Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis.

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Protein-protein interactions in fatty acid elongase complexes are important for very-long-chain fatty acid synthesis.

Fatty acid elongase (FAE), which catalyzes the synthesis of very-long-chain fatty acids (VLCFAs), is a multiprotein complex; however, little is known about its quaternary structure. In this study, bimolecular fluorescence complementation and/or yeast two-hybrid assays showed that homo-interactions were observed in ß-ketoacyl-CoA synthases (KCS2, KCS9, and KCS6), Eceriferum2-like proteins [CER2 and CER2-Like2 (C2L2)], and FAE complex proteins (KCR1, PAS2, ECR, and PAS1), except for CER2-Like1 (C2L1). Hetero-interactions were observed between KCSs (KCS2, KCS9, and KCS6), between CER2-LIKEs (CER2, C2L2, and C2L1), and between FAE complex proteins (KCR1, PAS2, ECR, and PAS1). PAS1 interacts with FAE complex proteins (KCR1, PAS2, and ECR), but not with KCSs (KCS2, KCS9, and KCS6) and CER2-LIKEs (CER2, C2L2, and C2L1). Asp308 and Arg309-Arg311 of KCS9 were essential for the homo-interactions of KCS9 and hetero-interactions between KCS9 and PAS2 or ECR. Asp339 of KCS9 is involved in its homo- and hetero-interactions with ECR. Complementation analysis of the Arabidopsis kcs9 mutant by the expression of amino acid-substituted KCS9 mutant genes showed that Asp308 and Asp339 of KCS9 are involved in the synthesis of C24 VLCFAs from C22. This study suggests that protein-protein interaction in FAE complexes is important for VLCFA synthesis and provides insight into the quaternary structure of FAE complexes for efficient synthesis of VLCFAs.

Interaction of ONION2 ketoacyl CoA synthase with ketoacyl CoA reductase of rice.

BACKGROUND: Fatty acid elongases (FAEs), which catalyse elongation reactions of a carbon chain of very-long-chain fatty acids, play an important role in shoot development in rice. The elongation reactions consist of four sequential reactions catalysed by distinct enzymes, which are assumed to form an elongation complex. However, no interacting proteins of ONION1 (ONI1) and ONI2, which are ketoacyl CoA synthase catalyzing the first step and are required for shoot development in rice, are reported. METHODS AND RESULTS: In this study ketoacyl CoA reductase (KCR) that interacts with ONI1 and ONI2 was searched. A database search identified 10 KCR genes in the rice genome. Among the genes, the expression pattern of KCR1 was similar to that of ONI2. Yeast two-hybrid analysis showed interaction of ONI2 with KCR1, which was confirmed by GST pull-down assay. No interacting partner of ONI1 was identified. CONCLUSIONS: Our results suggest that ONI2 and KCR1 form an FAE complex that may play a role in biosynthesizing VLCFAs during shoot development.

Dominant Elongase Activity of Elovl5a but Higher Expression of Elovl5b in Common Carp (Cyprinus carpio).

Most diploid freshwater and marine fish encode one elovl5 elongase, having substrate specificity and activities towards C18, C20 and C22 polyunsaturated fatty acids (PUFAs). The allo-tetraploid common carp is hypothesized to encode two duplicated elovl5 genes. How these two elovl5 genes adapt to coordinate the PUFA biosynthesis through elongase function and expression divergence requires elucidation. In this study, we obtained the full-length cDNA sequences of two elovl5 genes in common carp, named as elovl5a and elovl5b. Functional characterization showed that both enzymes had elongase activity towards C18, C20 and C22 PUFAs. Especially, the activities of these two enzymes towards C22 PUFAs ranged from 3.87% to 8.24%, higher than those in most freshwater and marine fish. The Elovl5a had higher elongase activities than Elovl5b towards seven substrates. The spatial-temporal expression showed that both genes co-transcribed in all tissues and development stages. However, the expression levels of elovl5b were significantly higher than those of elovl5a in all examined conditions, suggesting that elovl5b would be the dominantly expressed gene. These two genes had different potential transcriptional binding sites. These results revealed the complicated roles of elovl5 on PUFA synthesis in common carp. The data also increased the knowledge of co-ordination between two homoeologs of the polyploid fish through function and expression divergence.

Functional analysis of ß-ketoacyl-CoA synthase from biofuel feedstock Thlaspi arvense reveals differences in the triacylglycerol biosynthetic pathway among Brassicaceae.

KEY MESSAGE: Differences in FAE1 enzyme affinity for the acyl-CoA substrates, as well as the balance between the different pathways involved in their incorporation to triacylglycerol might be determinant of the different composition of the seed oil in Brassicaceae. Brassicaceae present a great heterogeneity of seed oil and fatty acid composition, accumulating Very Long Chain Fatty Acids with industrial applications. However, the molecular determinants of these differences remain elusive. We have studied the ß-ketoacyl-CoA synthase from the high erucic feedstock Thlaspi arvense (Pennycress). Functional characterization of the Pennycress FAE1 enzyme was performed in two Arabidopsis backgrounds; Col-0, with less than 2.5% of erucic acid in its seed oil and the fae1-1 mutant, deficient in FAE1 activity, that did not accumulate erucic acid. Seed-specific expression of the Pennycress FAE1 gene in Col-0 resulted in a 3 to fourfold increase of erucic acid content in the seed oil. This increase was concomitant with a decrease of eicosenoic acid levels without changes in oleic ones. Interestingly, only small changes in eicosenoic and erucic acid levels occurred when the Pennycress FAE1 gene was expressed in the fae1-1 mutant, with high levels of oleic acid available for elongation, suggesting that the Pennycress FAE1 enzyme showed higher affinity for eicosenoic acid substrates, than for oleic ones in Arabidopsis. Erucic acid was incorporated to triacylglycerol in the transgenic lines without significant changes in their levels in the diacylglycerol fraction, suggesting that erucic acid was preferentially incorporated to triacylglycerol via DGAT1. Expression analysis of FAE1, AtDGAT1, AtLPCAT1 and AtPDAT1 genes in the transgenic lines further supported this conclusion. Differences in FAE1 affinity for the oleic and eicosenoic substrates among Brassicaceae, as well as their incorporation to triacylglycerol might explain the differences in composition of their seed oil.

Identification and characterization of two fatty acid elongases in Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is a potential cost-effective source for the production of microbial lipids. Fatty acid elongases have vital roles in the syntheses of long-chain fatty acids. In this study, two genes encoding fatty acid elongases of L. starkeyi, LsELO1, and LsELO2 were identified and characterized. Heterologous expression of these genes in Saccharomyces cerevisiae revealed that LsElo1 is involved in the production of saturated long-chain fatty acids with 24 carbon atoms (C24:0) and that LsElo2 is involved in the conversion of C16 fatty acids to C18 fatty acids. In addition, both LsElo1 and LsElo2 were able to elongate polyunsaturated fatty acids. LsElo1 elongated linoleic acid (C18:2) to eicosadienoic acid (C20:2), and LsElo2 elongated α-linolenic acid (C18:3) to eicosatrienoic acid (C20:3). Overexpression of LsElo2 in L. starkeyi caused a reduction in C16 fatty acids, such as palmitic and palmitoleic acids, and an accumulation of C18 fatty acids such as oleic and linoleic acids. Our findings have the potential to contribute to the remodeling of fatty acid composition and the production of polyunsaturated long-chain fatty acids in oleaginous yeasts.

Functional characterization and structural modelling of Helianthus annuus (sunflower) ketoacyl-CoA synthases and their role in seed oil composition.

MAIN CONCLUSION: The enzymes HaKCS1 and HaKCS2 are expressed in sunflower seeds and contribute to elongation of C18 fatty acids, resulting in the C20-C24 fatty acids in sunflower oil. Most plant fatty acids are produced by plastidial soluble fatty acid synthases that produce fatty acids of up to 18 carbon atoms. However, further acyl chain elongations can take place in the endoplasmic reticulum, catalysed by membrane-bound synthases that act on acyl-CoAs. The condensing enzymes of these complexes are the ketoacyl-CoA synthase (KCSs), responsible for the synthesis of very long chain fatty acids (VLCFAs) and their derivatives in plants, these including waxes and cuticle hydrocarbons, as well as fatty aldehydes. Sunflower seeds accumulate oil that contains around 2-3% of VLCFAs and studies of the fatty acid elongase activity in developing sunflower embryos indicate that two different KCS isoforms drive the synthesis of these fatty acids. Here, two cDNAs encoding distinct KCSs were amplified from RNAs extracted from developing sunflower embryos and named HaKCS1 and HaKCS2. These genes are expressed in developing seeds during the period of oil accumulation and they are clear candidates to condition sunflower oil synthesis. These two KCS cDNAs complement a yeast elongase null mutant and when expressed in yeast, they alter the host's fatty acid profile, proving the encoded KCSs are functional. The structure of these enzymes was modelled and their contribution to the presence of VLCFAs in sunflower oil is discussed based on the results obtained.

In Human and Mouse Spino-Cerebellar Tissue, Ataxin-2 Expansion Affects Ceramide-Sphingomyelin Metabolism.

Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide-sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.

Evolutionarily conserved function of the sacred lotus (Nelumbo nucifera Gaertn.) CER2-LIKE family in very-long-chain fatty acid elongation.

MAIN CONCLUSION: Identification of NnCER2 and NnCER2-LIKE from Nelumbo nucifera, which are required for the very-long-chain fatty acid elongation, provides new evidence that CER2 proteins are evolutionarily conserved across the eudicots. CER2-LIKE family proteins have been described as core components of the fatty acid elongase complex in Arabidopsis, maize, and rice, having specific function in synthesis of the C30 to C34 fatty acyl-CoA precursors of cuticular waxes. Little is known about the functional conservation in this gene family across species. In this study, two CER2-LIKE family proteins, NnCER2 and NnCER2-LIKE, were characterized from sacred lotus (Nelumbo nucifera), which is an ancient basal eudicot. The transcriptional expression of NnCER2 and NnCER2-LIKE was found in floating leaf blades, emergent petioles and vertical leaves, petals, and anthers. The NnCER2 and NnCER2-LIKE proteins were localized to the endoplasmic reticulum and nucleus. Overexpressing NnCER2 and NnCER2-LIKE in Arabidopsis led to alteration of cuticle wax structure in inflorescence stems, and this was associated with elevated 30, 32, and 34 carbon length wax compounds, and their derivatives. The different substrate specificities of NnCER2 and NnCER2-LIKE were explored using co-expression with AtCER6 in yeast cells. These findings provide clear evidence that the function of CER2 family proteins in producing VLCFAs is highly conserved across the eudicots.

Design of a Seed-Specific Chimeric Promoter with a Modified Expression Profile to Improve Seed Oil Content.

Increasing the yield of plant oil is an important objective to meet the demand for sustainable resources and energy. Some attempts to enhance the expression of genes involved in oil synthesis in seeds have succeeded in increasing oil content. In many cases, the promoters of seed-storage protein genes have been used as seed-specific promoters. However, conventional promoters are developmentally regulated and their expression periods are limited. We constructed a chimeric promoter that starts to express in the early stage of seed development, and high-level expression is retained until the later stage by connecting the promoters of the biotin carboxyl carrier protein 2 (BCCP2) gene encoding the BCCP2 subunit of acetyl-CoA carboxylase and the fatty acid elongase 1 (FAE1) gene from Arabidopsis. The constructed promoter was ligated upstream of the TAG1 gene encoding diacylglycerol acyltransferase 1 and introduced into Arabidopsis. Seeds from transgenic plants carrying AtTAG1 under the control of the chimeric promoter showed increased oil content (up by 18⁻73%) compared with wild-type seeds. The novel expression profile of the chimeric promoter showed that this could be a promising strategy to manipulate the content of seed-storage oils and other compounds.

Changes in cuticular wax coverage and composition on developing Arabidopsis leaves are influenced by wax biosynthesis gene expression levels and trichome density.

MAIN CONCLUSION: Wax coverage on developing Arabidopsis leaf epidermis cells is constant and thus synchronized with cell expansion. Wax composition shifts from fatty acid to alkane dominance, mediated by CER6 expression. Epidermal cells bear a wax-sealed cuticle to hinder transpirational water loss. The amount and composition of the cuticular wax mixture may change as organs develop, to optimize the cuticle for specific functions during growth. Here, morphometrics, wax chemical profiling, and gene expression measurements were integrated to study developing Arabidopsis thaliana leaves and, thus, further our understanding of cuticular wax ontogeny. Before 5 days of age, cells at the leaf tip ceased dividing and began to expand, while cells at the leaf base switched from cycling to expansion at day 13, generating a cell age gradient along the leaf. We used this spatial age distribution together with leaves of different ages to determine that, as leaves developed, their wax compositions shifted from C24/C26 to C30/C32 and from fatty acid to alkane constituents. These compositional changes paralleled an increase in the expression of the elongase enzyme CER6 but not of alkane pathway enzymes, suggesting that CER6 transcriptional regulation is responsible for both chemical shifts. Leaves bore constant numbers of trichomes between 5 and 21 days of age and, thus, trichome density was higher on young leaves. During this time span, leaves of the trichome-less gl1 mutant had constant wax coverage, while wild-type leaf coverage was initially high and then decreased, suggesting that high trichome density leads to greater apparent coverage on young leaves. Conversely, wax coverage on pavement cells remained constant over time, indicating that wax accumulation is synchronized with cell expansion throughout leaf development.

Wax Crystal-Sparse Leaf 4, encoding a ß-ketoacyl-coenzyme A synthase 6, is involved in rice cuticular wax accumulation.

KEY MESSAGE: WSL4 encodes a KCS6 protein which is required for cuticular wax accumulation in rice. Very long chain fatty acids (VLCFAs) are essential precursors for cuticular wax biosynthesis. VLCFA biosynthesis occurs in the endoplasmic reticulum and requires the fatty acid elongase (FAE) complex. The ß-ketoacyl-coenzyme A synthase (KCS) catalyzes the first step of FAE-mediated VLCFA elongation. Here we characterized the Wax Crystal-Sparse Leaf 4 (WSL4) gene involved in leaf cuticular wax accumulation in rice. The wsl4 mutant displayed a pleiotropic phenotype including dwarfism, less tiller numbers and reduced surface wax load. Map-based cloning and nucleotide sequencing results revealed that wsl4 carried a single nucleotide substitution in the second exon of a putative KCS6 gene, encoding one subunit of the FAE complex for VLCFAs. Genetic complementation confirmed that the mutation in WSL4 was responsible for the phenotype of wsl4. WSL4 was constitutively expressed in various rice tissues and localized in the endoplasmic reticulum. Both WSL4-RNAi transgenic lines and WSL4 knocked-out mutants exhibited wax-deficient phenotypes similar to the wsl4 mutant. These data indicate that WSL4 is required for cuticular wax accumulation in rice.

Wax crystal-sparse leaf 3 encoding a ß-ketoacyl-CoA reductase is involved in cuticular wax biosynthesis in rice.

KEY MESSAGE: WSL3 encodes ß-ketoacyl-CoA reductase (KCR) in rice, in a similar way to YBR159w in yeast, and is essential for VLCFA biosynthesis and leaf wax accumulation. Cuticular waxes on plant surfaces limit non-stomatal water loss, protect plants against deposits of dust and impose a physical barrier to pathogen infection. We identified a wax-deficient mutant of rice, wax crystal-sparse leaf 3 (wsl3), which exhibits a pleiotropic phenotype that includes reduced epicuticular wax crystals on the leaf surface and altered wax composition. Map-based cloning demonstrated that defects in the mutant were caused by two adjacent single-nucleotide changes in a gene encoding ß-ketoacyl-CoA reductase (KCR) that catalyzes the second step of the fatty acid elongation reaction. The identity of WSL3 was further confirmed by genetic complementation. Transient assays of fluorescent protein-tagged WSL3 in tobacco protoplasts showed that WSL3 localizes to the endoplasmic reticulum, the compartment of fatty acid elongation in cells. Quantitative PCR and histochemical staining indicated that WSL3 is universally expressed in tissues. RNA interference of WSL3 caused a phenotype that mimicked the wsl3 mutant. Very long-chain fatty acids (VLCFAs) 20:0 and 22:0, or 20:1Δ(11) and 22:1Δ(13), were detected when WSL3 and Arabidopsis fatty acid elongation 1 (FAE1) were co-expressed in a yeast ybr159wΔ mutant strain. Our results indicated that WSL3 affects rice cuticular wax production by participating in VLCFA elongation.

Production of cis-11-eicosenoic acid by Mortierella fungi.

AIM: To find cis-11-eicosenoic acid (20:1ω9, EA)-producing micro-organisms. METHODS AND RESULTS: We found EA-producing fungi by screening about 300 fungal strains and identified a major fatty acid accumulated in the Mortierella fungi as EA by means of GC-MS analysis. In particular, Mortierella chlamydospora CBS 529.75 produced a high amount of EA (36.3 mg g(-1) of dried cells) on cultivation at 28°C for 4 days and then at 12°C for 3 days. In the result of lipid analysis, most of the EA was a component of triacylglycerols, not phospholipids. CONCLUSION: We found that M. chlamydospora CBS 529.75 was the best producer for the microbial production of EA. SIGNIFICANCE AND IMPACT OF THE STUDY: EA is beneficial as a raw material for medical supplies and a moisturizing component of cosmetic creams. This is the first report of microbial production of EA.

Elovl2 ablation demonstrates that systemic DHA is endogenously produced and is essential for lipid homeostasis in mice.

The potential role of endogenously synthesized PUFAs is a highly overlooked area. Elongation of very long-chain fatty acids (ELOVLs) in mammals is catalyzed by the ELOVL enzymes to which the PUFA elongase ELOVL2 belongs. To determine its in vivo function, we have investigated how ablation of ELOVL2, which is highly expressed in liver, affects hepatic lipid composition and function in mice. The Elovl2(-/-) mice displayed substantially decreased levels of 22:6(n-3), DHA, and 22:5(n-6), docosapentaenoic acid (DPA) n-6, and an accumulation of 22:5(n-3) and 22:4(n-6) in both liver and serum, showing that ELOVL2 primarily controls the elongation process of PUFAs with 22 carbons to produce 24-carbon precursors for DHA and DPAn-6 formation in vivo. The impaired PUFA levels positively influenced hepatic levels of the key lipogenic transcriptional regulator sterol-regulatory element binding protein 1c (SREBP-1c), as well as its downstream target genes. Surprisingly, the Elovl2(-/-) mice were resistant to hepatic steatosis and diet-induced weight gain, implying that hepatic DHA synthesis via ELOVL2, in addition to controlling de novo lipogenesis, also regulates lipid storage and fat mass expansion in an SREBP-1c-independent fashion. The changes in fatty acid metabolism were reversed by dietary supplementation with DHA.

The IGF2 mRNA binding protein p62/IGF2BP2-2 induces fatty acid elongation as a critical feature of steatosis.

Liver-specific overexpression of the insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IGF2BP2-2 induces a fatty liver, which highly expresses IGF2 Because IGF2 expression is elevated in patients with steatohepatitis, the aim of our study was to elucidate the role and interconnection of p62 and IGF2 in lipid metabolism. Expression of p62 and IGF2 highly correlated in human liver disease. p62 induced an elevated ratio of C18:C16 and increased fatty acid elongase 6 (ELOVL6) protein, the enzyme catalyzing the elongation of C16 to C18 fatty acids and promoting nonalcoholic steatohepatitis in mice and humans. The p62 overexpression induced the activation of the ELOVL6 transcriptional activator sterol regulatory element binding transcription factor 1 (SREBF1). Recombinant IGF2 induced the nuclear translocation of SREBF1 and a neutralizing IGF2 antibody reduced ELOVL6 and mature SREBF1 protein levels. Concordantly, p62 and IGF2 correlated with ELOVL6 in human livers. Decreased palmitoyl-CoA levels, as found in p62 transgenic livers, can explain the lipogenic action of ELOVL6. Accordingly, p62 represents an inducer of hepatic C18 fatty acid production via a SREBF1-dependent induction of ELOVL6. These findings underline the detrimental role of p62 in liver disease.

Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma.

BACKGROUND & AIMS: Epigenetic silencing of tumor suppressor genes contributes to the pathogenesis of hepatocellular carcinoma (HCC). To identify clinically relevant tumor suppressor genes silenced by DNA methylation in HCC, we integrated DNA methylation data from human primary HCC samples with data on up-regulation of gene expression after epigenetic unmasking. METHODS: We performed genome-wide methylation analysis of 71 human HCC samples using the Illumina HumanBeadchip27K array; data were combined with those from microarray analysis of gene re-expression in 4 liver cancer cell lines after their exposure to reagents that reverse DNA methylation (epigenetic unmasking). RESULTS: Based on DNA methylation in primary HCC and gene re-expression in cell lines after epigenetic unmasking, we identified 13 candidate tumor suppressor genes. Subsequent validation led us to focus on functionally characterizing 2 candidates, sphingomyelin phosphodiesterase 3 (SMPD3) and neurofilament, heavy polypeptide (NEFH), which we found to behave as tumor suppressor genes in HCC. Overexpression of SMPD3 and NEFH by stable transfection of inducible constructs into an HCC cell line reduced cell proliferation by 50% and 20%, respectively (SMPD3, P = .003 and NEFH, P = .003). Conversely, knocking down expression of these genes with small hairpin RNA promoted cell invasion and migration in vitro (SMPD3, P = .0001 and NEFH, P = .022), and increased their ability to form tumors after subcutaneous injection or orthotopic transplantation into mice, confirming their role as tumor suppressor genes in HCC. Low levels of SMPD3 were associated with early recurrence of HCC after curative surgery in an independent patient cohort (P = .001; hazard ratio = 3.22; 95% confidence interval: 1.6-6.5 in multivariate analysis). CONCLUSIONS: Integrative genomic analysis identified SMPD3 and NEFH as tumor suppressor genes in HCC. We provide evidence that SMPD3 is a potent tumor suppressor gene that could affect tumor aggressiveness; a reduced level of SMPD3 is an independent prognostic factor for early recurrence of HCC.

Novel elongase of Pythium sp. with high specificity on Δ(6)-18C desaturated fatty acids.

We identified a novel elongase gene from a selected strain of the Oomycete, Pythium sp. BCC53698. Using a PCR approach, the cloned gene (PyElo) possessed an open reading frame (ORF) of 834 bp encoding 277 amino acid residues. A similarity search showed that it had homology with the PUFA elongases of several organisms. In addition, the signature characteristics, including four conserved motifs, a histidine-rich catalytic motif and membrane-associated feature were present in the Pythium gene. Heterologous expression in Saccharomyces cerevisiae showed that it was specific for fatty acid substrates, having a double bond at Δ(6)-position, which included γ-linolenic acid (GLA) and stearidonic acid (STA), and preferentially elongated the n3-18C PUFA. This is an elongase in Oomycete fungi, which displays very high specificity on Δ(6)-18C desaturated fatty acids. This will be a powerful tool to engineer PUFA biosynthesis in organisms of interest through the n-6 series pathway for producing value-added fatty acids.

Advanced fructo-oligosaccharides improve itching and aberrant epidermal lipid composition in children with atopic dermatitis.

Introduction: The effects of fructo-oligosaccharides (FOS) on atopic dermatitis (AD) have not been determined. Methods: In a randomized, double-blind, placebo-controlled trial, children with AD aged 24 months to 17 years received either advanced FOS containing 4.25 g of 1-kestose or a placebo (maltose) for 12 weeks. Results: The SCORAD and itching scores were reduced in patients treated with both FOS (all p < 0.01) and maltose (p < 0.05 and p < 0.01). Sleep disturbance was improved only in the FOS group (p < 0.01). The FOS group revealed a decreased proportion of linoleic acid (18:2) esterified omega-hydroxy-ceramides (EOS-CERs) with amide-linked shorter chain fatty acids (C28 and C30, all p < 0.05), along with an increased proportion of EOS-CERs with longer chain fatty acids (C32, p < 0.01). Discussion: FOS may be beneficial in alleviating itching and sleep disturbance, as well as improving skin barrier function in children with AD.

A fatty acid elongase complex regulates cell membrane integrity and septin-dependent host infection by the rice blast fungus.

Very-long-chain fatty acids (VLCFAs) regulate biophysical properties of cell membranes to determine growth and development of eukaryotes, such as the pathogenesis of the rice blast fungus Magnaporthe oryzae. The fatty acid elongase Elo1 regulates pathogenesis of M. oryzae by modulating VLCFA biosynthesis. However, it remains unknown whether and how Elo1 associates with other factors to regulate VLCFA biosynthesis in fungal pathogens. Here, we identified Ifa38, Phs1 and Tsc13 as interacting proteins of Elo1 by proximity labelling in M. oryzae. Elo1 associated with Ifa38, Phs1 and Tsc13 on the endoplasmic reticulum (ER) membrane to control VLCFA biosynthesis. Targeted gene deletion mutants Δifa38, Δphs1 and Δtsc13 were all similarly impaired as Δelo1 in vegetative growth, conidial morphology, stress responses in ER, cell wall and membrane. These deletion mutants also displayed severe damage in cell membrane integrity and failed to organize the septin ring that is essential for penetration peg formation and pathogenicity. Our study demonstrates that M. oryzae employs a fatty acid elongase complex to regulate VLCFAs for maintaining or remodelling cell membrane structure, which is important for septin-mediated host penetration.