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Lemuth K.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | Steuer K.,University of Stuttgart | Albermann C.,University of Stuttgart
Microbial Cell Factories | Year: 2011

Background: The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.Results: Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).Conclusions: By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance. © 2011 Lemuth et al; licensee BioMed Central Ltd. Source


Surmenev R.A.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | Surmeneva M.A.,Tomsk Polytechnic University | Ivanova A.A.,Tomsk Polytechnic University
Acta Biomaterialia | Year: 2014

A systematic analysis of results available from in vitro, in vivo and clinical trials on the effects of biocompatible calcium phosphate (CaP) coatings is presented. An overview of the most frequently used methods to prepare CaP-based coatings was conducted. Dense, homogeneous, highly adherent and biocompatible CaP or hybrid organic/inorganic CaP coatings with tailored properties can be deposited. It has been demonstrated that CaP coatings have a significant effect on the bone regeneration process. In vitro experiments using different cells (e.g. SaOS-2, human mesenchymal stem cells and osteoblast-like cells) have revealed that CaP coatings enhance cellular adhesion, proliferation and differentiation to promote bone regeneration. However, in vivo, the exact mechanism of osteogenesis in response to CaP coatings is unclear; indeed, there are conflicting reports of the effectiveness of CaP coatings, with results ranging from highly effective to no significant or even negative effects. This review therefore highlights progress in CaP coatings for orthopaedic implants and discusses the future research and use of these devices. Currently, an exciting area of research is in bioactive hybrid composite CaP-based coatings containing both inorganic (CaP coating) and organic (collagen, bone morphogenetic proteins, arginylglycylaspartic acid etc.) components with the aim of promoting tissue ingrowth and vascularization. Further investigations are necessary to reveal the relative influences of implant design, surgical procedure, and coating characteristics (thickness, structure, topography, porosity, wettability etc.) on the long-term clinical effects of hybrid CaP coatings. In addition to commercially available plasma spraying, other effective routes for the fabrication of hybrid CaP coatings for clinical use still need to be determined and current progress is discussed. © 2013 Acta Materialia Inc. Source


Surmeneva M.A.,Tomsk Polytechnic University | Surmenev R.A.,Fraunhofer Institute for Interfacial Engineering and Biotechnology
Vacuum | Year: 2015

The present study reports the fabrication of a nano-hydroxyapatite (HA) film on an AZ31 magnesium alloy via radio frequency (RF) magnetron sputtering. The phase composition, microstructure, and surface morphology of the HA coatings were investigated using X-ray diffraction and atomic force microscopy. The polarization tests in a 3.5 wt.% NaCl solution were performed to examine the corrosion behaviour of the HA-coated magnesium alloy. Two HA coating thicknesses of 700 nm and 1500 nm were studied. The coatings homogeneously covered the entire surface of the substrates. In the case of a greater coating thickness, larger crystallites were formed. The potentiodynamic polarization test demonstrated that a 1500-nm thick nanocrystalline HA coating significantly improved the corrosion resistance of the bare AZ31 magnesium alloy. © 2015 Elsevier Ltd. Source


Rupp S.,Fraunhofer Institute for Interfacial Engineering and Biotechnology
Engineering in Life Sciences | Year: 2013

Within the last decade, biotechnology gained pace in substituting petro-based products for the chemical industries. This is visible with the appearance of bio-based products in the market, from biosurfactants to bio-based polymers like polylactic acid to bio-ethylene. These technologies are mainly based on established fermentation technologies fostered by the use of renewable resources, culminating in the establishment of biorefineries that may be connected directly to the existing chemical infrastructure. Besides these large-scale technologies, the combination of molecular technologies, microfluidic devices, and enzymatic and cell-free conversions are currently developed to create new bioproduction systems enabling the production of compounds that may not be produced within a cell. This article summarizes some of the current ideas that are currently in development paving the way for a next generation of biotechnology. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source


Purschke F.G.,University of Stuttgart | Hiller E.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | Trick I.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | Rupp S.,Fraunhofer Institute for Interfacial Engineering and Biotechnology
Molecular and Cellular Proteomics | Year: 2012

The majority of microorganisms persist in nature as surface-attached communities often surrounded by an extracellular matrix, called biofilms. Most natural biofilms are not formed by a single species but by multiple species. Microorganisms not only cooperate as in some multispecies biofilms but also compete for available nutrients. The Gram-negative bacterium Pseudomonas aeruginosa and the polymorphic fungus Candida albicans are two opportunistic pathogens that are often found coexisting in a human host. Several models of mixed biofilms have been reported for these organisms showing antagonistic behavior. To investigate the interaction of P. aeruginosa and C. albicans in more detail, we analyzed the secretome of single and mixed biofilms of both organisms using MALDI-TOF MS/MS at several time points. Overall 247 individual proteins were identified, 170 originated from P. aeruginosa and 77 from C. albicans. Only 39 of the 131 in mixed biofilms identified proteins were assigned to the fungus whereby the remaining 92 proteins belonged to P. aeruginosa. In single-species biofilms, both organisms showed a higher diversity of proteins with 73 being assigned to C. albicans and 154 to P. aeruginosa. Most interestingly, P. aeruginosa in the presence of C. albicans secreted 16 proteins in significantly higher amounts or exclusively among other virulence factors such as exotoxin A and iron acquisition systems. In addition, the high affinity iron-binding siderophore pyoverdine was identified in mixed biofilms but not in bacterial biofilms, indicating that P. aeruginosa increases its capability to sequester iron in competition with C. albicans. In contrast, C. albicans metabolism was significantly reduced, including a reduction in detectable iron acquisition proteins. The results obtained in this study show that microorganisms not only compete with the host for essential nutrients but also strongly with the present microflora in order to gain a competitive advantage. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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