IMPATIENT-qPCR: monitoring SELEX success during in vitro aptamer evolution.

SELEX (Systematic Evolution of Ligands by Exponential enrichment) processes aim on the evolution of high-affinity aptamers as binding entities in diagnostics and biosensing. Aptamers can represent game-changers as constituents of diagnostic assays for the management of instantly occurring infectious diseases or other health threats. Without in-process quality control measures SELEX suffers from low overall success rates. We present a quantitative PCR method for fast and easy quantification of aptamers bound to their targets. Simultaneous determination of melting temperatures (Tm) of each SELEX round delivers information on the evolutionary success via the correlation of increasing GC content and Tm alone with a round-wise increase of aptamer affinity to the respective target. Based on nine successful and published previous SELEX processes, in which the evolution/selection of aptamer affinity/specificity was demonstrated, we here show the functionality of the IMPATIENT-qPCR for polyclonal aptamer libraries and resulting individual aptamers. Based on the ease of this new evolution quality control, we hope to introduce it as a valuable tool to accelerate SELEX processes in general. IMPATIENT-qPCR SELEX success monitoring. Selection and evolution of high-affinity aptamers using SELEX technology with direct aptamer evolution monitoring using melting curve shifting analyses to higher Tm by quantitative PCR with fluorescence dye SYBR Green I. KEY POINTS: • Fast and easy analysis. • Universal applicability shown for a series of real successful projects.

Robust Fluorometric Aptamer Assay for Direct and Rapid Detection of Clinical Isolates of Candida spec.

Infections caused by yeasts of the genus Candida are likely to occur not only in immunocompromised patients but also in healthy individuals, leading to infections of the gastrointestinal tract, urinary tract, and respiratory tract. Due to the rapid increase in the frequency of reported Candidiasis cases in recent years, diagnostic research has become the subject of many studies, and therefore, we developed a polyclonal aptamer library-based fluorometric assay with high specificity and affinity towards Candida spec. to quantify the pathogens in clinical samples with high sensitivity. We recently obtained the specific aptamer library R10, which explicitly recognized Candida and evolved it by mimicking an early skin infection model caused by Candida using the FluCell-SELEX system. In the follow-up study presented here, we demonstrate that the aptamer library R10-based bioassay specifically recognizes invasive clinical Candida isolates, including not only C. albicans but also strains like C. tropcialis, C. krusei, or C. glabrata. The next-generation fluorometric bioassay presented here can reliably and easily detect an early Candida infection and could be used for further clinical research or could even be developed into a full in vitro diagnostic tool.

Robust Protocol for the Synthesis of BSA Nanohydrogels by Inverse Nanoemulsion for Drug Delivery.

In a highly efficient and reproducible process, bovine serum albumin (BSA) nanogels are prepared from inverse nanoemulsions. The concept of independent nanoreactors of the individual droplets in the nanoemulsions allows high protein concentrations of up to 0.6% in the inverse total system. The BSA gel networks are generated by the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride coupling strategy widely used in protein chemistry. In a robust work-up protocol, the hydrophobic continuous phase of the inverse emulsion is stepwise replaced by water without compromising the colloidal stability and non-toxicity of the nanogel particles. Further, the simple process allows the loading of the nanogels with various cargos like a dye (Dy-495), a drug (ibuprofen), another protein [FMN-binding fluorescent protein (EcFbFP)], and oligonucleotides [plasmid DNA for enhanced GFP expression in mammalian cells (pEGFP c3) and a synthetic anti-Pseudomonas aeruginosa aptamer library]. These charged nanoobjects work efficiently as carriers for staining and transfection of cells. This is exemplarily shown for a phalloidin dye and a plasmid DNA as cargo with adenocarcinomic human alveolar basal epithelial cells (A549), a cell revertant of the SV-40 cancer rat cell line SV-52 (Rev2), and human breast carcinoma cells (MDA-MB-231), respectively.

Short Photoperiod-Dependent Enrichment of Akkermansia spec. as the Major Change in the Intestinal Microbiome of Djungarian Hamsters (Phodopus sungorus).

The Djungarian hamster (Phodopus sungorus) is a prominent model organism for seasonal acclimatization, showing drastic whole-body physiological adjustments to an energetically challenging environment, which are considered to also involve the gut microbiome. Fecal samples of hamsters in long photoperiod and again after twelve weeks in short photoperiod were analyzed by 16S-rRNA sequencing to evaluate seasonal changes in the respective gut microbiomes. In both photoperiods, the overall composition was stable in the major superordinate phyla of the microbiota, with distinct and delicate changes of abundance in phyla representing each <1% of all. Elusimicrobia, Tenericutes, and Verrucomicrobia were exclusively present in short photoperiod hamsters. In contrast to Elusimicrobium and Aneroplasma as representatives of Elusimicrobia and Tenericutes, Akkermansia muciniphila is a prominent gut microbiome inhabitant well described as important in the health context of animals and humans, including neurodegenerative diseases and obesity. Since diet was not changed, Akkermansia enrichment appears to be a direct consequence of short photoperiod acclimation. Future research will investigate whether the Djungarian hamster intestinal microbiome is responsible for or responsive to seasonal acclimation, focusing on probiotic supplementation.

Identification and Characterization of Three New Antimicrobial Peptides from the Marine Mollusk Nerita versicolor (Gmelin, 1791).

Mollusks have been widely investigated for antimicrobial peptides because their humoral defense against pathogens is mainly based on these small biomolecules. In this report, we describe the identification of three novel antimicrobial peptides from the marine mollusk Nerita versicolor. A pool of N. versicolor peptides was analyzed with nanoLC-ESI-MS-MS technology, and three potential antimicrobial peptides (Nv-p1, Nv-p2 and Nv-p3) were identified with bioinformatical predictions and selected for chemical synthesis and evaluation of their biological activity. Database searches showed that two of them show partial identity to histone H4 peptide fragments from other invertebrate species. Structural predictions revealed that they all adopt a random coil structure even when placed near a lipid bilayer patch. Nv-p1, Nv-p2 and Nv-p3 exhibited activity against Pseudomonas aeruginosa. The most active peptide was Nv-p3 with an inhibitory activity starting at 1.5 µg/mL in the radial diffusion assays. The peptides were ineffective against Klebsiella pneumoniae, Listeria monocytogenes and Mycobacterium tuberculosis. On the other hand, these peptides demonstrated effective antibiofilm action against Candida albicans, Candida parapsilosis and Candida auris but not against the planktonic cells. None of the peptides had significant toxicity on primary human macrophages and fetal lung fibroblasts at effective antimicrobial concentrations. Our results indicate that N. versicolor-derived peptides represent new AMP sequences and have the potential to be optimized and developed into antibiotic alternatives against bacterial and fungal infections.

Aptamers as Novel Binding Molecules on an Antimicrobial Peptide-Armored Composite Hydrogel Wound Dressing for Specific Removal and Efficient Eradication of Pseudomonas aeruginosa.

Here we present for the first time a potential wound dressing material implementing aptamers as binding entities to remove pathogenic cells from newly contaminated surfaces of wound matrix-mimicking collagen gels. The model pathogen in this study was the Gram-negative opportunistic bacterium Pseudomonas aeruginosa, which represents a considerable health threat in hospital environments as a cause of severe infections of burn or post-surgery wounds. A two-layered hydrogel composite material was constructed based on an established eight-membered focused anti-P. aeruginosa polyclonal aptamer library, which was chemically crosslinked to the material surface to form a trapping zone for efficient binding of the pathogen. A drug-loaded zone of the composite released the C14R antimicrobial peptide to deliver it directly to the bound pathogenic cells. We demonstrate that this material combining aptamer-mediated affinity and peptide-dependent pathogen eradication can quantitatively remove bacterial cells from the "wound" surface, and we show that the surface-trapped bacteria are completely killed. The drug delivery function of the composite thus represents an extra safeguarding property and thus probably one of the most important additional advances of a next-generation or smart wound dressing ensuring the complete removal and/or eradication of the pathogen of a freshly infected wound.

Cm-p5 Peptide Dimers Inhibit Biofilms of Candida albicans Clinical Isolates, C. parapsilosis and Fluconazole-Resistant Mutants of C. auris.

Antimicrobial peptides (AMPs) represent a promising class of therapeutic biomolecules that show antimicrobial activity against a broad range of microorganisms, including life-threatening pathogens. In contrast to classic AMPs with membrane-disrupting activities, new peptides with a specific anti-biofilm effect are gaining in importance since biofilms could be the most important way of life, especially for pathogens, as the interaction with host tissues is crucial for the full development of their virulence in the event of infection. Therefore, in a previous study, two synthetic dimeric derivatives (parallel Dimer 1 and antiparallel Dimer 2) of the AMP Cm-p5 showed specific inhibition of the formation of Candida auris biofilms. Here we show that these derivatives are also dose-dependently effective against de novo biofilms that are formed by the widespread pathogenic yeasts C. albicans and C. parapsilosis. Moreover, the activity of the peptides was demonstrated even against two fluconazole-resistant strains of C. auris.

Polyclonal Aptamer Libraries from a FluRoot-SELEX for the Specific Labeling of the Apical and Elongation/Differentiation Zones of Arabidopsis thaliana Roots.

In more than 30 years of aptamer research, it has become widely accepted that aptamers are fascinating binding molecules for a vast variety of applications. However, the majority of targets have been proteins, although special variants of the so-called SELEX process for the molecular evolution of specific aptamers have also been developed, allowing for the targeting of small molecules as well as larger structures such as cells and even cellular networks of human (tumor) tissues. Although the provocative thesis is widely accepted in the field, that is, in principle, any level of complexity for SELEX targets is possible, the number of studies on whole organs or at least parts of them is limited. To pioneer this thesis, and based on our FluCell-SELEX process, here, we have developed polyclonal aptamer libraries against apices and the elongation/differentiation zones of plant roots as examples of organs. We show that dedicated libraries can specifically label the respective parts of the root, allowing us to distinguish them in fluorescence microscopy. We consider this achievement to be an initial but important evidence for the robustness of this SELEX variant. These libraries may be valuable tools for plant research and a promising starting point for the isolation of more specific individual aptamers directed against root-specific epitopes.

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.

A Polyclonal Selex Aptamer Library Directly Allows Specific Labelling of the Human Gut Bacterium Blautia producta without Isolating Individual Aptamers.

Recent studies have demonstrated that changes in the abundance of the intestinal bacterium Blautia producta, a potential probiotic, are closely associated with the development of various diseases such as obesity, diabetes, some neurodegenerative diseases, and certain cancers. However, there is still a lack of an effective method to detect the abundance of B. producta in the gut rapidly. Especially, DNA aptamers are now widely used as biometric components for medical testing due to their unique characteristics, including high chemical stability, low production cost, ease of chemical modification, low immunogenicity, and fast reproducibility. We successfully obtained a high-affinity nucleic acid aptamer library (B.p-R14) after 14 SELEX rounds, which efficiently discriminates B. producta in different analysis techniques including fluorometric suspension assays or fluorescence microscopy from other major gut bacteria in complex mixtures and even in human stool samples. These preliminary findings will be the basis towards aptamer-based biosensing applications for the fast and reliable monitoring of B. producta in the human gut microbiome.

FluCell-SELEX Aptamers as Specific Binding Molecules for Diagnostics of the Health Relevant Gut Bacterium Akkermansia muciniphila.

Based on their unique properties, oligonucleotide aptamers have been named a gift of biological chemistry to life science. We report the development of DNA aptamers as the first high-affinity binding molecules available for fast and rapid labeling of the human gut bacterium Akkermansia muciniphila with a certain impact on Alzheimer´s disease. Fast and reliable analyses of the composition of microbiomes is an emerging field in microbiology. We describe the molecular evolution and biochemical characterization of a specific aptamer library by a FluCell-SELEX and the characterization of specific molecules from the library by bioinformatics. The aptamer AKK13.1 exerted universal applicability in different analysis techniques in modern microbiology, including fluorimetry, confocal laser scanning microscopy and flow cytometry. It was also functional as a specific binding entity hybridized to anchor primers chemically coupled via acrydite-modification to the surface of a polyacrylamide-hydrogel, which can be prototypically used for the construction of affinity surfaces in sensor chips. Together, the performance and methodological flexibility of the aptamers presented here may open new routes not only to develop novel Akkermansia-specific assays for clinical microbiology and the analyses of human stool samples but may also be an excellent starting point for the construction of novel electronic biosensors.

BSA Hydrogel Beads Functionalized with a Specific Aptamer Library for Capturing Pseudomonas aeruginosa in Serum and Blood.

Systemic blood stream infections are a major threat to human health and are dramatically increasing worldwide. Pseudomonas aeruginosa is a WHO-alerted multi-resistant pathogen of extreme importance as a cause of sepsis. Septicemia patients have significantly increased survival chances if sepsis is diagnosed in the early stages. Affinity materials can not only represent attractive tools for specific diagnostics of pathogens in the blood but can prospectively also serve as the technical foundation of therapeutic filtration devices. Based on the recently developed aptamers directed against P. aeruginosa, we here present aptamer-functionalized beads for specific binding of this pathogen in blood samples. These aptamer capture beads (ACBs) are manufactured by crosslinking bovine serum albumin (BSA) in an emulsion and subsequent functionalization with the amino-modified aptamers on the bead surface using the thiol- and amino-reactive bispecific crosslinker PEG4-SPDP. Specific and quantitative binding of P. aeruginosa as the dedicated target of the ACBs was demonstrated in serum and blood. These initial but promising results may open new routes for the development of ACBs as a platform technology for fast and reliable diagnosis of bloodstream infections and, in the long term, blood filtration techniques in the fight against sepsis.

The Diversity of a Polyclonal FluCell-SELEX Library Outperforms Individual Aptamers as Emerging Diagnostic Tools for the Identification of Carbapenem Resistant Pseudomonas aeruginosa.

Textbook procedures require the use of individual aptamers enriched in SELEX libraries which are subsequently chemically synthesized after their biochemical characterization. Here we show that this reduction of the available sequence space of large libraries and thus the diversity of binding molecules reduces the labelling efficiency and fidelity of selected single aptamers towards different strains of the human pathogen Pseudomonas aeruginosa compared to a polyclonal aptamer library enriched by a whole-cell-SELEX involving fluorescent aptamers. The library outperformed single aptamers in reliable and specific targeting of different clinically relevant strains, allowed to inhibit virulence associated cellular functions and identification of bound cell surface targets by aptamer based affinity purification and mass spectrometry. The stunning ease of this FluCell-SELEX and the convincing performance of the P. aeruginosa specific library may pave the way towards generally new and efficient diagnostic techniques based on polyclonal aptamer libraries not only in clinical microbiology.

Azulitox-A Pseudomonas aeruginosa P28-Derived Cancer-Cell-Specific Protein Photosensitizer.

Azulitox as a new fusion polypeptide with cancer cell specificity and phototoxicity was generated and is composed of a photosensitizer domain and the cell-penetrating peptide P28. The photosensitizer domain (EcFbFP) was derived from a bacterial blue-light receptor, which belongs to the family of light-oxygen-voltage proteins and produces reactive oxygen species (ROS) upon excitation. P28 is derived from the cupredoxin protein azurin that is known to specifically penetrate cancer cells and bind to the tumor suppressor protein p53. We show that the P28 domain specifically directs and translocates the fused photosensitizer into cancer cells. Under blue-light illumination, Azulitox significantly induced cytotoxicity. Compared to the extracellular application of EcFbFP, Azulitox caused death to about 90% of cells, as monitored by flow cytometry, which also directly correlated with the amount of ROS produced in the cells. Azulitox may open new avenues toward targeted polypeptide-photosensitizer-based photodynamic therapies with reduced systemic toxicity compared to conventional photosensitizers.

Lectin-Functionalized Composite Hydrogels for "Capture-and-Killing" of Carbapenem-Resistant Pseudomonas aeruginosa.

Infections with multiresistant pathogens are a leading cause for mortality worldwide. Just recently, the World Health Organization (WHO) increased the threat rating for multiresistant Pseudomonas aeruginosa to the highest possible level. With this background, it is crucial to develop novel materials and procedures in the fight against multiresistant pathogens. In this study, we present a novel antimicrobial material, which could find applications as a wound dressing or antimicrobial coating. Lectins are multivalent sugar-binding proteins, which can be found in a variety of plants and bacteria, where they are associated with biofilm formation. By immobilizing lectin B on a protein-based hydrogel surface, we provided the hydrogel with the ability to immobilize ("catch") pathogens upon contact. Furthermore, another hydrogel layer was added which inhibits biofilm formation and releases a highly potent antimicrobial peptide to eradicate microorganisms ("kill"). The composite hydrogel showed a high antimicrobial activity against the reference strain Pseudomonas aeruginosa PAO1 as well as against a carbapenem-resistant clinical isolate (multiresistant Gram-negative class 4) and may thus represent a novel material to develop a new type of antimicrobial wound dressings to prevent infections with this problematic pathogen of burn or other large wounds.

On the road towards tailor-made rhamnolipids: current state and perspectives.

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.

Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida-a step forward to tailor-made rhamnolipids.

Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a ß-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.

Designer rhamnolipids by reduction of congener diversity: production and characterization.

BACKGROUND: 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. RESULTS: 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 ß-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 ß-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. CONCLUSIONS: 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.

Beyond bread and beer: whole cell protein extracts from baker's yeast as a bulk source for 3D cell culture matrices.

Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 µm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 µM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors.

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.