. This suggests that the system we made did not achieve the desired goal of linking growth of E. coli to hydrogenase activity and we were not able to isolate an O2-resistant hydrogenase. However, as a first attempt to engineer a selection for hydrogenases and one of the first attempts at engineering Fe-Fe hydrogenase at all our approach uses innovative methods and our findings provide valuable insight into the challenges and obstacles to be overcome to ultimately be successful in engineering new function in hydrogenases, as well as biological systems at large

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

<p>Abstract</p> <p>Background</p> <p>FeFe-hydrogenases are the most active class of H₂-producing enzymes known in nature and may have important applications in clean H₂energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O₂.</p> <p>Results</p> <p>We have developed a synthetic metabolic pathway in <it>E. coli </it>that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O₂levels eliminate growth. This pathway forms the basis for a genetic selection for O₂tolerance. Genetically selected hydrogenases did not show improved stability in O₂and in many cases had lost H₂production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.</p> <p>Conclusions</p> <p>Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient <it>in vivo </it>assays to aid in the engineering of synthetic H₂metabolism. Our results also indicate a H₂-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H₂-activating catalysts.</p

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography–mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance

Depot specific differences in the adipogenic potential of precursors are mediated by collagenous extracellular matrix and Flotillin 2 dependent signaling

Objective: Adipose tissue shows a high degree of plasticity, and adipocyte hyperplasia is an important mechanism for adipose tissue expansion. Different adipose depots respond differently to an increased demand for lipid storage. Orchestrating cellular expansion in vivo requires extracellular matrix (ECM) remodeling and a high degree of interaction between cells and ECM. Methods: We studied decellularized primary adipose stromal cell derived ECM of different adipose depots and reseeded them with primary adipose precursors. We tested ECM effect on adipocyte differentiation and analyzed ECM composition using proteomic and immunohistochemical approaches to identify factors in the ECM influencing adipogenesis. Results: We show that the ECM of an adipose depot is the major determinant for the differentiation capacity of primary preadipocytes. Visceral adipose tissue stromal cells differentiate less than subcutaneous cells, which, in turn, are less adipogenic than BAT-derived cells. This effect is based on the ECM composition of the respective depot and not dependent on the precursor origin. Addition of vitamin C pronounces the pro-adipogenic effects of the ECM, indicating the importance of collagenous ECM in mediating the effect. Using a proteomic global and a targeted downstream analysis, we identify Flotillin 2 as a protein enriched in pro-adipogenic ECM, which is involved in orchestrating ECM to preadipocyte signaling. Conclusions: We show that adipose tissue SVF secretes collagenous ECM, which directly modulates terminal differentiation of adipocyte precursors in a depot specific manner. These data demonstrate the importance of the tissue microenvironment in preadipocyte differentiation. Keywords: Adipocyte precursors, Extracellular matrix, Collagen, Stem cell niche, Flotillin

Depot specific differences in the adipogenic potential of precursors are mediated by collagenous extracellular matrix and Flotillin 2 dependent signaling

Objective Adipose tissue shows a high degree of plasticity, and adipocyte hyperplasia is an important mechanism for adipose tissue expansion. Different adipose depots respond differently to an increased demand for lipid storage. Orchestrating cellular expansion in vivo requires extracellular matrix (ECM) remodeling and a high degree of interaction between cells and ECM. Methods We studied decellularized primary adipose stromal cell derived ECM of different adipose depots and reseeded them with primary adipose precursors. We tested ECM effect on adipocyte differentiation and analyzed ECM composition using proteomic and immunohistochemical approaches to identify factors in the ECM influencing adipogenesis. Results We show that the ECM of an adipose depot is the major determinant for the differentiation capacity of primary preadipocytes. Visceral adipose tissue stromal cells differentiate less than subcutaneous cells, which, in turn, are less adipogenic than BAT-derived cells. This effect is based on the ECM composition of the respective depot and not dependent on the precursor origin. Addition of vitamin C pronounces the pro-adipogenic effects of the ECM, indicating the importance of collagenous ECM in mediating the effect. Using a proteomic global and a targeted downstream analysis, we identify Flotillin 2 as a protein enriched in pro-adipogenic ECM, which is involved in orchestrating ECM to preadipocyte signaling. Conclusions We show that adipose tissue SVF secretes collagenous ECM, which directly modulates terminal differentiation of adipocyte precursors in a depot specific manner. These data demonstrate the importance of the tissue microenvironment in preadipocyte differentiation

Short-term feeding of a ketogenic diet induces more severe hepatic insulin resistance than an obesogenic high-fat diet

KEY POINTS A ketogenic diet is known to lead to weight loss and is considered metabolically healthy; however there are conflicting reports on its effect on hepatic insulin sensitivity. KD fed animals appear metabolically healthy in the fasted state after 3 days of dietary challenge, whereas obesogenic high-fat diet (HFD) fed animals show elevated insulin levels. A glucose challenge reveals that both KD and HFD fed animals are glucose intolerant. Glucose intolerance correlates with increased lipid oxidation and lower respiratory exchange ratio (RER); however, all animals respond to glucose injection with an increase in RER. Hyperinsulinaemic-euglycaemic clamps with double tracer show that the effect of KD is a result of hepatic insulin resistance and increased glucose output but not impaired glucose clearance or tissue glucose uptake in other tissues. ABSTRACT Despite being a relevant healthcare issue and heavily investigated, the aetiology of type 2 diabetes (T2D) is still incompletely understood. It is well established that increased endogenous glucose production (EGP) leads to a progressive increase in glucose levels, causing insulin resistance and eventual loss of glucose homeostasis. The consumption of high carbohydrate, high-fat, western style diet (HFD) is linked to the development of T2D and obesity, whereas the consumption of a low carbohydrate, high-fat, ketogenic diet (KD) is considered healthy. However, several days of carbohydrate restriction are known to cause selective hepatic insulin resistance. In the present study, we compare the effects of short-term HFD and KD feeding on glucose homeostasis in mice. We show that, even though KD fed animals appear to be healthy in the fasted state, they exhibit decreased glucose tolerance to a greater extent than HFD fed animals. Furthermore, we show that this effect originates from blunted suppression of hepatic glucose production by insulin, rather than impaired glucose clearance and tissue glucose uptake. These data suggest that the early effects of HFD consumption on EGP may be part of a normal physiological response to increased lipid intake and oxidation, and that systemic insulin resistance results from the addition of dietary glucose to EGP-derived glucose

Short-term feeding of a ketogenic diet induces more severe hepatic insulin resistance than an obesogenic high-fat diet

Despite being a relevant healthcare issue and heavily investigated, the aetiology of type 2 diabetes (T2D) is still incompletely understood. It is well established that increased endogenous glucose production (EGP) leads to a progressive increase in glucose levels, causing insulin resistance and eventual loss of glucose homeostasis. The consumption of high carbohydrate, high‐fat, western style diet (HFD) is linked to the development of T2D and obesity, whereas the consumption of a low carbohydrate, high‐fat, ketogenic diet (KD) is considered healthy. However, several days of carbohydrate restriction are known to cause selective hepatic insulin resistance. In the present study, we compare the effects of short‐term HFD and KD feeding on glucose homeostasis in mice. We show that, even though KD fed animals appear to be healthy in the fasted state, they exhibit decreased glucose tolerance to a greater extent than HFD fed animals. Furthermore, we show that this effect originates from blunted suppression of hepatic glucose production by insulin, rather than impaired glucose clearance and tissue glucose uptake. These data suggest that the early effects of HFD consumption on EGP may be part of a normal physiological response to increased lipid intake and oxidation, and that systemic insulin resistance results from the addition of dietary glucose to EGP‐derived glucose.ISSN:0022-3751ISSN:1469-779

Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity

Aims/hypothesis In the context of diabetes, the health benefit of antioxidant treatment has been widely debated. In this study, we investigated the effect of antioxidant treatment during the development of insulin resistance and hyperphagia in obesity and partial lipodystrophy. Methods We studied the role of antioxidants in the regulation of insulin resistance using the tamoxifen-inducible fat-specific insulin receptor knockout (iFIRKO) mouse model, which allowed us to analyse the antioxidant’s effect in a time-resolved manner. In addition, leptin-deficient ob/ob mice were used as a hyperphagic, chronically obese and diabetic mouse model to validate the beneficial effect of antioxidants on metabolism. Results Acute induction of insulin receptor knockout in adipocytes changed the substrate preference to fat before induction of a diabetic phenotype including hyperinsulinaemia and hyperglycaemia. In healthy chow-fed animals as well as in morbidly obese mice, this diabetic phase could be reversed within a few weeks. Furthermore, after the induction of insulin receptor knockout in mature adipocytes, iFIRKO mice were protected from subsequent obesity development through high-fat diet feeding. By genetic tracing we show that the persistent fat mass loss in mice after insulin receptor knockout in adipocytes is not caused by the depletion of adipocytes. Treatment of iFIRKO mice with antioxidants postponed and reduced hyperglycaemia by increasing insulin sensitivity. In ob/ob mice, antioxidants rescued both hyperglycaemia and hyperphagia. Conclusions/interpretation We conclude that fat mass reduction through insulin resistance in adipocytes is not reversible. Furthermore, it seems unlikely that adipocytes undergo apoptosis during the process of extreme lipolysis, as a consequence of insulin resistance. Antioxidants have a beneficial health effect not only during the acute phase of diabetes development, but also in a temporary fashion once chronic obesity and diabetes have been established