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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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culture medium optimization for production of Rhamnolipids by burkholderia glumae
Colloids and Interfaces, 2018Co-Authors: Arvin Nickzad, Claude Guertin, Eric DezielAbstract:Burkholderia glumae is a biosafety level 1 bacterium capable of producing rhamnolipid biosurfactant with longer 3-hydroxy fatty acid chains moieties than those produced by the prototypal producer, the opportunistic pathogen Pseudomonas aeruginosa. Although the capacity of production of rhamnolipid, and the parameters affecting this production, are well established for P. aeruginosa, little is known about the factors that may affect their production in B. glumae. Hence, to evaluate and enhance the production of Rhamnolipids in B. glumae, following the selection of best carbon and nitrogen sources, a two-level fractional factorial design experiment was performed to identify the limiting factors significantly affecting the production of Rhamnolipids in this bacterial species. Effects of six inorganic nutrients and two physical parameters were studied, and mannitol, urea, CaCl2, and potassium phosphate buffer were selected for further optimization by applying a response surface methodology (RSM). Under the identified optimized conditions, a rhamnolipid production of 1.66 g/L was obtained, about five times higher than that of the initial non-optimized conditions. This represents a key step in the development of large-scale production processes.
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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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Structure, properties and applications of Rhamnolipids produced by Pseudomonas aeruginosa L2-1 from cassava wastewater
Process Biochemistry, 2010Co-Authors: Siddhartha G.v.a.o. Costa, Francois Lepine, Eric Deziel, Marcia Nitschke, Jonas ContieroAbstract:The properties and applications of rhamnolipid surfactants produced by Pseudomonas aeruginosa L2-1 from cassava wastewater added with waste cooking oil (CWO) as low-cost substrate, were investigated and compared with the commercial rhamnolipid mixture JBR599 (Jeneil Biosurfactant Co., Saukville, USA). The Rhamnolipids produced by strain L2-1 were characterized by high performance liquid chromatography-mass spectrometry. Sixteen different rhamnolipid congeners were detected, with Rha-C10-C10 and Rha-Rha-C10-C10 being the most abundant. The L2-1 Rhamnolipids from CWO showed similar or better tensioactive properties than those from JBR599, with a minimal surface tension of 30 mN/m and a critical micelle concentration (CMC) of 30 mg/l. The L2-1 biosurfactants formed stable emulsions with several hydrocarbons and showed excellent emulsification of soybean oil (100%). These Rhamnolipids removed 69% of crude oil present in contaminated sand samples at the CMC and presented antimicrobial activity against Bacillus cereus (32 μg/ml), Micrococcus luteus (32 μg/ml) and Staphylococcus aureus (128 μg/ml). These results demonstrate that the Rhamnolipids produced in CWO can be useful for industrial applications, such as the bioremediation of oil spills.
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burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of Rhamnolipids
BMC Microbiology, 2009Co-Authors: Danielle Dubeau, Eric Deziel, Donald E Woods, Francois LepineAbstract:Rhamnolipids are surface active molecules composed of rhamnose and β-hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of Rhamnolipids by Burkholderia species. Orthologs of rhlA, rhlB and rhlC, which are responsible for the biosynthesis of Rhamnolipids in P. aeruginosa, have been found in the non-infectious Burkholderia thailandensis, as well as in the genetically similar important pathogen B. pseudomallei. In contrast to P. aeruginosa, both Burkholderia species contain these three genes necessary for rhamnolipid production within a single gene cluster. Furthermore, two identical, paralogous copies of this gene cluster are found on the second chromosome of these bacteria. Both Burkholderia spp. produce Rhamnolipids containing 3-hydroxy fatty acid moieties with longer side chains than those described for P. aeruginosa. Additionally, the Rhamnolipids produced by B. thailandensis contain a much larger proportion of diRhamnolipids versus monoRhamnolipids when compared to P. aeruginosa. The Rhamnolipids produced by B. thailandensis reduce the surface tension of water to 42 mN/m while displaying a critical micelle concentration value of 225 mg/L. Separate mutations in both rhlA alleles, which are responsible for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid, prove that both copies of the rhl gene cluster are functional, but one contributes more to the total production than the other. Finally, a double ΔrhlA mutant that is completely devoid of rhamnolipid production is incapable of swarming motility, showing that both gene clusters contribute to this phenotype. Collectively, these results add another Burkholderia species to the list of bacteria able to produce Rhamnolipids and this, by the means of two identical functional gene clusters. Our results also demonstrate the very impressive tensio-active properties these long-chain Rhamnolipids possess in comparison to the well-studied short-chain ones from P. aeruginosa.
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rhla is required for the production of a novel biosurfactant promoting swarming motility in pseudomonas aeruginosa 3 3 hydroxyalkanoyloxy alkanoic acids haas the precursors of Rhamnolipids
Microbiology, 2003Co-Authors: Eric Deziel, Francois Lepine, Sylvain Milot, Richard VillemurAbstract:Pseudomonas aeruginosa produces extracellular glycolipids composed of l-rhamnose and 3-hydroxyalkanoic acid called Rhamnolipids. Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood. Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by the rhlAB operon, which catalyses the transfer of TDP-l-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths. RhlB is the catalytic protein of the rhamnosyltransferase. rhlA is indispensable for rhamnolipid synthesis, but its function is unknown. Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs by P. aeruginosa was detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis. In this report, evidence is presented indicating that rhlA is required for production of HAAs and that these HAAs display potent surface-active properties. P. aeruginosa can colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents. Using rhlA and rhlB mutants it was observed that swarming requires the expression of the rhlA gene but does not necessitate rhamnolipid production, as HAAs act as surfactants. Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decrease rhlA expression and swarming motility.
Ibrahim M. Banat - One of the best experts on this subject based on the ideXlab platform.
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identification and characterisation of short chain rhamnolipid production in a previously uninvestigated non pathogenic marine pseudomonad
Applied Microbiology and Biotechnology, 2018Co-Authors: Matthew S. Twigg, Roger Marchant, Lakshmi Tripathi, Aikaterini A Zompra, Karina Salek, Victor U Irorere, Tony Gutierrez, Georgios A Spyroulias, Ibrahim M. BanatAbstract:This study aimed to identify and characterise biosurfactant compounds produced by bacteria associated with a marine eukaryotic phytoplankton bloom. One strain, designated MCTG214(3b1), was isolated by enrichment with polycyclic aromatic hydrocarbons and based on 16S rDNA, and gyrB sequencing was found to belong to the genus Pseudomonas, however not related to P. aeruginosa. Cell-free supernatant samples of strain MCTG214(3b1) at stationary phase showed significant reductions in surface tension. HPLC-MS and NMR analysis of these samples indicated the presence of five different rhamnolipid (RL) congeners. Di-Rhamnolipids accounted for 87% relative abundance and all congeners possessed fatty acid moieties consisting of 8-12 carbons. PCR screening of strain MCTG214(3b1) DNA revealed homologues to the P. aeruginosa RL synthesis genes rhlA and rhlB; however, no rhlC homologue was identified. Using the Galleria mellonella larvae model, strain MCTG214(3b1) was demonstrated to be far less pathogenic than P. aeruginosa. This study identifies for the first time a significantly high level of synthesis of short chain di-Rhamnolipids by a non-pathogenic marine Pseudomonas species. We postulate that RL synthesis in Pseudomonas sp. MCTG214(3b1) is carried out by enzymes expressed from rhlA/B homologues similar to those of P. aeruginosa; however, a lack of rhlC potentially indicates the presence of a second novel rhamnosyltransferase responsible for the di-rhamnolipid congeners identified by HPLC-MS.
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Enhanced rhamnolipid production in Burkholderia thailandensis transposon knockout strains deficient in polyhydroxyalkanoate (PHA) synthesis
Applied Microbiology and Biotechnology, 2017Co-Authors: Scott J. Funston, Roger Marchant, Konstantina Tsaousi, Thomas J. Smyth, Matthew S. Twigg, Ibrahim M. BanatAbstract:Microbially produced Rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa , is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces Rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of Rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa . Multiple knockout strains for the phbA , phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (Δ phbA 1, Δ phbB 1 and Δ phbC 1) with the best enhancement of rhamnolipid production were selected for detailed study. Δ phbB 1 produced the highest level of purified RL (3.78 g l^−1) compared to the wild-type strain (1.28 g l^−1). In Δ phbB 1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa , knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway.
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Rhamnolipids from non pathogenic burkholderia thailandensis e264 physicochemical characterization antimicrobial and antibiofilm efficacy against oral hygiene related pathogens
New Biotechnology, 2017Co-Authors: Mohamed Elshikh, Roger Marchant, Scott J. Funston, Alif Chebbi, Syed Ahmed, Ibrahim M. BanatAbstract:Biosurfactants are naturally occurring surface active compounds that have mainly been exploited for environmental applications and consumer products, with their biomedical efficacy an emerging area of research. Rhamnolipids area major group of biosurfactants that have been reported for their antimicrobial and antibiofilm efficacy. One of the main limiting factors for scaled up production and downstream applications of Rhamnolipids is the fact that they are predominantly produced from the opportunistic pathogen Pseudomonas aeruginosa. In this article, we have reported the production and characterisation of long chain Rhamnolipids from non-pathogenic Burkholderia thailandensis E264 (ATCC 700388). We have also investigated the antibacterial and antibiofilm properties of these Rhamnolipids against some oral pathogens (Streptococcus oralis, Actinomyces naeslundii, Neisseria mucosa and Streptococcus sanguinis), important for oral health and hygiene. Treating these bacteria with different concentrations of long chain Rhamnolipids resulted in a reduction of 3–4 log of bacterial viability, placing these Rhamnolipids close to being classified as biocidal. Investigating long chain rhamnolipid efficacy as antibiofilm agents for prospective oral-related applications revealed good potency against oral-bacteria biofilms in a co-incubation experiments, in a pre-coated surface format, in disrupting immature biofilms and has shown excellent combination effect with Lauryl Sodium Sulphate which resulted in a drastic decrease in its minimal inhibitory concentration against different bacteria. Investigating the rhamnolipid permeabilization effect along with their ability to induce the formation of reactive oxygen species has shed light on the mechanism through which inhibition/killing of bacteria may occur.
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Rhamnolipids from pseudomonas aeruginosa strain w10 as antibiofilm antibiofouling products for metal protection
Journal of Basic Microbiology, 2017Co-Authors: Alif Chebbi, Roger Marchant, Mohamed Elshikh, Syed Ahmed, Farazul Haque, Sara Dobbin, Sami Sayadi, Mohamed Chamkha, Ibrahim M. BanatAbstract:Industrial biofouling-problems associated with the accumulation of microorganisms from flowing water and fluids on processing surfaces can cause severe problems. A Pseudomonas aeruginosa strain W10 was isolated from industrial setting and found to produce predominantly di-Rhamnolipids (Rha-Rha-C10-C10) with a yield of around 10 g L-1 and a critical micelle concentration (CMC) of 80 mg L-1 . P. aeruginosa W10 Rhamnolipids were able to disrupt up to 99% of 48 h pre-formed biofilms of the Gram-positive organisms Bacillus licheniformis CAN55, Staphylococcus capitis SH6, and a mixed culture (strains CAN55, SH6, and W10), under static conditions, at concentrations of 0.1, 0.5, and 1 mg ml-1 on a stainless steel surface commonly used in industrial process pipelines. CFU measurements and LIVE/DEAD BacLight staining confirmed these observations. Furthermore, a purified di-rhamnolipid fraction was found to be responsible for the microbial inhibition of B. licheniformis strain CAN55. This study provides evidence that Rhamnolipids may have valuable applications in preventing biofilms and biofouling in industrial plants and, in a wider context, may also apply to metal medical devices.
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Rhamnolipids are conserved biosurfactants molecules implications for their biotechnological potential
Applied Microbiology and Biotechnology, 2013Co-Authors: Amedea Perfumo, Roger Marchant, Thomas J. Smyth, Michelle Rudden, Paul Simon Stevenson, Neil James Parry, Ibrahim M. BanatAbstract:A range of isolates of Pseudomonas aeruginosa from widely different environmental sources were examined for their ability to synthesise rhamnolipid biosurfactants. No significant differences in the quantity or composition of the rhamnolipid congeners could be produced by manipulating the growth conditions. Sequences for the rhamnolipid genes indicated low levels of strain variation, and the majority of polymorphisms did lead to amino acid sequence changes that had no evident phenotypic effect. Expression of the rhlB and rhlC rhamnosyltransferase genes showed a fixed sequential expression pattern during growth, and no significant up-regulation could be induced by varying producer strains or growth media. The results indicated that Rhamnolipids are highly conserved molecules and that their gene expression has a rather stringent control. This leaves little opportunity to manipulate and greatly increase the yield of Rhamnolipids from strains of P. aeruginosa for biotechnological applications.
Luyan Z. - One of the best experts on this subject based on the ideXlab platform.
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analysis of biosurfactants from industrially viable pseudomonas strain isolated from crude oil suggests how Rhamnolipids congeners affect emulsification property and antimicrobial activity
Frontiers in Microbiology, 2014Co-Authors: Palashpriya Das, Xinping Yang, Luyan Z.Abstract:Rhamnolipid biosurfactants produced mainly by Pseudomonas sp. had been reported to possess a wide range of potential industrial application. These biosurfactants are produced as monorhamnolipid (MRL) and di-rhamnolipid (DRL) congeners. The present study deals with rhamnolipid biosurfactants produced by three bacterial isolates from crude oil. Biosurfactants produced by one of the strains (named as IMP67) was found to be very efficacious based on its critical micelle concentration value and hydrocarbon emulsification property. Strikingly, antimicrobial, and anti-biofilm potential of this biosurfactant were higher than biosurfactants produced by other two strains. Thin layer chromatography analysis and rhamnose quantification showed that the Rhamnolipids of IMP67 had more MRL congeners than biosurfactants of the other two strains. Emulsification and antimicrobial actions were affected by manual change of MRL and DRL congener proportions. Increase of MRL proportion enhanced emulsification index and antimicrobial property to Gram negative bacteria. This result indicated that the ratio of MRL and DRL affected the emulsification potentials of Rhamnolipids, and suggested that high emulsification potentials might enhance Rhamnolipids to penetrate the cell wall of Gram negative bacteria. In line with this finding, Rhamnolipids of IMP67 also reduced the MIC of some antibiotics against bacteria, suggesting their synergistic role with the antibiotics.
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analysis of biosurfactants from industrially viable pseudomonas strain isolated from crude oil suggests how Rhamnolipids congeners affect emulsification property and antimicrobial activity
Frontiers in Microbiology, 2014Co-Authors: Palashpriya Das, Xinping Yang, Luyan Z.Abstract:Rhamnolipid biosurfactants produced mainly by Pseudomonas sp. had been reported to possess a wide range of potential industrial application. These biosurfactants are produced as monorhamnolipid (MRL) and di-rhamnolipid (DRL) congeners. The present study deals with rhamnolipid biosurfactants produced by three bacterial isolates from crude oil. Biosurfactants produced by one of the strains (named as IMP67) was found to be very efficacious based on its critical micelle concentration (CMC) value and hydrocarbon emulsification property. Strikingly, antimicrobial and anti-biofilm potential of this biosurfactant were higher than biosurfactants produced by other two strains. Thin layer chromatography (TLC) analysis and rhamnose quantification showed that the Rhamnolipids of IMP67 had more MRL congeners than biosurfactants of the other two strains. Emulsification and antimicrobial actions were affected by manual change of MRL and DRL congener proportions. Increase of MRL proportion enhanced emulsification index and antimicrobial property to Gram negative bacteria. This result indicated that the ratio of MRL and DRL affect the emulsification potentials of Rhamnolipids, and suggested that high emulsification potentials might enhance Rhamnolipids to penetrate the cell wall of Gram negative bacteria. In consistent, Rhamnolipids of IMP67 reduced the MIC of some antibiotics against bacteria, suggesting the potential of biosurfactant as antibiotics synergist.
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coordination of swarming motility biosurfactant synthesis and biofilm matrix exopolysaccharide production in pseudomonas aeruginosa
Applied and Environmental Microbiology, 2014Co-Authors: Shiwei Wang, Shan Yu, Zhenyin Zhang, Guomin Ai, Luyan Z.Abstract:Biofilm formation is a complex process in which many factors are involved. Bacterial swarming motility and exopolysaccharides both contribute to biofilm formation, yet it is unclear how bacteria coordinate swarming motility and exopolysaccharide production. Psl and Pel are two key biofilm matrix exopolysaccharides in Pseudomonas aeruginosa. This opportunistic pathogen has three types of motility, swimming, twitching, and swarming. In this study, we found that elevated Psl and/or Pel production reduced the swarming motility of P. aeruginosa but had little effect on swimming and twitching. The reduction was due to decreased rhamnolipid production with no relation to the transcription of rhlAB, two key genes involved in the biosynthesis of Rhamnolipids. Rhamnolipid-negative rhlR and rhlAB mutants synthesized more Psl, whereas exopolysaccharide-deficient strains exhibited a hyperswarming phenotype. These results suggest that competition for common sugar precursors catalyzed by AlgC could be a tactic for P. aeruginosa to balance the synthesis of exopolysaccharides and Rhamnolipids and to control bacterial motility and biofilm formation inversely because the biosynthesis of Rhamnolipids, Psl, and Pel requires AlgC to provide the sugar precursors and an additional algC gene enhances the biosynthesis of Psl and Rhamnolipids. In addition, our data indicate that the increase in RhlI/RhlR expression attenuated Psl production. This implied that the quorum-sensing signals could regulate exopolysaccharide biosynthesis indirectly in bacterial communities. In summary, this study represents a mechanism that bacteria utilize to coordinate swarming motility, biosurfactant synthesis, and biofilm matrix exopolysaccharide production, which is critical for biofilm formation and bacterial survival in the environment.
Till Tiso - 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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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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characterization of Rhamnolipids by liquid chromatography mass spectrometry after solid phase extraction
Analytical and Bioanalytical Chemistry, 2016Co-Authors: Beate Behrens, Till Tiso, Lars M. Blank, Jeannine Engelen, Heiko HayenAbstract:Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-Rhamnolipids, mono-Rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three Rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.