The oleaginous yeast Starmerella bombicola reveals limitations of Saccharomyces cerevisiae as a model for fatty acid transport studies.

Saccharomyces cerevisiae is the model organism to most yeast researchers, and information obtained from its physiology is generally extrapolated to other yeasts. Studies on fatty acid transport in S. cerevisiae are based on the expression of both native fatty acid export genes as well as heterologous proteins. Starmerella bombicola, on the other hand, is an oleaginous yeast of industrial relevance but its fatty acid transport mechanisms are unknown. In this study, we attempt to use existing knowledge from S. cerevisiae to study fatty acid transport in S. bombicola, but the obtained results differ from those observed in S. cerevisiae. First, we observed that deletion of SbPRY1 in S. bombicola leads to higher fatty acid export, the opposite effect to the one previously observed for the Pry homologues in S. cerevisiae. Second, following reports that human FATP1 could export fatty acids and alcohols in S. cerevisiae, we expressed FATP1 in a fatty acid-accumulating S. bombicola strain. However, FATP1 reduced fatty acid export in S. bombicola, most likely due to its acyl-CoA synthetase activity. These results not only advance knowledge on fatty acid physiology of S. bombicola, but also improve our understanding of S. cerevisiae and its limitations as a model organism.

Exploring the transportome of the biosurfactant producing yeast Starmerella bombicola.

Starmerella bombicola is a non-conventional yeast mainly known for its capacity to produce high amounts of the glycolipids 'sophorolipids'. Although its product has been used as biological detergent for a couple of decades, the genetics of S. bombicola are still largely unknown. Computational analysis of the yeast's genome enabled us to identify 254 putative transporter genes that make up the entire transportome. For each of them, a potential substrate was predicted using homology analysis, subcellular localization prediction and RNA sequencing in different stages of growth. One transporter family is of exceptional importance to this yeast: the ATP Binding Cassette (ABC) transporter Superfamily, because it harbors the main driver behind the highly efficient sophorolipid export. Furthermore, members of this superfamily translocate a variety of compounds ranging from antibiotics to hydrophobic molecules. We conducted an analysis of this family by creating deletion mutants to understand their role in the export of hydrophobic compounds, antibiotics and sophorolipids. Doing this, we could experimentally confirm the transporters participating in the efflux of medium chain fatty alcohols, particularly decanol and undecanol, and identify a second sophorolipid transporter that is located outside the sophorolipid biosynthetic gene cluster.

Identification and importance of mitochondrial citrate carriers and ATP citrate lyase for glycolipid production in Starmerella bombicola.

Starmerella bombicola is a non-conventional yeast commercially used as a microbial cell factory for sophorolipid production. Sophorolipids are glycolipid biosurfactants composed of a glucose disaccharide sophorose and a fatty acid. In de novo sophorolipid synthesis, the fatty acid moiety is derived from the fatty acid synthesis (FAS) complex; therefore, the yeast's lipid metabolism plays a crucial role in sophorolipid biosynthesis. As a fatty acid precursor, citric acid is a key primary metabolite that connects carbohydrate and lipid metabolism, and in S. bombicola, it also has a regulatory effect on sophorolipid composition and productivity. We aimed to identify the mitochondrial transporters involved in citrate shuttling and the ATP citrate lyase (Acl), the enzyme that converts citric acid into acetyl-CoA. Subsequently, we studied their role in the citric acid shuttle and glycolipid synthesis and the potential of citrate metabolism as a genetic manipulation target for increased glycolipid synthesis. Bioinformatics analyses predicted 32 mitochondrial carriers of which two were identified as citrate transporters, named SbCtp1 and SbYhm2. Deletion of these mitochondrial carriers led to a lesser sophorolipid yield and a shift in the lactonic/acidic sophorolipid ratio. However, only the knockout of SbYhm2 caused a decrease of citric and an increase of malic acid extracellular concentrations. Additionally, deletion of SbAcl1 had a negative effect on S. bombicola's specific growth rate and sophorolipid synthesis and contributed to extra- and intracellular citric acid accumulation. Unexpectedly, SbAcl1 overexpression also decreased glycolipid production.Key Points• Starmerella bombicola is an industrially relevant microbial cell factory for biosurfactant production.• There are 32 predicted mitochondrial carriers in S. bombicola.• Citrate mitochondrial carriers SbYhm2 and SbCtp1 are essential for glycolipid synthesis in S. bombicola.• Deletion of SbAcl1 negatively affects growth and sophorolipid production in S. bombicola. Graphical abstract.

Redirecting the lipid metabolism of the yeast Starmerella bombicola from glycolipid to fatty acid production.

Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and cosmetics. Microbial fatty acid biosynthesis has gained much attention as it provides a sustainable alternative for petrol- and plant oil-derived chemicals. The yeast Starmerella bombicola is a microbial cell factory that naturally employs its powerful lipid metabolism for the production of the biodetergents sophorolipids (> 300 g/L). However, in this study we exploit the lipidic potential of S. bombicola and convert it from the glycolipid production platform into a free fatty acid cell factory. We used several metabolic engineering strategies to promote extracellular fatty acid accumulation which include blocking competing pathways (sophorolipid biosynthesis and ß-oxidation) and preventing free fatty acid activation. The best producing mutant (Δcyp52m1Δfaa1Δmfe2) secreted 0.933 g/L (± 0.04) free fatty acids with a majority of C18:1 (43.8%) followed by C18:0 and C16:0 (40.0 and 13.2%, respectively). Interestingly, deletion of SbFaa1 in a strain still producing sophorolipids also resulted in 25% increased de novo sophorolipid synthesis (P = 0.0089) and when oil was supplemented to the same strain, a 50% increase in sophorolipid production was observed compared to the wild type (P = 0.03). We believe that our work is pivotal for the further development and exploration of S. bombicola as a platform for synthesis of environmentally friendly oleochemicals.

Protein-facilitated transport of hydrophobic molecules across the yeast plasma membrane.

In yeasts, the plasma membrane forms the barrier that protects the cell from the outside world, but also gathers and keeps valuable compounds inside. Although it is often suggested that hydrophobic molecules surpass this checkpoint by simple diffusion, it now becomes evident that protein-facilitated transport mechanisms allow for selective import and export of triglycerides, fatty acids, alkanes, and sterols in yeasts. During biomass production, hydrophobic carbon sources enter and exit the cell efficiently in a strictly regulated manner that helps avoid toxicity. Furthermore, various molecules, such as yeast pheromones, secondary metabolites and xenobiotics, are exported to ensure cell-cell communication, or increase chances of survival. This review summarizes the current knowledge on how hydrophobic compounds interact with protein-facilitated transport systems on the plasma membrane and how selective import and export across the yeast plasma membrane is achieved. Both the model organism Saccharomyces cerevisiae, as well as unconventional yeasts are discussed.

Enteral nutrition in cystic fibrosis

Introduction: Cystic fibrosis is a genetically determined disease. It is currently detected right after birth thanks to a screening program. This early detection allows for quick treatment inclusion. Cystic fibrosis therapy has a comprehensive character. The way of nutrition is also very important. The aim of this study was to evaluate the benefits and risks of enteral nutrition in patients with cystic fibrosis. Material and methods: 53 people with cystic fibrosis participated in the study (15 men, 38 women). Qualification for the study required the fulfillment of three conditions: enteral nutrition used for at least one month, no increase in body weight when using a high energy diet, underweight. The research was carried with use of self-constructed questionnaire. Results: The mean age of the respondents was 19.9 ± 4 years. The median weight gain after enteral nutrition was 7 kg. Most (n = 42, 79.2%) did not report any side effects. Few reported local infections (n = 9; 17%) or prolapse of the balloon (n = 2, 3.8%). On the other hand, everyone reported benefits - it was mainly weight gain and improved quality of life. In some cases (n = 22, 41.5%) with the use of enteral nutrition, a decrease in the frequency of respiratory infections was observed. Conclusions: Enteral nutrition is a beneficial method in those patients with cystic fibrosis, in whom high-energy oral nutrition is inefficient.

Yeast glycolipid biosurfactants.

Various yeasts, both conventional and exotic ones, are known to produce compounds useful to mankind. Ethanol is the most known of these compounds, but more complex molecules such as amphiphilic biosurfactants can also be derived from eukaryotic microorganisms at an industrially and commercially relevant scale. Among them, glycolipids are the most promising, due to their attractive properties and high product titers. Many of these compounds can be considered as secondary metabolites with a specific function for the host. Hence, a dedicated biosynthetic process enables regulation and combines pathways delivering the lipidic moiety and the hydrophilic carbohydrate part of the glycolipid. In this Review, we will discuss the biosynthetic and regulatory aspects of the yeast-derived sophorolipids, mannosylerythritol lipids, and cellobiose lipids, with special emphasis on the relation between glycolipid synthesis and the general lipid metabolism.

Crossing boundaries: the importance of cellular membranes in industrial biotechnology.

How small molecules cross cellular membranes is an often overlooked issue in an industrial microbiology and biotechnology context. This is to a large extent governed by the technical difficulties to study these transport systems or by the lack of knowledge on suitable efflux pumps. This review emphasizes the importance of microbial cellular membranes in industrial biotechnology by highlighting successful strategies of membrane engineering towards more resistant and hence better performing microorganisms, as well as transporter and other engineering strategies for increased efflux of primary and secondary metabolites. Furthermore, the benefits and limitations of eukaryotic subcellular compartmentalization are discussed, as well as the biotechnological potential of membrane vesicles.