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Andreas Wittgens - One of the best experts on this subject based on the ideXlab platform.
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Heterologous Rhamnolipid Biosynthesis: Advantages, Challenges, and the Opportunity to Produce Tailor-Made Rhamnolipids.
Frontiers in bioengineering and biotechnology, 2020Co-Authors: Andreas Wittgens, Frank RosenauAbstract:The first heterologous expression of genes responsible for the production of Rhamnolipids was already implemented in the mid-1990s during the functional identification of the rhlAB operon. This was the starting shot for multiple approaches to establish the Rhamnolipid biosynthesis in different host organisms. Since most of the native Rhamnolipid producing organisms are human or plant pathogens, the intention for these ventures was the establishment of non-pathogenic organisms as heterologous host for the production of Rhamnolipids. The pathogenicity of producing organisms is one of the bottlenecks for applications of Rhamnolipids in many industrial products especially foods and cosmetics. The further advantage of heterologous Rhamnolipid production is the circumvention of the complex regulatory network, which regulates the Rhamnolipid biosynthesis in wild type production strains. Furthermore, a suitable host with an optimal genetic background to provide sufficient amounts of educts allows the production of tailor-made Rhamnolipids each with its specific physico-chemical properties depending on the contained numbers of rhamnose sugar residues and the numbers, chain length and saturation degree of 3-hydroxyfatty acids. The heterologous expression of rhl genes can also enable the utilization of unusual carbon sources for the production of Rhamnolipids depending on the host organism.
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On the road towards tailor-made Rhamnolipids: current state and perspectives
Applied Microbiology and Biotechnology, 2018Co-Authors: Andreas Wittgens, Frank RosenauAbstract:Rhamnolipids are biosurfactants with an enormous potential to replace or complement classic surfactants in industrial applications. They consist of one or two L-rhamnose residues linked to one or two 3-hydroxyfatty acids of various chain lengths, which can also contain unsaturated carbon-carbon bonds, yielding a wide variety of different structures each with its specific physicochemical properties. Since different applications of surfactants require specific tenside characteristics related to surface tension reduction, emulsification, and foaming etc., Rhamnolipids represent a platform molecule which harbors an enormous potential to adopt tailor-made properties to meet a huge variety of demands of surfactants for food-, healthcare-, and biotechnological applications. We are here giving an overview on current technology to synthesize tailor-made Rhamnolipids based on the biotechnological use of different enzymes responsible for Rhamnolipid biosynthesis originating from different naturally Rhamnolipid-producing microorganism. Furthermore, we present future strategies to determine the number of L-rhamnose and 3-hydroxyfatty acids as well as their specific chain lengths and unsaturations to produce customized Rhamnolipids perfectly tuned for every application.
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heterologous production of long chain Rhamnolipids from burkholderia glumae in pseudomonas putida a step forward to tailor made Rhamnolipids
Applied Microbiology and Biotechnology, 2018Co-Authors: Andreas Wittgens, Beatrix Santiagoschuebel, Till Tiso, Diana Hofmann, Marius Henkel, Susanne Wilhelm, Rudolf Hausmann, Lars M. Blank, Karl-erich JaegerAbstract:Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native Rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain Rhamnolipids mainly consisting of a Rha-Rha-C10-C10 congener. Bacteria from the genus Burkholderia are also able to produce Rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C14-C14 congener. These long-chain Rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of Rhamnolipids are still hampered by the pathogenicity of producer strains and—even more important—by the complexity of regulatory networks controlling Rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for Rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain Rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-Rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in Rhamnolipids.
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Designer Rhamnolipids by reduction of congener diversity: production and characterization.
Microbial cell factories, 2017Co-Authors: Till Tiso, Andreas Wittgens, Beate Behrens, Frank Rosenau, Heiko Hayen, Rabea Zauter, Hannah Tulke, Bernd Leuchtle, Lars M. BlankAbstract:Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes Rhamnolipids by decoupling production from host-intrinsic regulations and cell growth. Here, the molecular structure of the Rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural Rhamnolipid producers can synthesize mono- and di-Rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of Rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main Rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-Rhamnolipids, mono-Rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of Rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate Rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics. The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.
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Novel insights into biosynthesis and uptake of Rhamnolipids and their precursors.
Applied microbiology and biotechnology, 2016Co-Authors: Andreas Wittgens, Till Tiso, Diana Hofmann, Marius Henkel, Lars M. Blank, Markus Muller, Filip Kovacic, Melanie Gerlitzki, Beatrix Santiago-schübel, Rudolf HausmannAbstract:The human pathogenic bacterium Pseudomonas aeruginosa produces Rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-Rhamnolipids are converted to di-Rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of Rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in Rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in Rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-Rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-Rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of Rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical Rhamnolipids containing two fatty acids, valuable for the production of designer Rhamnolipids with desired physicochemical properties.
Eric Deziel - One of the best experts on this subject based on the ideXlab platform.
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Comparative Analysis of Rhamnolipids from Novel Environmental Isolates of Pseudomonas aeruginosa
Journal of Surfactants and Detergents, 2015Co-Authors: Milena G. Rikalović, Gordana Dj. Gojgic-Cvijovic, Zorica Nestorovic, Ahmad Mohammad Abdel-Mawgoud, Miroslav M. Vrvić, Eric Deziel, Ivanka M. KaradžićAbstract:A comparative analysis of Rhamnolipids from environmental isolates of Pseudomonas aeruginosa was undertaken to evaluate strain-specific Rhamnolipid fingerprints obtained under different growth conditions. Environmental isolates of P. aeruginosa produced Rhamnolipids on different types of substrates, including cheap and renewable sources like sunflower oil from deep fryers and sunflower oil mill effluent. Rhamnolipids were monitored by high-performance liquid chromatography–electrospray ionization interface mass spectrometry, which allowed fast and reliable identification and quantification of the congeners present. The highest concentration of total Rhamnolipids of 3.33 g/l was obtained by the strain P. aeruginosa 67, recovered from petroleum contaminated soil, and strains D1 (1.73 g/l) and D2 (1.70 g/l), recovered from natural microbial consortia originated from mazut-contaminated soil, grown on sunflower oil as a carbon source. Di- to mono-Rhamnolipids ratios were in the range of 0.90–5.39 for different media composition and from 1.12 to 4.17 for different producing strains. Rhamnolipid profiles of purified mixtures of all tested strains are similar with chain length from C8–C12, pronounced abundance of Rha–C10–C10 and Rha–Rha–C10–C10 congeners, and a low content of 3-(3-hydroxyalkanoyloxy)-alkanoic acids. Concentrations of major congeners of RLs were found to slightly vary, depending on strain and growth conditions, while variations in minor congeners were more pronounced. Statistically significant increase of critical micelle concentration values was observed with lowering the ratio of total mono- to di-Rhamnolipids ratio indicating that mono-Rhamnolipids start to form micelles at lower concentration than di-Rhamnolipids.
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quorum sensing controls swarming motility of burkholderia glumae through regulation of Rhamnolipids
PLOS ONE, 2015Co-Authors: Arvin Nickzad, Francois Lepine, Eric DezielAbstract:Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active Rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, Rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of Rhamnolipids in B. glumae and that Rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce Rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous Rhamnolipids. Impaired Rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.
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characterization of Rhamnolipid production by burkholderia glumae
Letters in Applied Microbiology, 2011Co-Authors: S G V A O Costa, Eric Deziel, Francois LepineAbstract:Aims: To investigate if Burkholderia glumae can produce Rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Methods and Results: Burkholderia glumae AU6208 produces a large spectrum of mono- and di-Rhamnolipid congeners with side chains varying between C(12) -C(12) and C(16) -C(16) , the most abundant being Rha-Rha-C(14) -C(14) .The effects on Rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000.ovrhdot.7 mg l(-1) was reached after 6 days. These Rhamnolipids display a critical micelle concentration of 25-27 mg l(-1) and decrease the interfacial tension against hexadecane from 40 to 1.ovrhdot.8 mN m(-1) . They also have excellent emulsifying properties against long chain alkanes. Conclusions: Burkholderia glumae AU6208 can produce considerable amounts of Rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. Significance and Impact of the Study: In contrast with the classical Rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of Rhamnolipids with this species could be easier than with Ps. aeruginosa
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Characterization of Rhamnolipid production by Burkholderia glumae.
Letters in applied microbiology, 2011Co-Authors: S G V A O Costa, Eric Deziel, Francois LepineAbstract:To investigate if Burkholderia glumae can produce Rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Burkholderia glumae AU6208 produces a large spectrum of mono- and di-Rhamnolipid congeners with side chains varying between C(12)-C(12) and C(16)-C(16), the most abundant being Rha-Rha-C(14)-C(14).The effects on Rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000.7 mg l(-1) was reached after 6 days. These Rhamnolipids display a critical micelle concentration of 25-27 mg l(-1) and decrease the interfacial tension against hexadecane from 40 to 1.8 mN m(-1). They also have excellent emulsifying properties against long chain alkanes. Burkholderia glumae AU6208 can produce considerable amounts of Rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. In contrast with the classical Rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of Rhamnolipids with this species could be easier than with Ps. aeruginosa. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.
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Rhamnolipids: diversity of structures, microbial origins and roles
Applied Microbiology and Biotechnology, 2010Co-Authors: Ahmad Mohammad Abdel-Mawgoud, Francois Lepine, Eric DezielAbstract:Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α - L -rhamnopyranosyl- α-L -rhamnopyranosyl- β -hydroxydecanoyl- β -hydroxydecanoate (Rha-Rha-C_10-C_10), α - L -rhamnopyranosyl- α-L -rhamnopyranosyl- β -hydroxydecanoate (Rha-Rha-C_10), as well as their mono-Rhamnolipid congeners Rha-C_10-C_10 and Rha-C_10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of Rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce Rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa . In P. aeruginosa , three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of Rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC . Genes highly similar to rhlA , rhlB , and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for Rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, Rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.
Rudolf Hausmann - One of the best experts on this subject based on the ideXlab platform.
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heterologous production of long chain Rhamnolipids from burkholderia glumae in pseudomonas putida a step forward to tailor made Rhamnolipids
Applied Microbiology and Biotechnology, 2018Co-Authors: Andreas Wittgens, Beatrix Santiagoschuebel, Till Tiso, Diana Hofmann, Marius Henkel, Susanne Wilhelm, Rudolf Hausmann, Lars M. Blank, Karl-erich JaegerAbstract:Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native Rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain Rhamnolipids mainly consisting of a Rha-Rha-C10-C10 congener. Bacteria from the genus Burkholderia are also able to produce Rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C14-C14 congener. These long-chain Rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of Rhamnolipids are still hampered by the pathogenicity of producer strains and—even more important—by the complexity of regulatory networks controlling Rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for Rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain Rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-Rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in Rhamnolipids.
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Novel insights into biosynthesis and uptake of Rhamnolipids and their precursors.
Applied microbiology and biotechnology, 2016Co-Authors: Andreas Wittgens, Till Tiso, Diana Hofmann, Marius Henkel, Lars M. Blank, Markus Muller, Filip Kovacic, Melanie Gerlitzki, Beatrix Santiago-schübel, Rudolf HausmannAbstract:The human pathogenic bacterium Pseudomonas aeruginosa produces Rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-Rhamnolipids are converted to di-Rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of Rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in Rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in Rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-Rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-Rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of Rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical Rhamnolipids containing two fatty acids, valuable for the production of designer Rhamnolipids with desired physicochemical properties.
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integrated foam fractionation for heterologous Rhamnolipid production with recombinant pseudomonas putida in a bioreactor
AMB Express, 2016Co-Authors: Janina Beuker, Andreas Wittgens, Marius Henkel, Frank Rosenau, Anke Steier, Rudolf HausmannAbstract:Heterologeous production of Rhamnolipids in Pseudomonas putida is characterized by advantages of a non-pathogenic host and avoidance of the native quorum sensing regulation in Pseudomonas aeruginosa. Yet, downstream processing is a major problem in Rhamnolipid production and increases in complexity at low Rhamnolipid titers and when using chemical foam control. This leaves the necessity of a simple concentrating and purification method. Foam fractionation is an elegant method for in situ product removal when producing microbial surfactants. However, up to now in situ foam fractionation is nearly exclusively reported for the production of surfactin with Bacillus subtilis. So far no cultivation integrated foam fractionation process for Rhamnolipid production has been reported. This is probably due to excessive bacterial foam enrichment in that system. In this article a simple integrated foam fractionation process is reported for heterologous Rhamnolipid production in a bioreactor with easily manageable bacterial foam enrichments. Rhamnolipids were highly concentrated in the foam during the cultivation process with enrichment factors up to 200. The described process was evaluated at different pH, media compositions and temperatures. Foam fractionation processes were characterized by calculating procedural parameter including Rhamnolipid and bacterial enrichment, Rhamnolipid recovery, YX/S, YP/X, and specific as well as volumetric productivities. Comparing foam fractionation parameters of the Rhamnolipid process with the surfactin process a high effectiveness of the integrated foam fractionation for Rhamnolipid production was demonstrated.
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Rhamnolipids as biosurfactants from renewable resources concepts for next generation Rhamnolipid production
Process Biochemistry, 2012Co-Authors: Marius Henkel, Jonas Contiero, Markus Muller, Johannes H Kugler, Roberta B Lovaglio, Christoph Syldatk, Rudolf HausmannAbstract:Several microorganisms are known to produce a wide variety of surface-active substances, which are referred to as biosurfactants. Interesting examples for biosurfactants are Rhamnolipids, glycolipids mainly known from Pseudomonas aeruginosa produced during cultivation on different substrates like vegetable oils, sugars, glycerol or hydrocarbons. However, besides costs for downstream processing of Rhamnolipids, relatively high raw-material prices and low productivities currently inhibit potential economical production of Rhamnolipids on an industrial scale. This review focuses on cost-effective and sustainable production of Rhamnolipids by introducing new possibilities and strategies regarding renewable substrates. Additionally, past and recent production strategies using alternative substrates such as agro-industrial byproducts or wastes are summarized. Requirements and concepts for next-generation Rhamnolipid producing strains are discussed and potential targets for strain-engineering are presented. The discussion of potential new strategies is supported by an analysis of the metabolism of different Pseudomonas species. According to calculations of theoretical substrate-to-product conversion yields and current world-market price analysis, different renewable substrates are compared and discussed from an economical point of view. A next-generation Rhamnolipid producing strain, as proposed within this review, may be engineered towards reduced formation of byproducts, increased metabolic spectrum, broadened substrate spectrum and controlled regulation for the induction of Rhamnolipid synthesis.
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Rhamnolipids--next generation surfactants?
Journal of biotechnology, 2012Co-Authors: Markus Muller, Marius Henkel, Johannes H Kugler, Christoph Syldatk, Melanie Gerlitzki, Barbara Hörmann, Martin Pöhnlein, Rudolf HausmannAbstract:The demand for bio-based processes and materials in the petrochemical industry has significantly increased during the last decade because of the expected running out of petroleum. This trend can be ascribed to three main causes: (1) the increased use of renewable resources for chemical synthesis of already established product classes, (2) the replacement of chemical synthesis of already established product classes by new biotechnological processes based on renewable resources, and (3) the biotechnological production of new molecules with new features or better performances than already established comparable chemically synthesized products. All three approaches are currently being pursued for surfactant production. Biosurfactants are a very promising and interesting substance class because they are based on renewable resources, sustainable, and biologically degradable. Alkyl polyglycosides are chemically synthesized biosurfactants established on the surfactant market. The first microbiological biosurfactants on the market were sophorolipids. Of all currently known biosurfactants, Rhamnolipids have the highest potential for becoming the next generation of biosurfactants introduced on the market. Although the metabolic pathways and genetic regulation of biosynthesis are known qualitatively, the quantitative understanding relevant for bioreactor cultivation is still missing. Additionally, high product titers have been exclusively described with vegetable oil as sole carbon source in combination with Pseudomonas aeruginosa strains. Competitive productivity is still out of reach for heterologous hosts or non-pathogenic natural producer strains. Thus, on the one hand there is a need to gain a deeper understanding of the regulation of Rhamnolipid production on process and cellular level during bioreactor cultivations. On the other hand, there is a need for metabolizable renewable substrates, which do not compete with food and feed. A sustainable bioeconomy approach should combine a holistic X-omics strategy with metabolic engineering to achieve the next step in Rhamnolipid production based on non-food renewable resources. This review discusses different approaches towards optimization of Rhamnolipid production and enhancement of product spectra. The optimization of Rhamnolipid production with P. aeruginosa strains, screening methods for new non-pathogenic natural Rhamnolipid producers and recombinant Rhamnolipid production are examined. Finally, biocatalysis with Rhamnolipids for the synthesis of l-rhamnose, β-hydroxyfatty acids, and tailor-made surfactants is discussed. Biosurfactants are still in the phase of initial commercialization. However, for next generation development of Rhamnolipid production processes and next generation biosurfactants there are still considerable obstacles to be surmounted, which are discussed here.
Till Tiso - One of the best experts on this subject based on the ideXlab platform.
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Interaction of Rhamnolipids with model biomembranes of varying complexity
Biochimica et biophysica acta. Biomembranes, 2020Co-Authors: Marius Herzog, Till Tiso, Lars M. Blank, Roland WinterAbstract:Rhamnolipids represent a large class of biologically produced surface-active compounds, which participate in various essential cellular functions. While many studies have reported on the antibacterial and antifungal effects of Rhamnolipids, only a few tried to describe the molecular mechanisms underlying these effects. Here, we first review the literature on Rhamnolipid-phospholipid interactions and then add own results on a prominent monoRhamnolipid congener, RhaC10C10. By focusing on the interactions between the Rhamnolipid and lipid model membranes of different complexity, up to heterogeneous raft-like model biomembranes, we gained new insights into changes of the lateral membrane organization and morphological changes of membrane vesicles induced by partitioning of the Rhamnolipid. To this end, AFM, confocal fluorescence microscopy, and Laurdan fluorescence spectroscopy analyses were employed. In summary, we provide a concise description of the physio-chemical effects Rhamnolipids impose on lipid membranes, which help us to understand their physiological role.
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heterologous production of long chain Rhamnolipids from burkholderia glumae in pseudomonas putida a step forward to tailor made Rhamnolipids
Applied Microbiology and Biotechnology, 2018Co-Authors: Andreas Wittgens, Beatrix Santiagoschuebel, Till Tiso, Diana Hofmann, Marius Henkel, Susanne Wilhelm, Rudolf Hausmann, Lars M. Blank, Karl-erich JaegerAbstract:Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a β-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native Rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain Rhamnolipids mainly consisting of a Rha-Rha-C10-C10 congener. Bacteria from the genus Burkholderia are also able to produce Rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C14-C14 congener. These long-chain Rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of Rhamnolipids are still hampered by the pathogenicity of producer strains and—even more important—by the complexity of regulatory networks controlling Rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for Rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain Rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-Rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in Rhamnolipids.
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Designer Rhamnolipids by reduction of congener diversity: production and characterization.
Microbial cell factories, 2017Co-Authors: Till Tiso, Andreas Wittgens, Beate Behrens, Frank Rosenau, Heiko Hayen, Rabea Zauter, Hannah Tulke, Bernd Leuchtle, Lars M. BlankAbstract:Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes Rhamnolipids by decoupling production from host-intrinsic regulations and cell growth. Here, the molecular structure of the Rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural Rhamnolipid producers can synthesize mono- and di-Rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of Rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main Rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-Rhamnolipids, mono-Rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of Rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate Rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics. The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.
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Novel insights into biosynthesis and uptake of Rhamnolipids and their precursors.
Applied microbiology and biotechnology, 2016Co-Authors: Andreas Wittgens, Till Tiso, Diana Hofmann, Marius Henkel, Lars M. Blank, Markus Muller, Filip Kovacic, Melanie Gerlitzki, Beatrix Santiago-schübel, Rudolf HausmannAbstract:The human pathogenic bacterium Pseudomonas aeruginosa produces Rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-Rhamnolipids are converted to di-Rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of Rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in Rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in Rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-Rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-Rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of Rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical Rhamnolipids containing two fatty acids, valuable for the production of designer Rhamnolipids with desired physicochemical properties.
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creating metabolic demand as an engineering strategy in pseudomonas putida Rhamnolipid synthesis as an example
Metabolic Engineering Communications, 2016Co-Authors: Till Tiso, Andreas Wittgens, Petra Sabelhaus, Beate Behrens, Frank Rosenau, Heiko Hayen, Lars M. BlankAbstract:Abstract Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In Pseudomonas putida , a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering. We tested this driven-by-demand metabolic engineering strategy using Rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, Rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis. Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes ( rhlAB ) for Rhamnolipid synthesis. The best Rhamnolipid-synthesizing strain had a yield of 40% Rhamnolipids on sugar [Cmol RL /Cmol Glc ], which is approximately 55% of the theoretical yield. The rate of Rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its flux by 300%, whereas the flux through de novo fatty acid synthesis increased by 50%. We show that the central carbon metabolism of P. putida is capable of meeting the metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling a significant rerouting of carbon flux toward the product of interest, in this case, Rhamnolipids of industrial interest.
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quorum sensing controls swarming motility of burkholderia glumae through regulation of Rhamnolipids
PLOS ONE, 2015Co-Authors: Arvin Nickzad, Francois Lepine, Eric DezielAbstract:Burkholderia glumae is a plant pathogenic bacterium that uses an acyl-homoserine lactone-mediated quorum sensing system to regulate protein secretion, oxalate production and major virulence determinants such as toxoflavin and flagella. B. glumae also releases surface-active Rhamnolipids. In Pseudomonas aeruginosa and Burkholderia thailandensis, Rhamnolipids, along with flagella, are required for the social behavior called swarming motility. In the present study, we demonstrate that quorum sensing positively regulates the production of Rhamnolipids in B. glumae and that Rhamnolipids are necessary for swarming motility also in this species. We show that a rhlA- mutant, which is unable to produce Rhamnolipids, loses its ability to swarm, and that this can be complemented by providing exogenous Rhamnolipids. Impaired Rhamnolipid production in a quorum sensing-deficient B. glumae mutant is the main factor responsible for its defective swarming motility behaviour.
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characterization of Rhamnolipid production by burkholderia glumae
Letters in Applied Microbiology, 2011Co-Authors: S G V A O Costa, Eric Deziel, Francois LepineAbstract:Aims: To investigate if Burkholderia glumae can produce Rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Methods and Results: Burkholderia glumae AU6208 produces a large spectrum of mono- and di-Rhamnolipid congeners with side chains varying between C(12) -C(12) and C(16) -C(16) , the most abundant being Rha-Rha-C(14) -C(14) .The effects on Rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000.ovrhdot.7 mg l(-1) was reached after 6 days. These Rhamnolipids display a critical micelle concentration of 25-27 mg l(-1) and decrease the interfacial tension against hexadecane from 40 to 1.ovrhdot.8 mN m(-1) . They also have excellent emulsifying properties against long chain alkanes. Conclusions: Burkholderia glumae AU6208 can produce considerable amounts of Rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. Significance and Impact of the Study: In contrast with the classical Rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of Rhamnolipids with this species could be easier than with Ps. aeruginosa
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Characterization of Rhamnolipid production by Burkholderia glumae.
Letters in applied microbiology, 2011Co-Authors: S G V A O Costa, Eric Deziel, Francois LepineAbstract:To investigate if Burkholderia glumae can produce Rhamnolipids, define a culture medium for good production yields, analyse their composition and determine their tensioactive properties. Burkholderia glumae AU6208 produces a large spectrum of mono- and di-Rhamnolipid congeners with side chains varying between C(12)-C(12) and C(16)-C(16), the most abundant being Rha-Rha-C(14)-C(14).The effects on Rhamnolipid production of the cultivation temperature, nitrogen and carbon source were investigated. With urea as the nitrogen source and canola oil as the carbon source, a production of 1000.7 mg l(-1) was reached after 6 days. These Rhamnolipids display a critical micelle concentration of 25-27 mg l(-1) and decrease the interfacial tension against hexadecane from 40 to 1.8 mN m(-1). They also have excellent emulsifying properties against long chain alkanes. Burkholderia glumae AU6208 can produce considerable amounts of Rhamnolipids. They are produced as diversified mixtures of congeners. Their side chains are longer than those normally produced by those of Pseudomonas aeruginosa. They also present excellent tensioactive properties. In contrast with the classical Rhamnolipid producer Ps. aeruginosa, B. glumae is not a pathogen to humans. This work shows that the industrial production of Rhamnolipids with this species could be easier than with Ps. aeruginosa. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.
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Rhamnolipids: diversity of structures, microbial origins and roles
Applied Microbiology and Biotechnology, 2010Co-Authors: Ahmad Mohammad Abdel-Mawgoud, Francois Lepine, Eric DezielAbstract:Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α - L -rhamnopyranosyl- α-L -rhamnopyranosyl- β -hydroxydecanoyl- β -hydroxydecanoate (Rha-Rha-C_10-C_10), α - L -rhamnopyranosyl- α-L -rhamnopyranosyl- β -hydroxydecanoate (Rha-Rha-C_10), as well as their mono-Rhamnolipid congeners Rha-C_10-C_10 and Rha-C_10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of Rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce Rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa . In P. aeruginosa , three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of Rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC . Genes highly similar to rhlA , rhlB , and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for Rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, Rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.
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Rhamnolipids: diversity of structures, microbial origins and roles.
Applied Microbiology and Biotechnology, 2010Co-Authors: Ahmad Mohammad Abdel-Mawgoud, Francois Lepine, Eric DezielAbstract:Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (Rha-Rha-C(10)-C(10)), alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecanoate (Rha-Rha-C(10)), as well as their mono-Rhamnolipid congeners Rha-C(10)-C(10) and Rha-C(10). The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of Rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce Rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of Rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for Rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, Rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.