Ω-hydroxy Fatty Acids Research Articles

Lipidomic, Transcriptomic, and BSA-660K Single Nucleotide Polymorphisms Profiling Reveal Characteristics of the Cuticular Wax in Wheat.

Plant epidermal wax helps protect plants from adverse environmental conditions, maintains the function of tissues and organs, and ensures normal plant development. However, the constituents of epidermal wax and the regulatory mechanism of their biosynthesis in wheat have not been fully understood. Wheat varieties with different wax content, Jinmai47 and Jinmai84, were selected to comparatively analyze their waxy components and genetic characteristics, using a combination of lipidomic, transcriptomic, and BSA-Wheat 660K chip analysis. Through lipidomic analysis, 1287 lipid molecules were identified representing 31 lipid subclasses. Among these, Diacylglycerols (DG), (O-acyl)-ω-hydroxy fatty acids (OAHFA), wax ester (WE), Triacylglycerols (TG), and Monoradylglycerols (MG) accounted for 96.4% of the total lipids in Jinmai84 and 94.5% in Jinmai47. DG, OAHFA, and WE were higher in Jinmai84 than in Jinmai47 with the content of OAHFA 2.88-fold greater and DG 1.66-fold greater. Transcriptome sequence and bioinformatics analysis revealed 63 differentially expressed genes related to wax biosynthesis. Differentially expressed genes (DEGs) were found to be involved with the OAHFA, DG, and MG of synthesis pathways, which enriched the wax metabolism pathway. Non-glaucous and glaucous bulks from a mapping population were used to identify single nucleotide polymorphisms (SNP) via 660K chip analysis. Two loci centered on chromosomes 2D and 4B were detected and the locus on 4B is likely novel. These data improve understanding of complex lipid metabolism for cuticular wax biosynthesis in wheat and lay the foundation for future detailed investigation of mechanisms regulating wax metabolism.

Human meibum and tear film derived cholesteryl and wax esters in meibomian gland dysfunction and tear film structure

PurposeThis study evaluated the presence and roles of cholesteryl esters (CEs) and wax esters (WEs) from human tear film and meibum in meibomian gland dysfunction (MGD). MethodsOut of 195 enrolled subjects, 164 and 179 subjects provided tear and meibum samples, respectively. Subjects were classified into normal, asymptomatic MGD, MGD, and mixed (MGD & aqueous deficient). The precorneal tear film (PCTF) thinning rate (evaporation) was measured using optical coherence tomography. Lipids extracted from tear and meibum samples were infused into a SCIEX 5600 TripleTOF mass spectrometer. CE and WE intensities quantified with Analyst 1.7 TF and LipidView 1.3 were compared across disease groups in MetaboAnalyst 5.0 and correlated with PCTF thinning rates. ResultsThe numbers of unique CEs and WEs identified in the samples were 125 and 86, respectively. Unsupervised Principal Component (PC) analysis and supervised Partial Least Square Discriminant analysis exhibited little separation among groups for both CEs and WEs in tears and meibum. Spearman's correlation analyses showed no association between either the first or second PC scores with PCTF thinning rates. ConclusionThe abundances of human PCTF and meibum-derived CEs and WEs were independent of MGD disease status and PCTF thinning (evaporation). CEs and WEs alterations do not contribute to alterations in tear film dynamics in MGD, such as has been demonstrated by the (O-acyl) ω-hydroxy fatty acids (OAHFAs).

Lipidomics Analysis of the Tears in the Patients Receiving LASIK, FS-LASIK, or SBK Surgery.

Purpose: Tear film lipid layer (TFLL) plays a vital role in maintaining the tear film stability and, thus, the lipid composition of the tears could greatly affect the physiological function and biophysical integrity of the tear film. The objective of this study is to assess the tear lipid composition of the patients receiving laser-assisted in situ keratomileusis (LASIK), femtosecond LASIK (FS-LASIK), or sub-Bowman's keratomileusis (SBK) surgery preoperatively and postoperatively.Methods: Tear samples were collected from the left eye of the patient who receiving LASIK (n = 10), FS-LASIK (n = 10), or SBK (n = 10) surgery in week 0, week 1, week 4, and week 52. A rapid direct injection shotgun lipidomics workflow, MS/MSALL (<2 min/sample), was applied to examine the tear lipidome.Results: In week 52, the SBK group demonstrated a similar lipidome profile compared to week 0, while the FS-LASIK and LASIK groups shifted away from week 0. Two lipids, ganglioside (GD3) 27:4 and triacylglycerol (TAG) 59:3, were found to be associated with the lipidome changes preoperatively and postoperatively. No statistical significance was found in the overall lipid classes from the FS-LASIK group. The LASIK group showed significant alteration in the phospholipid and sphingolipid over time, while the SBK group demonstrated a significant difference in the (O-acyl)-ω-hydroxy fatty acid (OAHFA) and phospholipid.Conclusion: LASIK showed the greatest impact on the tear lipidome changes over time, while SBK demonstrated minimal impact among the three types of refractive surgeries after 1 year.

Alteration in meibum lipid composition and subjective symptoms due to aging and meibomian gland dysfunction

PurposeTo investigate the alteration in lipid composition of meibum, objective clinical signs, and subjective symptoms associated with aging and meibomian gland (MG) dysfunction (MGD). MethodsIn 10 MGD patients [4 males/6 females, mean age: 65.6 ± 7.9 years (range: 50–79 years)] and 24 healthy volunteer subjects [young subjects: 6 males/6 females, mean age: 25.7 ± 3.8 years (range: 20–35 years), elderly subjects: 6 males/6 females, mean age: 58.4 ± 7.5 years (range: 50–79 years)], three objective clinical signs were evaluated: MG orifice obstruction, meibum score, and tear film lipid layer interference pattern. Subjective symptoms were analyzed via a 15-item questionnaire. After careful collection of meibum samples, comprehensive lipid analysis was performed via liquid chromatography-mass spectrometry. Data was analyzed via JMP® ver. 13 (SAS Institute, Inc., Cary, NC) statistical analysis software. ResultsIn the MGD patients and elderly subjects, there was a significant decrease in non-polar lipids such as cholesterol esters (ChEs), while a significant increase in polar lipids [cholesterol (Ch), (O-acyl)-ω-hydroxy fatty acid (OAHFA), and free fatty acid (FA)] in total lipids (Tukey-Kramer test: p < 0.05). Triglyceride was significantly increased only in MGD patients (p < 0.05). Symptom scores representative of vision quality (i.e., blurred vision/haziness) were significantly negatively-correlated with the ratio of the non-polar lipid ChE, while significantly positively correlated with the polar lipids Ch, OAHFA, and FA (Spearman's rank correlation coefficient: p < 0.05). ConclusionsOur findings revealed that both MGD and aging affect the composition ratio of major meibum lipids, resulting in the appearance of subjective symptoms.

Diverse meibum lipids produced by Awat1 and Awat2 are important for stabilizing tear film and protecting the ocular surface

SummaryA lipid layer consisting of meibum lipids exists in the tear film and functions in preventing dry eye disease. Although the meibum lipids include diverse lipid classes, the synthesis pathway and role of each class remain largely unknown. Here, we created single and double knockout (KO and DKO, respectively) mice for the two acyl-CoA wax alcohol acyltransferases (Awat1 and Awat2) and investigated their dry eye phenotypes and meibum lipid composition. Awat2 KO and DKO mice exhibited severe dry eye with meibomian gland dysfunction, whereas Awat1 KO mice had mild dry eye. In these mice, specific meibum lipid classes were reduced: (O-acyl)-ω-hydroxy fatty acids and type 1ω wax diesters in Awat1 KO mice, wax monoesters and types 1ω and 2ω wax diesters in Awat2 KO mice, and most of these in DKO mice. Our findings reveal that Awat1 and Awat2 show characteristic substrate specificity and together produce diverse meibum lipids.

A multi-enzyme cascade reaction for the production of α,ω-dicarboxylic acids from free fatty acids

α,ω-Dicarboxylic acids (α,ω-DCAs) have numerous applications, as raw materials for producing various commodities and polymers in chemical industry. For example, dodecanedioic acid (DDDA), a monomer of nylon 612 polymer is used for numerous applications such as plasticizers and adhesive. In this study, firstly the enzymatic production of DDDA from corresponding ω-hydroxy fatty acid was optimized using aldehyde reductase (AHR) and aldehyde dehydrogenase (ALDH), and the cofactor imbalance and proficient regeneration thereof, is a major hurdle for the effective biosynthesis. To circumvent this, NAD(P)H oxidases (NOXs) from Lactobacillus brevis (LbrNOX), Brevibacterium glutamicum (BreNOX) and Lactobacillus reuteri (LreNOX) were examined for the AHR/ALDH/NOX cascade and LreNOX was identified as suitable enzyme. Moreover, LreNOX was fused with AHR and employing this fusion protein in Escherichia. coli (E. coli) holding ALDH, nearly complete conversion (>99%) into DDDA was achieved from 30mM of 12-hydroxy dodecanoic acid within 12h of whole-cell biotransformation (2.2-fold improvement in productivity) turning out to be beneficial. Finally, the synthesis of DDDA from corresponding free fatty acid (dodecanoic acid), was performed by employing combination of AHR/ALDH/NOX cascade with CYP153AL.m, where 2.3mM (0.53g/L) of DDDA was produced in one-pot one step.

Enhancement of sophorolipids production in Candida batistae, an unexplored sophorolipids producer, by fed-batch fermentation.

Sophorolipids (SLs) from Candida batistae has a unique structure that contains ω-hydroxy fatty acids, which can be used as a building block in the polymer and fragrance industries. To improve the production of this industrially important SLs, we optimized the culture medium of C. batistae for the first time. Using an optimized culture medium composed of 50g/L glucose, 50g/L rapeseed oil, 5g/L ammonium nitrate and 5g/L yeast extract, SLs were produced at a concentration of 24.1g/L in a flask culture. Sophorolipids production increased by about 19% (28.6g/L) in a fed-batch fermentation using a 5L fermentor. Sophorolipids production more increased by about 121% (53.2g/L), compared with that in a flask culture, in a fed-batch fermentation using a 50 L fermentor, which was about 787% higher than that of the previously reported SLs production (6g/L). These results indicate that a significant increase in C. batistae-derived SLs production can be achieved by optimization of the culture medium composition and fed-batch fermentation. Finally, we successfully separated and purified the SLs from the culture medium. The improved production of SLs from C. batistae in this study will help facilitate the successful development of applications for the SLs.

Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica.

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

Extracellular lipids of Camelina sativa: Characterization of cutin and suberin reveals typical polyester monomers and unusual dicarboxylic fatty acids

Camelina sativa is relatively drought tolerant and requires less fertilizer than other oilseed crops. Various lipid- and phenolic-based extracellular barriers of plants help to protect them against biotic and abiotic stresses. These barriers, which consist of solvent-insoluble polymeric frameworks and solvent-extractable waxes, include the cuticle of aerial plant surfaces and suberized cell walls found, for example, in periderms and seed coats. Cutin, the polymeric matrix of the cuticle, and the aliphatic domain of suberin are fatty acid- and glycerol-based polyesters. These polyesters were investigated by base-catalyzed transesterification of C. sativa aerial and underground delipidated tissues followed by gas chromatographic analysis of the released monomer mixtures. Seed coat and root suberin had similar compositions, with 18-hydroxyoctadecenoic and 1,18-octadecenedioic fatty acids being the dominant species. Root suberin presented a typical lamellar ultrastructure, but seed coats showed almost imperceptible, faint dark bands. Leaf and stem lipid polyesters were composed of fatty acids (FA), 1,ω-dicarboxylic fatty acids (DCA), ω-hydroxy fatty acids (HFA) and hydroxycinnamic acids (HCA). Dihydroxypalmitic acid (DHP) and caffeic acid were the major constituents of leaf cutin, whereas stem cutin presented similar molar proportions in several monomers across the four classes. Unlike the leaf cuticle, the C. sativa stem cuticle presented lamellar structure by transmission electron microscopy. Flower cutin was dominated by DHP, did not contain aromatics, and presented substantial amounts (>30%) of hydroxylated 1,ω-dicarboxylic acids. We found striking differences between the lipid polyester monomer compositions of aerial tissues of C. sativa and that of its close relatives Arabidopsis thaliana and Brassica napus.

Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity.

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (kcat /KM ) 8.9-fold to 484 s-1 mM-1 for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMOC308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L-1 d-1 . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.

Chemical composition of leaf cutin in six Quercus suber provenances

The cutin content and composition of cork oak (Quercus suber) leaves was determined in six provenances with different seed geographical origin spreading across the species natural distribution. The cutin layer on the leaf surface was on average 518 μg/cm2 of leaf area and represented 6.7% of the leaf dry weight, with no significant differences among provenances.Cutin depolymerisation was carried out by transesterification on whole leaves. The cutin composition of cork oak leaves is presented here for the first time. It is essentially composed of long-chain aliphatic ω-hydroxy fatty acids (44.4% of the total monomers), mostly with mid-chain hydroxyl and epoxy groups, fatty acids (20.7%), and a smaller proportion of α,ω-dicarboxylic acids (6.5%). The predominant compounds are 10,16-dihydroxy hexadecanoic acid (17.7–25.2%) and 9,10,18-trihydroxyoctadecanoic acid (15.6–18.0%). Alkanols represent 2.8% and aromatic compounds 12.8%, mainly coumarates. Isolation of cuticles from Q. suber leaves was performed using an enzymatic separation procedure and the fragments were analysed. Cuticle isolation is difficult and direct depolymerisation applied to whole leaves proved a suitable method to study cutin monomeric composition, which did not differ substantially to that of the isolated cuticles. No differences between provenances were found regarding cutin content and composition, thereby ruling out a significant genetic determination of these traits, but rather a highly adaptive phenotypic plasticity of cork oak. Although overall similar in their chemical nature, cutin and suberin in cork oak differ in the proportion of the major chemical families, i.e. ω-hydroxy acids, α,ω-diacids, and fatty acids.

Supplementation with eicosapentaenoic acid and linoleic acid increases the production of epidermal ceramides in in vitro canine keratinocytes

Few studies have investigated the effects of essential fatty acids on the production of epidermal ceramide (CER) in canine keratinocytes. To investigate the effects of eicosapentaenoic acid (EPA) and linoleic acid (LA) supplementation on the production of CERs using an in vitro canine keratinocyte culture system. Canine keratinocyte cells (MSCEK) were incubated with high Ca2+ [1.8mM calcium chloride (CaCl2 )] serum-free medium, supplemented with 3µM EPA and 15µM LA when the cells showed confluency. On Day 8 of application, lipid analysis using high-performance thin layer chromatography and real-time PCR for detecting glucosylceramide synthase and ceramidase were performed. It was revealed that the amounts of CER (EOS) (combination of ω-hydroxy fatty acids and sphingosines), CER [EOP] (combination of ω-hydroxy fatty acids and phytosphingosines) and a mixture of CER [NS] (combination of nonhydroxy fatty acids and sphingosines) and [NDS] (combination of nonhydroxy fatty acids and dihydrosphingosines), as well as total CERs, were significantly increased in cells incubated with EPA and LA compared to those of the vehicle, with increased mRNA expression of glucosylceramide synthase. These findings suggest that EPA and LA can potentially alter the CER profile of the skin and this may contribute to its epidermal barrier function in canine skin.

Drought-Responsive ZmFDL1/MYB94 Regulates Cuticle Biosynthesis and Cuticle-Dependent Leaf Permeability.

In all land plants, the outer surface of aerial parts is covered by the cuticle, a complex lipid layer that constitutes a barrier against damage caused by environmental factors and provides protection against nonstomatal water loss. We show in this study that both cuticle deposition and cuticle-dependent leaf permeability during the juvenile phase of plant development are controlled by the maize (Zea mays) transcription factor ZmFUSED LEAVES 1 (FDL1)/MYB94. Biochemical analysis showed altered cutin and wax biosynthesis and deposition in fdl1-1 mutant seedlings at the coleoptile stage. Among cutin compounds, ω-hydroxy fatty acids and polyhydroxy-fatty acids were specifically affected, while the reduction of epicuticular waxes was mainly observed in primary long chain alcohols and, to a minor extent, in long-chain wax esters. Transcriptome analysis allowed the identification of candidate genes involved in lipid metabolism and the assembly of a proposed pathway for cuticle biosynthesis in maize. Lack of ZmFDL1/MYB94 affects the expression of genes located in different modules of the pathway, and we highlighted the correspondence between gene transcriptional variations and biochemical defects. We observed a decrease in cuticle-dependent leaf permeability in maize seedlings exposed to drought as well as abscisic acid treatment, which implies coordinated changes in the transcript levels of ZmFDL1/MYB94 and associated genes. Overall, our results suggest that the response to water stress implies the activation of wax biosynthesis and the involvement of both ZmFDL1/MYB94 and abscisic acid regulatory pathways.

Lipid metabolism is involved in male fertility regulation of the photoperiod- and thermo sensitive genic male sterile rice line Peiai 64S

Peiai 64S (PA64S) is a photoperiod- and thermo sensitive genic male sterile (PTGMS) rice line that has been widely applied in two-line hybrid rice breeding. The male fertility mechanism of PTGMS has always been the research focus. We obtained fertile PA64S (F) and sterile fertile PA64S (S) plants at 21℃ and 28℃, respectively. Here, we analyzed the development of anthers and pollen grains of PA64S (S) and found that the degradation of tapetum and sporopollenin accumulation of pollen exine was abnormal. The content of lipid components in PA64S (F) and PA64S (S) were different by LC–MS, among which sterols, (O-acyl) ω-hydroxy fatty acids, ceramide, and other lipid components were upregulated in PA64S (F). The results of transcriptome showed that many significantly different genes were enriched in the lipid metabolism pathways. Additionally, lipid synthesis and transport genes were downregulated in PA64S (S). In summary, the differences of the PA64S fertility under different temperatures were analyzed through multi-levels comparison. These results suggest that lipid synthesis and transport during PA64S anther development affects the lipid accumulation of pollen exine, and ultimately affected fertility. The differences in lipids content may also be a factor affecting PA64S pollen fertility.

Unique skin abnormality in patients with peanut allergy but no atopic dermatitis

The nonlesional skin of children with atopic dermatitis (AD) with peanut allergy (PA) is associated with increased transepidermal water loss; low urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), both of which are filaggrin breakdown products; and a reduced ratio of esterified ω-hydroxy fatty acid sphingosine ceramides (EOS-CERs) to nonhydroxy fatty acid sphingosine ceramides (NS-CERs) in the skin. The skin barrier of subjects with PA without AD (AD-PA+) has not been studied. Our aim was to explore whether AD-PA+ is associated with skin barrier abnormalities. A total of 33 participants were enrolled, including 13 AD-PA+, 9 AD+PA+, and 11 nonatopic (NA) participants. The PCA content in the stratum corneum of AD-PA+ subjects was significantly reduced versus that in NA subjects (median level, 67 vs 97 μg/mg protein [P= .028]). The ratio between cis- and trans-UCA decreased significantly from being highest in the NA group (1.62) to lowest in AD+PA+ group (0.07 [P< .001 vs in the NA group; P= .006 vs in the AD-PA+ group]), with the AD-PA+ group having an intermediate cis/trans-UCA ratio (1.17 [P= .024 vs in the NA group]). The TEWL in AD-PA+ subjects did not differ from that in the group with NA skin. Interestingly, AD-PA+ subjects had an increased EOS/NS-CER ratio versus that in the group of subjects with NA skin (1.9 vs 1.3 [P= .008]), whereas the AD+PA+ group had a decreased proportion of EOS-CERs (0.8 [P= .001] vs in the AD-PA+ group). Our data demonstrate that irrespective of AD, PA is associated with decreased skin cis-UCA and PCA content. An increase in skin EOS-CER/NS-CER ratio separates the AD-PA+ group from the AD+PA+ and NA groups.

Lipid polarity gradient formed by ω-hydroxy lipids in tear film prevents dry eye disease.

Meibum lipids form a lipid layer on the outermost side of the tear film and function to prevent water evaporation and reduce surface tension. (O-Acyl)-ω-hydroxy fatty acids (OAHFAs), a subclass of these lipids, are thought to be involved in connecting the lipid and aqueous layers in tears, although their actual function and synthesis pathway have to date remained unclear. Here, we reveal that the fatty acid ω-hydroxylase Cyp4f39 is involved in OAHFA production. Cyp4f39-deficient mice exhibited damaged corneal epithelium and shortening of tear film break-up time, both indicative of dry eye disease. In addition, tears accumulated on the lower eyelid side, indicating increased tear surface tension. In Cyp4f39-deficient mice, the production of wax diesters (type 1ω and 2ω) and cholesteryl OAHFAs was also impaired. These OAHFA derivatives show intermediate polarity among meibum lipids, suggesting that OAHFAs and their derivatives contribute to lipid polarity gradient formation for tear film stabilization.

Development of an in vitro submerged culture system to synthesize epidermal ceramides in canine keratinocytes

Ceramides (CERs) in the stratum corneum (SC) are known to play a crucial role in determining skin barrier function in dogs. We aimed to develop an in vitro culture system that synthesized epidermal CER classes to better understand the synthesis of CER classes in canine SC–. Canine keratinocyte cells (MSCEK) at appropriate confluency were incubated with high Ca2+ (1.8 mM CaCl2) supplemented serum-free medium. Eight days post Ca2+ application, the surface of cultured MSCEK was broadly stained with anti-loricrin antibody implying that the keratinocytes had stratified into stratum granulosum. MSCEK cells synthesized heterogenous epidermal CERs, similar to those seen during the stratification of canine keratinocytes. CER fractions obtained from MSCEK cells were comparable to those from canine SC, including CER[EOS] (combination of ω-hydroxy fatty acids and sphingosines), CER[NP] (combination of non-hydroxy fatty acids and phytosphingosines), and CER[EOP] (combination of ω-hydroxy fatty acids and phytosphingosines), all of which are lowered in the SC during canine atopic dermatitis. Thus, the present study provides a simple culture system as a tool for in-depth analysis of CER production in canine keratinocytes.

Lipidomic Analysis Reveals Specific Differences between Fibroblast and Keratinocyte Ceramide Profile of Patients with Psoriasis Vulgaris.

Ceramides are important lipid metabolites for primal skin functions. There is increasing evidence that alteration of the profile and metabolism of ceramides is associated with skin diseases, such as psoriasis vulgaris. Most studies have reported alteration in ceramide content in the stratum corneum, but these have been scarcely reported for other skin layers. In the present work, we aimed to explore changes in the ceramide profile of fibroblasts and keratinocytes in patients with psoriasis vulgaris and healthy subjects. Using the reversed-phase liquid chromatography-quadrupole-time-of-flight-tandem-mass spectrometry (RPLC-QTOF-MS/MS) platform, we identified ceramide containing non-hydroxy fatty acid ([N]), α-hydroxy fatty acid ([A]), and esterified ω-hydroxy fatty acid ([EO]) and 3 sphingoid bases, dihydrosphingosine ([DS]), sphingosine ([S]), and phytosphingosine ([P]). We found that in the keratinocytes of patients with psoriasis, CER[NS], CER[NP], CER[AS], CER[ADS], CER[AP] and CER[EOS] tended to be expressed at higher relative levels, whereas CER[NDS] tended to be expressed with lower levels than in healthy subjects. In the case of fibroblasts, significant differences were observed, mainly in the three ceramide classes (CER[AS], CER[ADS] and CER[EOS]), which were expressed at significantly higher levels in patients with psoriasis. The most significant alteration in the fibroblasts involved elevated levels of CER[EOS] that contained ester-linked fatty acids. Our findings provide insights into the ceramide profile in the dermis and epidermis of patients with psoriasis and contribute for the research in this field, focusing on the role of keratinocyte-fibroblast crosstalk in the development of psoriasis vulgaris.

Skin permeability barrier formation by the ichthyosis-causative gene FATP4 through formation of the barrier lipid ω-O-acylceramide

The epidermis-specific lipid acylceramide plays a pivotal role in the formation of the permeability barrier in the skin; abrogation of its synthesis causes the skin disorder ichthyosis. However, the acylceramide synthetic pathway has not yet been fully elucidated: Namely, the acyl-CoA synthetase (ACS) involved in this pathway remains to be identified. Here, we hypothesized it to be encoded by FATP4/ACSVL4, the causative gene of ichthyosis prematurity syndrome (IPS). In vitro experiments revealed that FATP4 exhibits ACS activity toward an ω-hydroxy fatty acid (FA), an intermediate of the acylceramide synthetic pathway. Fatp4 knockout (KO) mice exhibited severe skin barrier dysfunction and morphological abnormalities in the epidermis. The total amount of acylceramide in Fatp4 KO mice was reduced to ∼10% of wild-type mice. Decreased levels and shortening of chain lengths were observed in the saturated, nonacylated ceramides. FA levels were not decreased in the epidermis of Fatp4 KO mice. The expression levels of the FA elongase Elovl1 were reduced in Fatp4 KO epidermis, partly accounting for the reduction and shortening of saturated, nonacylated ceramides. A decrease in acylceramide levels was also observed in human keratinocytes with FATP4 knockdown. From these results, we conclude that skin barrier dysfunction observed in IPS patients and Fatp4 KO mice is caused mainly by reduced acylceramide production. Our findings further elucidate the molecular mechanism governing acylceramide synthesis and IPS pathology.

De novo biosynthesis of 8-hydroxyoctanoic acid via a medium-chain length specific fatty acid synthase and cytochrome P450 in Saccharomyces cerevisiae

Terminally hydroxylated fatty acids or dicarboxylic acids are industrially relevant compounds with broad applications. Here, we present the proof of principle for the de novo biosynthesis of 8-hydroxyoctanoic acid from glucose and ethanol in the yeast Saccharomyces cerevisiae. Toxicity tests with medium-chain length ω-hydroxy fatty acids and dicarboxylic acids revealed little or no growth impairments on yeast cultures even at higher concentrations. The ability of various heterologous cytochrome P450 enzymes in combination with their cognate reductases for ω-hydroxylation of externally fed octanoic acid were compared. Finally, the most efficient P450 enzyme system was expressed in a yeast strain, whose fatty acid synthase was engineered for octanoic acid production, resulting in de novo biosynthesis of 8-hydroxyoctanoic acid up to 3 ​mg/l. Accumulation of octanoic acid revealed that cytochromes P450 activities were limiting 8-hydroxyoctanoic acid synthesis. The hydroxylation of both externally added and intracellularly produced octanoic acid was strongly dependent on the carbon source used, with ethanol being preferred. We further identified the availability of heme, a cofactor needed for P450 activity, as a limiting factor of 8-hydroxyoctanoic acid biosynthesis.