Palmitic Acid Sophorolipid Biosurfactant: From Self-Assembled Fibrillar Network (SAFiN) To Hydrogels with Fast Recovery

Nanofibers are an interesting phase into which amphiphilic molecules can self-assemble. Described for a large number of synthetic lipids, they were seldom reported for natural lipids like microbial amphiphiles, known as biosurfactants. In this work, we show that the palmitic acid congener of sophorolipids (SLC16:0), one of the most studied families of biosurfactants, spontaneously forms a self-assembled fiber network (SAFiN) at pH below 6 through a pH jump process. pH-resolved in-situ Small Angle X-ray Scattering (SAXS) shows a continuous micelle-to-fiber transition, characterized by an enhanced core-shell contrast between pH 9 and pH 7 and micellar fusion into flat membrane between pH 7 and pH 6, approximately. Below pH 6, homogeneous, infinitely long nanofibers form by peeling off the membranes. Eventually, the nanofiber network spontaneously forms a thixotropic hydrogel with fast recovery rates after applying an oscillatory strain amplitude out of the linear viscoelastic regime (LVER): after being submitted to strain amplitudes during 5 min, the hydrogel recovers about 80% and 100% of its initial elastic modulus after, respectively, 20 s and 10 min. Finally, the strength of the hydrogel depends on the medium's final pH, with an elastic modulus fivefold higher at pH 3 than at pH 6.Comment: Philosophical Transactions of the Royal Society of London. A (1887--1895), Royal Society, The, In pres

Increasing uniformity of biosurfactant production in Starmerella bombicola via the expression of chimeric cytochrome P450s

Sophorolipids are one of the best known microbial biosurfactants and are produced by several yeast species. The best studied producer is Starmerella bombicola, a non-pathogenic yeast associated in nature with bumblebees. Sophorolipids are built up of the rare disaccharide sophorose, which is attached to a fatty acid through a glyosidic bound. Sophorolipids produced by S. bombicola mainly contain oleic acid as the incorporated hydrophobic group. Other chain lengths can, to a certain content, be incorporated by feeding the yeast with substrates of alternative chain lengths. However, the efficiency for such substrates is low as compared to the preferred C18 chain length and defined by the substrate specificity of the first enzymatic step in sophorolipid biosynthesis, i.e., the cytochrome P450 enzyme CYP52M1. To increase product uniformity and diversity at the same time, a new strain of S. bombicola was developed that produces sophorolipids with a palmitic acid acyl chain. This was achieved by heterologous expression of the cytochrome P450 cyp1 gene of Ustilago maydis and feeding with palmitic acid. Optimization of the production was done by protein and process engineering

Lipid analysis: a way to improve the production of sophorolipids by Starmerella bombicola

The yeast Starmerella bombicola is known for the commercial production of the biosurfactant sophorolipids. Sophorolipids are surface-active molecules consisting of a disaccharide and a fatty acyl chain. The yeast can utilize various lipophilic substrates such as vegetable oils, alkanes and fatty acid esters and converts them to free fatty acids; the substrate of the sophorolipid biosynthetic pathway. In addition, the yeast has a very active de novo fatty acid synthesis; also in the absence of a lipophilic carbon source reasonable amounts of sophorolipids are obtained. While the production level of sophorolipids is already high; some efforts are still necessary for the synthesis of new-to-nature sophorolipids. The production of these molecules by modified Starmerella bombicola strains or by using special substrates is in some cases less efficient and requires further optimization. On the one hand, the efficiency is lower because special substrates are lost due to catabolic pathways. On the other hand, some modified strains are less productive than the wild type yeast. The blockage of these catabolic pathways and a better availability of lipophilic carbon sources can be a solution. Because uptake, transport and storage of lipophilic molecules are very important in the production process of sophorolipids, the lipid composition of different compartments are examined and compared with other (oleaginous) yeasts. By gaining knowledge on how these molecules are converted, new metabolic engineering strategies can be found to target specific genes. The fundamental knowledge about the lipid metabolism of Starmerella bombicola will hence lead to a better production platform for the synthesis of economic valuable new-to-nature sophorolipids

Lipidomics for the production of biosurfactants

The yeast Starmerella bombicola is known for the commercial production of the biosurfactant sophorolipids. Sophorolipids are surface-active molecules consisting of a disaccharide and a fatty acyl chain. Furthermore, the yeast can utilize various lipophilic substrates such as vegetable oils, alkanes and fatty acid esters and converts them to free fatty acids; the substrate of the sophorolipids biosynthetic pathway. In addition, the yeast has a very active de novo fatty acid synthesis; also in the absence of a lipophilic carbon source reasonable amounts of sophorolipids are obtained. While the production level of sophorolipids is already high (400g/l); some efforts are still necessary for the synthesis of new-to-nature sophorolipids1. The production of these molecules by modified Starmerella bombicola strains or by using special substrates is in some cases less efficient and requires further optimization. On the one hand, the efficiency is lower because special substrates are lost due to catabolic pathways. On the other hand, some modified strains are less productive than the wild type yeast. The blockage of these catabolic pathways and a better availability of lipophilic carbon sources can be a solution. By means of lipidomics, the detailed lipid composition of the whole cell will be determined, as well as the effects of genetic modifications and different production conditions on the present lipophilic compounds. By merging lipidomics data to other information (genomics, transcriptomics and proteomics data), a metabolic network can be made. Based on this model new metabolic engineering strategies can be found to target specific genes. The new strains will then be evaluated based on growth and viability of the strain; and on the purity, stability, yield and absence of byproducts of the product. The fundamental knowledge about the lipidome of Starmerella bombicola will hence lead to a better production platform for the synthesis of economic valuable new-to-nature sophorolipids

Advancing pediatric precision healthcare : integrating rapid metabotyping as a fundamental component for 4P medicine

Introduction: The rising incidence of pediatric obesity and associated comorbidities prompts the search for effective therapeutic approaches. To address this problem, we propose 2D conventional and ambient ionization-based metabolomics and lipidomics, which possess tremendous potential in the context of weight stratification (metabotyping) and provides researchers with unparalleled insights into the complex metabolic processes of the human body. Approach: My study aims to conduct source-driven quantitative metabolite predictions, by integrating diet, microbiome, lifestyle and psychological parameters as relevant sources. The cornerstone of this framework rests upon the metabolome signatures obtained from three pediatric cohorts (MetaBEAse, FAME, and ENVIRONAGE), encompassing a total of ca. 1500 children. These signatures will be acquired using our innovative 2D-ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (2D-UHPLC-HRMS) methodology, constituting conventional consecutive metabolomics and lipidomics. When combined with the source data, these signatures will be integrated into machine learning models. Next, correlations between sources and their biomarker signatures can be obtained and serve as a basis for treatment individualization (i.e., 4P medicine). To demonstrate the feasibility of bringing 4P medicine to routine clinical practice, the potential of direct ambient ionization-based methods seems utterly suitable for large-scale point-of-care applications, especially combined with our patented customized electrospun nanofibrous sampling membrane (i.e., MetaSAMP®)1. This membrane will undergo further development into a kit design and will be integrated into our rapid laser-assisted rapid evaporative ionization MS (LA-REIMS) workflow. This rapid workflow will be correlated with our conventional 2D workflow, and a selection of source-relevant metabolites will be monitored during individualized interventions consisting of dietary and lifestyle counseling, pro-, pre- and/or synbiotic supplementation, and/or psychological therapy on a representative selection of children with overweight. Alterations in predicted metabolic signatures of source-relevant metabolites and clinical data will be used to assess the improvement in metabolic status. Results: Recent work of De Spiegeleer et al., demonstrated that the MetaSAMP®, as a short-term metabolome stabilizing substrate, offers superior metabolome coverage compared to crude biofluids, allowing significant differentiation according to weight status (normal weight vs. overweight)2. Consequently, this time- and cost-efficient rapid workflow is promising for on-the-spot health stratification and combined with the source-driven metabolome framework, may pave the way to effective routinely applicable 4P medicine. References: 1. Plekhova, V., De Windt, K., De Spiegeleer, M., De Graeve, M. & Vanhaecke, L. Recent advances in high-throughput biofluid metabotyping by direct infusion and ambient ionization mass spectrometry. TrAC Trends Anal. Chem. 117287, (2023). 2. De Spiegeleer, M., Plekhova, V., Geltmeyer, J., Schoolaert, E., Pomian, B., Singh, V., Wijnant, K., De Windt, K., Paukku, V., De Loof, A., Gies, I., Michels, N., Henauw, S. D., De Graeve, M., De Clerck, K., & Vanhaecke, L. Point-of-care applicable metabotyping using biofluid-specific electrospun MetaSAMPs directly amenable to ambient LA-REIMS. Sci. Adv. 9, (2023)