Der Kreuzenzian-Ameisenbläuling (Maculinea rebeli) : zur Situation und Entwicklung der Vorkommen im Kreis Höxter im Zeitraum von 1990 bis 2008

Mit den recht großen Vorkommen von Maculinea rebeli im Kreis Höxter – sie bilden zusammen mit den benachbarten niedersächsischen und nordhessischen Populationen einen Verbreitungsschwerpunkt, der im norddeutschen Raum einzigartig ist – weist der Kreis eine faunistische Besonderheit auf. Denn durch den Verlust seines Lebensraumes, des Kalkmagerrasens, zählt der Kreuzenzian-Ameisenbläuling mittlerweile zu den stark gefährdeten Arten: Fast alle Populationen von Maculinea rebeli (Hirschke, 1904), der wissenschaftlich auch als Phengaris rebeli (Hirschke, 1904) und in der Vergangenheit als Glaucopsyche rebeli (Hirschke, 1904) bzw. Maculinea alcon ssp. rebeli (Hirschke, 1904) bezeichnet wird bzw. wurde, sind in Deutschland heute hochgradig in ihrer Existenz bedroht! Die von der Autorin im Jahr 2008 erstellte Diplomarbeit „Der Kreuzenzian-Ameisenbläuling Maculinea rebeli – Auswertung faunistischer und populationsökologischer Untersuchungen im Hinblick auf die Entwicklung eines Artenschutzkonzeptes“ beschäftigt sich daher eingehend mit dem aktuellen Zustand der hiesigen Vorkommen und deren Entwicklung seit 1990. Ziel der Arbeit war die Erarbeitung geeigneter Schutzmaßnahmen in Form eines auf die Biologie des Bläulings und die jeweilige Fläche abgestimmten Managements. Die wichtigsten Ergebnisse der Arbeit sollen im Folgenden wiedergegeben werden

Cocoa butter-like lipid production ability of non-oleaginous and oleaginous yeasts under nitrogen-limited culture conditions

Cocoa butter (CB) extracted from cocoa beans is the main raw material for chocolate production. However, growing chocolate demands and limited CB production has resulted in a shortage of CB supply. CB is mainly composed of three different kinds of triacylglycerols (TAGs), POP (C16:0-C18:1-C16:0), POS (C16:0-C18:1-C18:0), and SOS (C18:0-C18:1-C18:0). The storage lipids of yeasts, mainly TAGs, also contain relative high-level of C16 and C18 fatty acids and might be used as CB-like lipids (CBL). In this study, we cultivated six different yeasts, including one non-oleaginous yeast strain, Saccharomyces cerevisiae CEN.PK113-7D, and five oleaginous yeast strains, Trichosporon oleaginosus DSM11815, Rhodotorula graminis DSM 27356, Lipomyces starkeyi DSM 70296, Rhodosporidium toruloides DSM 70398, and Yarrowia lipolytica CBS 6124, in nitrogen-limited medium and compared their CBL production ability. Under the same growth conditions, we found that TAGs were the main lipids in all six yeasts and that T. oleaginosus can produce more TAGs than the other five yeasts. Less than 3% of the total TAGs were identified as potential SOS in the six yeasts. However, T. oleaginosus produced 27.8% potential POP and POS at levels of 378 mg TAGs/g dry cell weight, hinting that this yeast may have potential as a CBL production host after further metabolic engineering in future

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8- fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories

"Manchmal ist der Hang zu steil" : von Praktikantinnen und Praktikanten der Landschaftsstation

"Es ist noch kein Meister vom Himmel gefallen", sagt das Sprichwort, aber schon manch Praktikant oder Praktikantin der Landschaftsstation hat die Steilheit und Viskosität eines Kalkmagerrasen("KMR")-Hanges oder auch "nur" einer Waldrandfläche unterschätzt. Jan-Eric REITH aus Wrexen zum Beispiel, Dipl.-Agraringenieur mit Abschluss an der Rheinischen Friedrich-Wilhelms-Universität zu Bonn, sowie Praktikant der Landschaftsstation im Jahr 2008, "fiel" im Frühjahr von der "Waldrand"-Projektfläche an den Weserhängen "unterhalb des Nierenberges" bei Beverungen und freute sich, dass hangabwärts Bäume standen. So blieben nur blaue Flecken, die von der mitkartierenden Kollegin Vera GLANERT, Studentin an der Hochschule Ostwestfalen-Lippe - University of applied sciences im Studiengang "Landschaftsarchitektur", mit viel Mitgefühl begutachtet und versorgt wurden. Zum Glück ist es guter Brauch in Borgentreich, auch beim Kartieren die Praktikanten im "Doppelpack" einzusetzen. In der praktischen Landschaftspflege, d. h. beim Arbeiten mit Werkzeug und Maschinen, gehen Praktikanten, Zivildienstleistende, Ehrenamtliche und sonstige Naturschützer mindestens zu zweit auf die Fläche, so wie es Berufsgenossenschaft und Unfallversicherung vorsehen. Aber Kalkmagerrasen-Hänge sind nun mal meist sehr steil und lassen häufig nur spezielle selbst fahrende Maschinen zu, geschweige denn solche mit "Sitz", wie der damalige kaufmännische Leiter der Landschaftsstation und weiterhin hauptberufliche Geschäftsführer des Maschinenrings Warburg-Höxter, Norbert HOFNAGEL, bei einer Praxiserprobung feststellen musste: „Wie will man da Maschinen einsetzen oder überhaupt erst auf die Fläche bringen?“ Besagter Norbert HOFNAGEL und die Kollegen der Landschaftsstation haben dann später eine passende Maschine auch für jene Fläche gefunden: "Rapid" mit Schlegelmulcher, inzwischen beim Maschinenring zu mieten für die Pflege für den Naturschutz wertvoller Flächen. Freundlicher Förderer war die Nordrhein-Westfalen-Stiftung. Hauptnutzer ist bisher die Landschaftsstation im Kreis Höxter - Wen wundert's? Zurück zu den Hängen, die vom wissenschaftlichen Personal im "Alleingang" auf ihre Artenausstattung hin überwacht (wir sagen: "gemonitort"), betreut und kartiert werden, denn sonst würde die Arbeit ja nie fertig. Sie haben selbstverständlich zur Sicherheit ein Handy und im Auto einen Verbandskasten dabei, plus die Erfahrung im "Sich-nicht-wehtun". Aber weshalb waren Vera und Jan-Eric eigentlich am Nierenberg

Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae

Phosphoketolases catalyze an energy-and redox-independent cleavage of certain sugar phosphates. Hereby, the two-carbon (C2) compound acetyl-phosphate is formed, which enzymatically can be converted into acetyl-CoA-a key precursor in central carbon metabolism. Saccharomyces cerevisiae does not demonstrate efficient phosphoketolase activity naturally. In this study, we aimed to compare and identify efficient heterologous phosphoketolase enzyme candidates that in yeast have the potential to reduce carbon loss compared to the native acetyl-CoA producing pathway by redirecting carbon flux directly from C5 and C6 sugars towards C2-synthesis. Nine phosphoketolase candidates were expressed in S. cerevisiae of which seven produced significant amounts of acetyl-phosphate after provision of sugar phosphate substrates in vitro. The candidates showed differing substrate specificities, and some demonstrated activity levels significantly exceeding those of candidates previously expressed in yeast. The conducted studies also revealed that S. cerevisiae contains endogenous enzymes capable of breaking down acetyl-phosphate, likely into acetate, and that removal of the phosphatases Gpp1 and Gpp2 could largely prevent this breakdown. An evaluation of in vivo function of a subset of phosphoketolases was conducted by monitoring acetate levels during growth, confirming that candidates showing high activity in vitro indeed showed increased acetate accumulation, but expression also decreased cellular fitness. The study shows that expression of several bacterial phosphoketolase candidates in S. cerevisiae can efficiently divert intracellular carbon flux toward C2-synthesis, thus showing potential to be used in metabolic engineering strategies aimed to increase yields of acetyl-CoA derived compounds

Engineering microbial fatty acid metabolism for biofuels and biochemicals

Traditional oleochemical industry chemically processes animal fats and plant oils to produce detergents, lubricants, biodiesel, plastics, coatings, and other products. Biotechnology offers an alternative process, where the same oleochemicals can be produced from abundant biomass feedstocks using microbial catalysis. This review summarizes the recent advances in the engineering of microbial metabolism for production of fatty acid-derived products. We highlight the efforts in engineering the central carbon metabolism, redox metabolism, controlling the chain length of the products, and obtaining metabolites with different functionalities. The prospects of commercializing microbial oleochemicals are also discussed

Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190. © 2017, The Author(s)

Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes

Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1–C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis. © 2017, The Author(s)

Enhanced ethanol production and reduced glycerol formation in fps1∆ mutants of Saccharomyces cerevisiae engineered for improved redox balancing

Ethanol is by volume the largest fermentation product. During ethanol production by Saccharomyces cerevisiae about 4-5% of the carbon source is lost to glycerol production. Different approaches have been proposed for improving the ethanol yield while reducing glycerol production. Here we studied the effect of reducing glycerol export/formation through deletion of the aquaglyceroporin gene FPS1 together with expressing gapN encoding NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans and overexpressing the ATP-NADH kinase gene UTR1 from S. cerevisiae. This strategy will allow reducing the redox balance problem observed when the glycerol pathway is blocked, and hereby improve ethanol production. We found that our strategy enabled increasing the ethanol yield by 4.6% in the case of the best producing strain, compared to the reference strain, without any major effect on the specific growth rate

Profiling of Cytosolic and Peroxisomal Acetyl-CoA Metabolism in Saccharomyces cerevisiae

As a key intracellular metabolite, acetyl-coenzyme A (acetyl-CoA) plays a major role in various metabolic pathways that link anabolism and catabolism. In the yeast Saccharomyces cerevisiae, acetyl-CoA involving metabolism is compartmentalized, and may vary with the nutrient supply of a cell. Membranes separating intracellular compartments are impermeable to acetyl-CoA and no direct transport between the compartments occurs. Thus, without carnitine supply the glyoxylate shunt is the sole possible route for transferring acetyl-CoA from the cytosol or the peroxisomes into the mitochondria. Here, we investigate the physiological profiling of different deletion mutants of ACS1, ACS2, CIT2 and MLS1 individually or in combination under alternative carbon sources, and study how various mutations alter carbon distribution. Based on our results a detailed model of carbon distribution about cytosolic and peroxisomal acetyl-CoA metabolism in yeast is suggested. This will be useful to further develop yeast as a cell factory for the biosynthesis of acetyl-CoA-derived products