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As microbial membranes are naturally impermeable to even the smallest biomolecules, transporter proteins are physiologically essential for normal cell functioning. This makes transporters a key target area for engineering enhanced cell factories. As part of the wider cellular transportome, aquaporins (AQPs) are responsible for transporting small polar solutes, encompassing many compounds which are of great interest for industrial biotechnology, including cell feedstocks, numerous commercially relevant polyols and even weak organic acids. In this review, examples of cell factory engineering by targeting AQPs are presented. These AQP modifications aid in redirecting carbon fluxes and boosting bioconversions either by enhanced feedstock uptake, improved intermediate retention, increasing product export into the media or superior cell viability against stressors with applications in both bacterial and yeast production platforms. Additionally, the future potential for AQP deployment and targeting is discussed, showcasing hurdles and considerations of this strategy as well as recent advances and future directions in the field. By leveraging the natural diversity of AQPs and breakthroughs in channel protein engineering, these transporters are poised to be promising tools capable of enhancing a wide variety of biotechnological processes.

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Cellobiose lipids are surface-active compounds or biological detergents produced by distinct Basidiomycetes yeasts, of which the most and best-described ones belong to the Ustilaginomycetes class. The molecules display slight variation in congener type, which is linked to the hydroxylation position of the long fatty acid, acetylation profile of the cellobiose unit, and presence or absence of the short fatty acid. In general, this variation is strain specific. Although cellobiose lipid biosynthesis has been described for about 11 yeast species, hitherto only two types of biosynthetic gene clusters are identified, and this for only three species. This work adds six more biosynthetic gene clusters and describes for the first time a novel type of cellobiose lipid biosynthetic cluster with a simplified architecture related to specific cellobiose lipids synthesized by Trichosporonaceae family members.

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Lipid binding domains and protein lipidations are essential features to recruit proteins to intracellular membranes, enabling them to function at specific sites within the cell. Membrane association can also be exploited to answer fundamental and applied research questions, from obtaining insights into the understanding of lipid metabolism to employing them for metabolic engineering to redirect fluxes. This review presents a broad catalog of membrane binding strategies focusing on the plasma membrane of Saccharomyces cerevisiae. Both lipid binding domains (pleckstrin homology, discoidin-type C2, kinase associated-1, basic-rich and bacterial phosphoinositide-binding domains) and co- and post-translational lipidations (prenylation, myristoylation and palmitoylation) are introduced as tools to target the plasma membrane. To provide a toolset of membrane targeting modules, respective candidates that facilitate plasma membrane targeting are showcased including their in vitro and in vivo properties. The relevance and versatility of plasma membrane targeting modules are further highlighted by presenting a selected set of use cases.

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Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.

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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.

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Oleaginous yeasts are typically defined as those able to accumulate more than 20% of their cell dry weight as lipids or triacylglycerides. Research on these yeasts has increased lately fuelled by an interest to use biotechnology to produce lipids and oleochemicals that can substitute those coming from fossil fuels or offer sustainable alternatives to traditional extractions (e.g., palm oil). Some oleaginous yeasts are attracting attention both in research and industry, with Yarrowia lipolytica one of the best-known and studied ones. Oleaginous yeasts can be found across several clades and different metabolic adaptations have been found, affecting not only fatty acid and neutral lipid synthesis, but also lipid particle stability and degradation. Recently, many novel oleaginous yeasts are being discovered, including oleaginous strains of the traditionally considered non-oleaginous Saccharomyces cerevisiae. In the face of this boom, a closer analysis of the definition of "oleaginous yeast" reveals that this term has instrumental value for biotechnology, while it does not give information about distinct types of yeasts. Having this perspective in mind, we propose to expand the term "oleaginous yeast" to those able to produce either intracellular or extracellular lipids, not limited to triacylglycerides, in at least one growth condition (including ex novo lipid synthesis). Finally, a critical look at Y. lipolytica as a model for oleaginous yeasts shows that the term "oleaginous" should be reserved only for strains and not species and that in the case of Y. lipolytica, it is necessary to distinguish clearly between the lipophilic and oleaginous phenotype.

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Sophorolipids are glycolipid biosurfactants consisting of a carbohydrate sophorose head with a fatty acid tail and exist in either an acidic or lactonic form. Sophorolipids are gaining interest as potential cancer chemotherapeutics due to their inhibitory effects on a range of tumour cell lines. Currently, most anti-cancer studies reporting the effects of sophorolipids have focused on lactonic preparations with the effects of acidic sophorolipids yet to be elucidated. We produced a 94% pure acidic sophorolipid preparation which proved to be non-toxic to normal human colonic and lung cells. In contrast, we observed a dose-dependent reduction in viability of colorectal cancer lines treated with the same preparation. Acidic sophorolipids induced apoptosis and necrosis, reduced migration, and inhibited colony formation in all cancer cell lines tested. Furthermore, oral administration of 50 mg kg-1 acidic sophorolipids over 70 days to Apcmin+/- mice was well tolerated and resulted in an increased haematocrit, as well as reducing splenic size and red pulp area. Oral feeding did not affect tumour numbers or sizes in this model. This is the first study to show that acidic sophorolipids dose-dependently and specifically reduces colon cancer cell viability in addition to reducing tumour-associated bleeding in the Apcmin+/- mouse model. KEY POINTS: • Acidic sophorolipids are produced by yeast species such as Starmerella bombicola. • Acidic sophorolipids selectively killed colorectal cells with no effect on healthy gut epithelia. • Acidic sophorolipids reduced tumour-associated gut bleed in a colorectal mouse model.

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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.

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Biological membranes are inherently complex, making transport processes in microbial cell factories a significant bottleneck. Lack of knowledge on transport proteins' characteristics and the need for advanced technical equipment often hamper transporter identification and optimization. For these reasons, moving away from individual characterization and towards high-throughput mining, engineering, and screening of transporters is an increasingly attractive approach. Superior transporters can be selected from large libraries by coupling their activity to growth, for substrates that function as feedstocks or toxic compounds. Other compounds can be screened thanks to recent advances in the design and deployment of synthetic genetic circuits (biosensors). Furthermore, novel strategies are rapidly increasing the repertoire of biomolecule transporters susceptible to high-throughput selection methods.

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The global pandemic of COVID-19 has forced educational provision to suddenly shift to a digital environment all around the globe. During these extraordinary times of teaching and learning both the challenges and the opportunities of embedding technologically enhanced education permanently became evident. Even though reinforced by constraints due to the pandemic, teaching through digital tools increases the portfolio of approaches to reach learning outcomes in general. In order to reap the full benefits, this Minireview displays various initiatives and tools for distance education in the area of Synthetic Biology in higher education while taking into account specific constraints of teaching Synthetic Biology from a distance, such as collaboration, laboratory and practical experiences. The displayed teaching resources can benefit current and future educators and raise awareness about a diversified inventory of teaching formats as a starting point to reflect upon one's own teaching and its further advancement.

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Fatty acid metabolism has been widely studied in various organisms. However, fatty acid transport has received less attention, even though it plays vital physiological roles, such as export of toxic free fatty acids or uptake of exogenous fatty acids. Hence, there are important knowledge gaps in how fatty acids cross biological membranes, and many mechanisms and proteins involved in these processes still need to be determined. The lack of information is more predominant in microorganisms, even though the identification of fatty acids transporters in these cells could lead to establishing new drug targets or improvements in microbial cell factories. This review provides a thorough analysis of the current information on fatty acid transporters in microorganisms, including bacteria, yeasts and microalgae species. Most available information relates to the model organisms Escherichia coli and Saccharomyces cerevisiae, but transport systems of other species are also discussed. Intracellular trafficking of fatty acids and their transport through organelle membranes in eukaryotic organisms is described as well. Finally, applied studies and engineering efforts using fatty acids transporters are presented to show the applied potential of these transporters and to stress the need for further identification of new transporters and their engineering.

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Several microorganisms are currently being used as production platform for glycolipid biosurfactants, providing a greener alternative to chemical biosurfactants. One of the reasons why these processes are commercially competitive is the fact that microbial producers can efficiently export their product to the extracellular environment, reaching high product titers. Glycolipid biosynthetic genes are often found in a dedicated cluster, amidst which genes encoding a dedicated transporter committed to shuttle the glycolipid to the extracellular environment are often found, as is the case for many other secondary metabolites. Knowing this, one can rely on gene clustering features to screen for novel putative transporters, as described and performed in this review. The above strategy proves to be very powerful to identify glycolipid transporters in fungi but is less valid for bacterial systems. Indeed, the genetics of these export systems are currently largely unknown, but some hints are given. Apart from the direct export of the glycolipid, several other transport systems have an indirect effect on glycolipid production. Specific importers dictate which hydrophilic and hydrophobic substrates can be used for production and influence the final yields. In eukaryotes, cellular compartmentalization allows the assembly of glycolipid building blocks in a highly specialized and efficient way. Yet, this requires controlled transport across intracellular membranes. Next to the direct export of glycolipids, the current state of the art regarding this indirect involvement of transporter systems in microbial glycolipid synthesis is summarized in this review. KEY POINTS: • Transporters are directly and indirectly involved in microbial glycolipid synthesis. • Yeast glycolipid transporters are found in their biosynthetic gene cluster. • Hydrophilic and hydrophobic substrate uptake influence microbial glycolipid synthesis.

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The demand for microbially produced surface-active compounds for use in industrial processes and products is increasing. As such, there has been a comparable increase in the number of publications relating to the characterization of novel surface-active compounds: novel producers of already characterized surface-active compounds and production processes for the generation of these compounds. Leading researchers in the field have identified that many of these studies utilize techniques are not precise and accurate enough, so some published conclusions might not be justified. Such studies lacking robust experimental evidence generated by validated techniques and standard operating procedures are detrimental to the field of microbially produced surface-active compound research. In this publication, we have critically reviewed a wide range of techniques utilized in the characterization of surface-active compounds from microbial sources: identification of surface-active compound producing microorganisms and functional testing of resultant surface-active compounds. We have also reviewed the experimental evidence required for process development to take these compounds out of the laboratory and into industrial application. We devised this review as a guide to both researchers and the peer-reviewed process to improve the stringency of future studies and publications within this field of science.

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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.

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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.

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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.

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Twelve new quaternary ammonium sophorolipids with long alkyl chains on the nitrogen atom were synthesized starting from oleic and petroselinic acid-based sophorolipids. These novel derivatives were evaluated for their antimicrobial activity against selected Gram-negative and Gram-positive bacteria and their transfection efficacies on three different eukaryotic cell lines in vitro as good activities were demonstrated for previously synthesized derivatives. Self-assembly properties were also evaluated. All compounds proved to possess antimicrobial and transfection properties, and trends could be observed based on the length of the nitrogen substituent and the total length of the sophorolipid tail. Moreover, all long-chain quaternary ammonium sophorolipids form micelles, which proved to be a prerequisite to induce antimicrobial activity and transfection capacity. These results are promising for future healthcare applications of long-chained quaternary ammonium sophorolipids.

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Conventional head-chain but also more exotic divalent, Gemini, or bolaform amphiphiles have in common well-defined hydrophilic and hydrophobic blocks with often a predictable self-assembly behavior. However, new categories of amphiphiles, such as microbial biosurfactants, challenge such conventional understanding because of the poorly defined boundaries between the hydrophilic and hydrophobic portions. Microbial glycolipids, such as sophorolipids, rhamnolipids, or cellobioselipids, interesting biodegradable, nontoxic, alternatives to synthetic surfactants, all represent interesting examples of atypical amphiphiles with partially predictable self-assembly properties. However, their limited molecular diversity strongly limits their application potential. For this reason, we used them as ready-made platform to prepare a whole class of new derivatives. In particular, a broad range of amino derivatives of sophorolipid biosurfactant was recently prepared with the goal of producing biobased antimicrobial and transfection agents, of which the efficiency strongly depends on their molecular structure and unpredictable self-assembly behavior. The new compounds contain a set of asymmetrical and symmetrical bolaamphiphiles, the latter with three or four hydrophilic centers, divalent amphiphiles with asymmetric polar headgroups and even Y-shaped amphiphiles, bearing two sophorose groups connected to one nitrogen atom. In this contribution, we employ small-angle X-ray scattering to establish a relationship between their peculiar molecular structures and the self-assembly properties in water. We find that all divalent and Y-shaped compounds form micelles, of which the hydrophilic shell is composed of a bulky sophorose-C x( x = 8,11)-amine moiety, with aggregation numbers between 30 and 100. On the contrary, most symmetrical and asymmetrical bolaamphiphiles display poor self-assembly properties, generally showing aggregation numbers below 20, especially in the presence of either short spacers or large spacers containing hydrophilic centers.

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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.

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Sophorolipids are well-known biosurfactants produced by yeasts, having potential applications ranging from nanomaterials, medicine, and cosmetics to large-volume applications such as cleaning and soil remediation. Because of their environmentally friendly nature, they attained much interest during the past decades as a sustainable and ecological alternative to petroleum-derived surfactants. Stronger yet, research activities and scientific publications on the topic are ever increasing. However, often these studies lack proper producer strain identification and detailed structural product characterization. Flaws regarding strain identity can have huge consequences when moving to valorization and moreover tend to persist quite long in scientific literature. Furthermore, too often sophorolipid production is proposed where other biosurfactant structures cannot be ruled out based on the chemical analysis. Finally, absolute quantitative yield determination frequently occurs with variable product purity and without proper calibration standards. This review aims to highlight and discuss these discrepancies and proposes some guidelines for good practice in future sophorolipid research.

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