Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

Bottlenecks in the Investigation of Retinal Sterol Homeostasis.

Sterol homeostasis in mammalian cells and tissues involves balancing three fundamental processes: de novo sterol biosynthesis; sterol import (e.g., from blood-borne lipoproteins); and sterol export. In complex tissues, composed of multiple different cell types (such as the retina), import and export also may involve intratissue, intercellular sterol exchange. Disruption of any of these processes can result in pathologies that impact the normal structure and function of the retina. Here, we provide a brief overview of what is known currently about sterol homeostasis in the vertebrate retina and offer a proposed path for future experimental work to further our understanding of these processes, with relevance to the development of novel therapeutic interventions for human diseases involving defective sterol homeostasis.

Fatty acid-binding proteins 3, 7, and 8 bind cholesterol and facilitate its egress from lysosomes.

Cholesterol from low-density lipoprotein (LDL) can be transported to many organelle membranes by non-vesicular mechanisms involving sterol transfer proteins (STPs). Fatty acid-binding protein (FABP) 7 was identified in our previous study searching for new regulators of intracellular cholesterol trafficking. Whether FABP7 is a bona fide STP remains unknown. Here, we found that FABP7 deficiency resulted in the accumulation of LDL-derived cholesterol in lysosomes and reduced cholesterol levels on the plasma membrane. A crystal structure of human FABP7 protein in complex with cholesterol was resolved at 2.7 Å resolution. In vitro, FABP7 efficiently transported the cholesterol analog dehydroergosterol between the liposomes. Further, the silencing of FABP3 and 8, which belong to the same family as FABP7, caused robust cholesterol accumulation in lysosomes. These two FABP proteins could transport dehydroergosterol in vitro as well. Collectively, our results suggest that FABP3, 7, and 8 are a new class of STPs mediating cholesterol egress from lysosomes.

Novel sterol binding domains in bacteria.

Sterol lipids are widely present in eukaryotes and play essential roles in signaling and modulating membrane fluidity. Although rare, some bacteria also produce sterols, but their function in bacteria is not known. Moreover, many more species, including pathogens and commensal microbes, acquire or modify sterols from eukaryotic hosts through poorly understood molecular mechanisms. The aerobic methanotroph Methylococcus capsulatus was the first bacterium shown to synthesize sterols, producing a mixture of C-4 methylated sterols that are distinct from those observed in eukaryotes. C-4 methylated sterols are synthesized in the cytosol and localized to the outer membrane, suggesting that a bacterial sterol transport machinery exists. Until now, the identity of such machinery remained a mystery. In this study, we identified three novel proteins that may be the first examples of transporters for bacterial sterol lipids. The proteins, which all belong to well-studied families of bacterial metabolite transporters, are predicted to reside in the inner membrane, periplasm, and outer membrane of M. capsulatus, and may work as a conduit to move modified sterols to the outer membrane. Quantitative analysis of ligand binding revealed their remarkable specificity for 4-methylsterols, and crystallographic structures coupled with docking and molecular dynamics simulations revealed the structural bases for substrate binding by two of the putative transporters. Their striking structural divergence from eukaryotic sterol transporters signals that they form a distinct sterol transport system within the bacterial domain. Finally, bioinformatics revealed the widespread presence of similar transporters in bacterial genomes, including in some pathogens that use host sterol lipids to construct their cell envelopes. The unique folds of these bacterial sterol binding proteins should now guide the discovery of other proteins that handle this essential metabolite.

Direct binding to sterols accelerates endoplasmic reticulum-associated degradation of HMG CoA reductase.

The maintenance of cholesterol homeostasis is crucial for normal function at both the cellular and organismal levels. Two integral membrane proteins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and Scap, are key targets of a complex feedback regulatory system that operates to ensure cholesterol homeostasis. HMGCR catalyzes the rate-limiting step in the transformation of the 2-carbon precursor acetate to 27-carbon cholesterol. Scap mediates proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a membrane-bound transcription factor that controls expression of genes involved in the synthesis and uptake of cholesterol. Sterol accumulation triggers binding of HMGCR to endoplasmic reticulum (ER)-localized Insig proteins, leading to the enzyme's ubiquitination and proteasome-mediated ER-associated degradation (ERAD). Sterols also induce binding of Insigs to Scap, which leads to sequestration of Scap and its bound SREBP-2 in the ER, thereby preventing proteolytic activation of SREBP-2 in the Golgi. The oxygenated cholesterol derivative 25-hydroxycholesterol (25HC) and the methylated cholesterol synthesis intermediate 24,25-dihydrolanosterol (DHL) differentially modulate HMGCR and Scap. While both sterols promote binding of HMGCR to Insigs for ubiquitination and subsequent ERAD, only 25HC inhibits the Scap-mediated proteolytic activation of SREBP-2. We showed previously that 1,1-bisphosphonate esters mimic DHL, accelerating ERAD of HMGCR while sparing SREBP-2 activation. Building on these results, our current studies reveal specific, Insig-independent photoaffinity labeling of HMGCR by photoactivatable derivatives of the 1,1-bisphosphonate ester SRP-3042 and 25HC. These findings disclose a direct sterol binding mechanism as the trigger that initiates the HMGCR ERAD pathway, providing valuable insights into the intricate mechanisms that govern cholesterol homeostasis.

Combining Metabolic Engineering and Lipid Droplet Assembly to Achieve Campesterol Overproduction in Saccharomyces cerevisiae.

Campesterol is a kind of important functional food additive. Therefore, stable and efficient campesterol biosynthesis is significant. Herein, we first knocked out the sterol 22-desaturase gene in Saccharomyces cerevisiae and expressed sterol Δ7-reductase from Pangasianodon hypophthalmus, obtaining a strain that produced 6.6 mg/L campesterol. Then, the modular expression of campesterol synthesis enzymes was performed, and a campesterol titer of 88.3 mg/L was achieved. Because campesterol is a lipid-soluble macromolecule, we promoted lipid droplet formation by exploring regulatory factors, and campesterol production was improved to 169.20 mg/L. Next, triacylglycerol lipase was used to achieve compartment campesterol synthesis. After enhancing the expression of sterol Δ7-reductase and screening cations, the campesterol titer reached 438.28 mg/L in a shake flask and 1.44 g/L in a 5 L bioreactor, which represents the highest campesterol titer reported to date. Metabolic regulation combined with lipid droplet engineering may be useful for the synthesis of other steroids as well.

Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Identification of the aldolase responsible for the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from natural sterols in Mycolicibacterium smegmatis.

Mycobacterial mutants blocked in ring degradation constructed to achieve C19 synthons production, also accumulate by-products such as C22 intermediates throughout an alternative pathway reducing the production yields and complicating the downstream purification processing of final products. In this work, we have identified the MSMEG_6561 gene, encoding an aldolase responsible for the transformation of 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA (22-OH-BCN-CoA) into the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) precursor (20S)-3-oxopregn-4-ene-20-carboxaldehyde (3-OPA). The deletion of this gene increases the production yield of the C-19 steroidal synthon 4-androstene-3,17-dione (AD) from natural sterols, avoiding the production of 4-HBC as by-product and the drawbacks in the AD purification. The molar yield of AD production using the MS6039-5941-6561 triple mutant strain was checked in flasks and bioreactor improving very significantly compared with the previously described MS6039-5941 strain.

Membrane lipid modulations by methyl-ß-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating.

Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-ß-cyclodextrin (MßCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cß (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MßCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MßCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MßCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MßCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MßCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MßCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MßCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating.

Estimating the Cholesterol Affinity of Integral Membrane Proteins from Experimental Data.

The cholesterol affinities of many integral plasma membrane proteins have been estimated by molecular computation. However, these values lack experimental confirmation. We therefore developed a simple mathematical model to extract sterol affinity constants and stoichiometries from published isotherms for the dependence of the activity of such proteins on the membrane cholesterol concentration. The binding curves for these proteins are sigmoidal, with strongly lagged thresholds attributable to competition for the cholesterol by bilayer phospholipids. The model provided isotherms that matched the experimental data using published values for the sterol association constants and stoichiometries of the phospholipids. Three oligomeric transporters were found to bind cholesterol without cooperativity, with dimensionless association constants of 35 for Kir3.4* and 100 for both Kir2 and a GAT transporter. (The corresponding ΔG° values were -8.8, -11.4, and -11.4 kJ/mol, respectively). These association constants are significantly lower than those for the phospholipids, which range from ∼100 to 6000. The BK channel, the nicotinic acetylcholine receptor, and the M192I mutant of Kir3.4* appear to bind multiple cholesterol molecules cooperatively (n = 2 or 4), with subunit affinities of 563, 950, and 700, respectively. The model predicts that the three less avid transporters are approximately half-saturated in their native plasma membranes; hence, they are sensitive to variations in cholesterol in vivo. The more avid proteins would be nearly saturated in vivo. The method can be applied to any integral protein or other ligands in any bilayer for which there are reasonable estimates of the sterol affinities and stoichiometries of the phospholipids.

Surfactin and Capric Acid Affect the Posaconazole Susceptibility of Candida albicans Strains with Altered Sterols and Sphingolipids Biosynthesis.

Infections caused by Candida spp. pose a continuing challenge for modern medicine, due to widespread resistance to commonly used antifungal agents (e.g., azoles). Thus, there is considerable interest in discovering new, natural compounds that can be used in combination therapy with conventional antibiotics. Here, we investigate whether the natural compounds surfactin and capric acid, in combination with posaconazole, enhance the growth inhibition of C. albicans strains with alterations in sterols and the sphingolipids biosynthesis pathway. We demonstrate that combinations of posaconazole with surfactin or capric acid correspond with the decreased growth of C. albicans strains. Moreover, surfactin and capric acid can independently contribute to the reduced adhesion of C. albicans strains with altered ergosterol biosynthesis to abiotic surfaces (up to 90% reduction in adhesion). A microscopic study of the C. albicans plasma membrane revealed that combinations of those compounds do not correspond with the increased permeabilization of the plasma membrane when compared to cells treated with posaconazole alone. This suggests that the fungistatic effect of posaconazole in combination with surfactin or capric acid is related to the reduction in adhesion of C. albicans.

Application of Miscanthus substrates in the cultivation of Ganoderma lingzhi.

Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.

[Identification of a new C-23 metabolite in sterol degradation of Mycobacterium neoaurum HGMS2 and analysis of its metabolic pathways].

Mycobacterium neoaurum has the ability to produce steroidal intermediates known as 22-hydroxy-23, 24-bisnorchol-4-en-3-one (BA) upon the knockout of the genes for either the hydroxyacyl-CoA dehydrogenase (Hsd4A) or acyl-CoA thiolase (FadA5). In a previous study, we discovered a novel metabolite in the fermentation products when the fadA5 gene was deleted. This research aims to elucidate the metabolic pathway of this metabolite through structural identification, homologous sequence analysis of the fadA5 gene, phylogenetic tree analysis of M. neoaurum HGMS2, and gene knockout. Our findings revealed that the metabolite is a C23 metabolic intermediate, named 24-norchol-4-ene-3, 22-dione (designated as 3-OPD). It is formed when a thioesterase (TE) catalyzes the formation of a ß-ketonic acid by removing CoA from the side chain of 3, 22-dioxo-25, 26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA), followed by spontaneously undergoing decarboxylation. These results have the potential to contribute to the development of novel steroid intermediates.

Fully activated structure of the sterol-bound Smoothened GPCR-Gi protein complex.

Smoothened (SMO) is an oncoprotein and signal transducer in the Hedgehog signaling pathway that regulates cellular differentiation and embryogenesis. As a member of the Frizzled (Class F) family of G protein-coupled receptors (GPCRs), SMO biochemically and functionally interacts with Gi family proteins. However, key molecular features of fully activated, G protein-coupled SMO remain elusive. We present the atomistic structure of activated human SMO complexed with the heterotrimeric Gi protein and two sterol ligands, equilibrated at 310 K in a full lipid bilayer at physiological salt concentration and pH. In contrast to previous experimental structures, our equilibrated SMO complex exhibits complete breaking of the pi-cation interaction between R4516.32 and W5357.55, a hallmark of Class F receptor activation. The Gi protein couples to SMO at seven strong anchor points similar to those in Class A GPCRs: intracellular loop 1, intracellular loop 2, transmembrane helix 6, and helix 8. On the path to full activation, we find that the extracellular cysteine-rich domain (CRD) undergoes a dramatic tilt, following a trajectory suggested by positions of the CRD in active and inactive experimental SMO structures. Strikingly, a sterol ligand bound to a shallow transmembrane domain (TMD) site in the initial structure migrates to a deep TMD pocket found exclusively in activator-bound SMO complexes. Thus, our results indicate that SMO interacts with Gi prior to full activation to break the molecular lock, form anchors with Gi subunits, tilt the CRD, and facilitate migration of a sterol ligand in the TMD to an activated position.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Sterol interactions influence the function of Wsc sensors.

The Wsc1, Wsc2, and Wsc3 proteins are essential cell surface sensors that respond to cell wall perturbation by activating the cell wall integrity pathway (CWIP). We show here that in situ production of cholesterol (in place of ergosterol) induces hyper-phosphorylation of Slt2, the MAPK of the CWIP, and upregulates cell wall biosynthesis. Deletion of all three Wsc genes in K. phaffii reverts these phenotypes. In the cholesterol-producing strain, both Wsc1 and Wsc3 accumulate in the plasma membrane. Close inspection of the transmembrane domains of all three Wsc proteins predicted by AlphaFold2 revealed the presence of CRAC sterol-binding motifs. Experiments using a photoreactive cholesterol derivative indicate intimate interaction of this sterol with the Wsc transmembrane domain, and this apparent sterol binding was abrogated in Wsc mutants with substitutions in the CRAC motif. We also observed cholesterol interaction with CRAC-like motifs in the transmembrane domains of mammalian integrins, analogs of Wsc proteins. Our results suggest that proper signaling of the Wsc sensors requires highly specific binding of the native endogenous terminal sterol, ergosterol.

Chitosan-Based Sustained Expression of Sterol Regulatory Element-Binding Protein 1a Stimulates Hepatic Glucose Oxidation and Growth in Sparus aurata.

The administration of a single dose of chitosan nanoparticles driving the expression of sterol regulatory element-binding protein 1a (SREBP1a) was recently associated with the enhanced conversion of carbohydrates into lipids. To address the effects of the long-lasting expression of SREBP1a on the growth and liver intermediary metabolism of carnivorous fish, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid expressing the N terminal active domain of hamster SREBP1a (pSG5-SREBP1a) were injected intraperitoneally every 4 weeks (three doses in total) to gilthead sea bream (Sparus aurata) fed high-protein-low-carbohydrate and low-protein-high-carbohydrate diets. Following 70 days of treatment, chitosan-TPP-pSG5-SREBP1a nanoparticles led to the sustained upregulation of SREBP1a in the liver of S. aurata. Independently of the diet, SREBP1a overexpression significantly increased their weight gain, specific growth rate, and protein efficiency ratio but decreased their feed conversion ratio. In agreement with an improved conversion of dietary carbohydrates into lipids, SREBP1a expression increased serum triglycerides and cholesterol as well as hepatic glucose oxidation via glycolysis and the pentose phosphate pathway, while not affecting gluconeogenesis and transamination. Our findings support that the periodical administration of chitosan-TPP-DNA nanoparticles to overexpress SREBP1a in the liver enhanced the growth performance of S. aurata through a mechanism that enabled protein sparing by enhancing dietary carbohydrate metabolisation.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Secretory production of 7-dehydrocholesterol by engineered Saccharomyces cerevisiae.

BACKGROUND: 7-Dehydrocholesterol (7-DHC) can be directly converted to vitamin D3 by UV irradiation and de novo synthesis of 7-DHC in engineered Saccharomyces cerevisiae has been recognized as an attractive substitution to traditional chemical synthesis. Introduction of sterol extracellular transport pathway for the secretory production of 7-DHC is a promising approach to achieve higher titer and simplify the downstream purification processing. METHODS AND RESULTS: A series of genes involved in ergosterol pathway were combined reinforced and reengineered in S. cerevisiae. A biphasic fermentation system was introduced and 7-DHC was found to be enriched in oil-phase with an increased titer by 1.5-folds. Quantitative PCR revealed that say1, atf2, pdr5, pry1-3 involved in sterol storage and transport were all significantly induced in sterol overproduced strain. To enhance the secretion capacity, lipid transporters of pathogen-related yeast proteins (Pry), Niemann-Pick disease type C2 (NPC2), ATP-binding cassette (ABC)-family, and their homologues were screened. Both individual and synergetic overexpression of Plant pathogenesis Related protein-1 (Pr-1) and Sterol transport1 (St1) largely increased the de novo biosynthesis and secretory productivity of 7-DHC, and the final titer reached 28.2 mg g-1 with a secretion ratio of 41.4%, which was 26.5-folds higher than the original strain. In addition, the cooperation between Pr-1 and St1 in sterol transport was further confirmed by confocal microscopy, molecular docking, and directed site-mutation. CONCLUSION: Selective secretion of different sterol intermediates was characterized in sterol over-produced strain and the extracellular export of 7-DHC developed in present study significantly improved the cell biosynthetic capacity, which offered a novel modification idea for 7-DHC de novo biosynthesis by S. cerevisiae cell factory.

Simultaneous structural replacement of the sphingoid long-chain base and sterol in budding yeast.

The basic structures of membrane lipids that compose biomembranes differ among species; i.e., in mammals, the primary structure of long-chain base (LCB), the common backbone of ceramides and complex sphingolipids, is sphingosine, whereas, in yeast Saccharomyces cerevisiae, it is phytosphingosine, and S. cerevisiae does not have sphingosine. In addition, the sterol, which is coordinately involved in various functions with complex sphingolipids, is cholesterol in mammals, while in yeast it is ergosterol. Previously, it was found that yeast cells are viable when the structure of LCBs is replaced by sphingosine by supplying an exogenous LCB to cells lacking LCB biosynthesis. Here, we characterized yeast cells having sphingosine instead of phytosphingosine (sphingosine cells). Sphingosine cells exhibited a strong growth defect when biosynthesis of ceramides or complex sphingolipids was inhibited, indicating that, in the sphingosine cells, exogenously added sphingosine is required to be further metabolized. The sphingosine cells exhibited hypersensitivity to various environmental stresses and had abnormal plasma membrane and cell wall properties. Furthermore, we also established a method for simultaneous replacement of both LCB and sterol structures with those of mammals (sphingosine/cholesterol cells). The multiple stress hypersensitivity and abnormal plasma membrane and cell wall properties observed in sphingosine cells were also observed in sphingosine/cholesterol cells, suggesting that simultaneous replacement of both LCB and sterol structures with those of mammals cannot prevent these abnormal phenotypes. This is the first study to our knowledge showing that S. cerevisiae can grow even if LCB and sterol structures are simultaneously replaced with mammalian types.