Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids.

Odd-numbered fatty acids (FAs) have been widely used in nutrition, agriculture, and chemical industries. Recently, some studies showed that they could be produced from bacteria or yeast, but the products are almost exclusively odd-numbered long-chain FAs. Here we report the design and construction of two biosynthetic pathways in Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids (OMFAs) via ricinoleic acid and 10-hydroxystearic acid, respectively. The production of OMFAs was enabled by introducing a hydroxy fatty acid cleavage pathway, including an alcohol dehydrogenase from Micrococcus luteus, a Baeyer-Villiger monooxygenase from Pseudomonas putida, and a lipase from Pseudomonas fluorescens. These OMFA biosynthetic pathways were optimized by eliminating the rate-limiting step, generating heptanoic acid, 11-hydroxyundec-9-enoic acid, nonanoic acid, and 9-hydroxynonanoic acid at 7.83 mg/L, 9.68 mg/L, 9.43 mg/L and 13.48 mg/L, respectively. This work demonstrates the biological production of OMFAs in a sustainable manner in S. cerevisiae.

Dietary habits and plasma lipid concentrations in a general Japanese population.

INTRODUCTION: Accumulating data on the associations between food consumption and lipid composition in the body is essential for understanding the effects of dietary habits on health. OBJECTIVES: As part of omics research in the Tohoku Medical Megabank Community-Based Cohort Study, this study sought to reveal the dietary impact on plasma lipid concentration in a Japanese population. METHODS: We conducted a correlation analysis of food consumption and plasma lipid concentrations measured using mass spectrometry, for 4032 participants in Miyagi Prefecture, Japan. RESULTS: Our analysis revealed 83 marked correlations between six food categories and the concentrations of plasma lipids in nine subclasses. Previously reported associations, including those between seafood consumption and omega-3 fatty acids, were validated, while those between dairy product consumption and odd-carbon-number fatty acids (odd-FAs) were validated for the first time in an Asian population. Further analysis suggested that dairy product consumption is associated with odd-FAs via sphingomyelin (SM), which suggests that SM is a carrier of odd-FAs. These results are important for understanding odd-FA metabolism with regards to dairy product consumption. CONCLUSION: This study provides insight into the dietary impact on plasma lipid concentration in a Japanese population.

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens.

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.

Ultrastable and Responsive Foams Based on 10-Hydroxystearic Acid Soap for Spore Decontamination.

Currently, there is renewed interest in using fatty acid soaps as surfactants. Hydroxylated fatty acids are specific fatty acids with a hydroxyl group in the alkyl chain, giving rise to chirality and specific surfactant properties. The most famous hydroxylated fatty acid is 12-hydroxystearic acid (12-HSA), which is widely used in industry and comes from castor oil. A very similar and new hydroxylated fatty acid, 10-hydroxystearic acid (10-HSA), can be easily obtained from oleic acid by using microorganisms. Here, we studied for the first time the self-assembly and foaming properties of R-10-HSA soap in an aqueous solution. A multiscale approach was used by combining microscopy techniques, small-angle neutron scattering, wide-angle X-ray scattering, rheology experiments, and surface tension measurements as a function of temperature. The behavior of R-10-HSA was systematically compared with that of 12-HSA soap. Although multilamellar micron-sized tubes were observed for both R-10-HSA and 12-HSA, the structure of the self-assemblies at the nanoscale was different, which is probably due to the fact that the 12-HSA solutions were racemic mixtures, while the 10-HSA solutions were obtained from a pure R enantiomer. We also demonstrated that stable foams based on R-10-HSA soap can be used for cleaning applications, by studying spore removal on model surfaces in static conditions via foam imbibition.

The Urinary Bladder is Rich in Glycosphingolipids Composed of Phytoceramides.

Glycosphingolipids (GSLs) are composed of a polar glycan chain and a hydrophobic tail known as ceramide. Together with variation in the glycan chain, ceramides exhibit tissue-specific structural variation in the long-chain base (LCB) and N-acyl chain moieties in terms of carbon chain length, degree of desaturation, and hydroxylation. Here, we report the structural variation in GSLs in the urinary bladders of mice and humans. Using TLC, we showed that the major GSLs are hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, Neu5Ac-Gal-Glc-Ceramide, and Neu5Ac-Neu5Ac-Gal-Glc-Ceramide. Our LC-MS analysis indicated that phytoceramide structures with a 20-carbon LCB (4-hydroxyeicosasphinganine) and 2-hydroxy fatty acids are abundant in hexosylceramide and Neu5Ac-Gal-Glc-Ceramide in mice and humans. In addition, quantitative PCR demonstrated that DES2 and FA2H, which are responsible for the generation of 4-hydroxysphinganine and 2-hydroxy fatty acid, respectively, and SPTLC3 and SPTSSB, which are responsible for the generation of 20-carbon LCBs, showed significant expressions in the epithelial layer than in the subepithelial layer. Immunohistochemically, dihydroceramide:sphinganine C4-hydroxylase (DES2) was expressed exclusively in urothelial cells of the urinary bladder. Our findings suggest that these ceramide structures have an impact on membrane properties of the stretching and shrinking in transitional urothelial cells.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall.

The marine bacterium Alcanivorax borkumensis produces a surface-active glycine-glucolipid during growth with long-chain alkanes. A high-performance liquid chromatography (HPLC) method was developed for absolute quantification. This method is based on the conversion of the glycine-glucolipid to phenacyl esters with subsequent measurement by HPLC with diode array detection (HPLC-DAD). Different molecular species were separated by HPLC and identified as glucosyl-tetra(3-hydroxy-acyl)-glycine with varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups via mass spectrometry. The growth rate of A. borkumensis cells with pyruvate as the sole carbon source was elevated compared to hexadecane as recorded by the increase in cell density as well as oxygen/carbon dioxide transfer rates. The amount of the glycine-glucolipid produced per cell during growth on hexadecane was higher compared with growth on pyruvate. The glycine-glucolipid from pyruvate-grown cells contained considerable amounts of 3-hydroxy-octanoic acid, in contrast to hexadecane-grown cells, which almost exclusively incorporated 3-hydroxy-decanoic acid into the glycine-glucolipid. The predominant proportion of the glycine-glucolipid was found in the cell pellet, while only minute amounts were present in the cell-free supernatant. The glycine-glucolipid isolated from the bacterial cell broth, cell pellet, or cell-free supernatant showed the same structure containing a glycine residue, in contrast to previous reports, which suggested that a glycine-free form of the glucolipid exists which is secreted into the supernatant. In conclusion, the glycine-glucolipid of A. borkumensis is resident to the cell wall and enables the bacterium to bind and solubilize alkanes at the lipid-water interface. IMPORTANCE Alcanivorax borkumensis is one of the most abundant marine bacteria found in areas of oil spills, where it degrades alkanes. The production of a glycine-glucolipid is considered an essential element for alkane degradation. We developed a quantitative method and determined the structure of the A. borkumensis glycine-glucolipid in different fractions of the cultures after growth in various media. Our results show that the amount of the glycine-glucolipid in the cells by far exceeds the amount measured in the supernatant, confirming the proposed cell wall localization. These results support the scenario that the surface hydrophobicity of A. borkumensis cells increases by producing the glycine-glucolipid, allowing the cells to attach to the alkane-water interface and form a biofilm. We found no evidence for a glycine-free form of the glucolipid.

A multigene approach secures hydroxy fatty acid production in Arabidopsis.

A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from more than a dozen wild species, progress has been limited because expression of these enzymes in transgenic plants produces only low yields of the desired products. For example, fatty acid hydroxylase 12 (FAH12) from castor (Ricinus communis) produces only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis (Arabidopsis thaliana), compared with 90% HFAs in castor seeds. The transgenic plants also have reduced oil content and seed vigor. Here, we review experiments that have provided for steady increased HFA accumulation and oil content. This research has led to exciting new discoveries of enzymes and regulatory processes in the pathways of both seed oil synthesis and lipid metabolism in other parts of the plant. Recent investigations have revealed that HFA-accumulating seeds are unable to rapidly mobilize HFA-containing triacylglycerol (TAG) storage lipid after germination to provide carbon and energy for seedling development, resulting in reduced seedling establishment. These findings present a new opportunity to investigate a different, key area of lipid metabolism-the pathways of TAG lipolysis and ß-oxidation in germinating seedlings.

Chemical characterization of ceramides isolated from a gliding marine bacterium Aureispira marina.

Ceramides containing 2-hydroxy fatty acids were purified from a gliding marine bacterium Aureispira marina, and their chemical structure was investigated. The ceramide molecules contained 2-hydroxy-15-methyl-hexadecanoic acid and 2-hydroxy-15-methyl-hexadecenoic acid, and the double bond of the latter fatty acid was proved to be located between the positions C3 and C4. The major portion of these 2-hydroxy fatty acids was determined to have D-configuration (S-configuration) after diastereomeric derivatization. Three carbon skeletons were found in sphingosines from ceramides, ie (1) 1,3-dihydroxy-2-amino-4-octadecen, (2) 1,3-dihydroxy-2-amino-17-methyl-4-octadecen, and (3) 1,3-dihydroxy-2-amino-9-methyl-4-octadecen. Molecules with additional double bonds were found in sphingosines with structures 1 and 3. The presence of ceramides with these chemical characteristics would be a significant feature for the taxonomy of A. marina and related bacteria.

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.

Uncovering potential interspecies signaling factors in plant-derived mixed microbial culture.

Microbes use signaling factors for intraspecies and interspecies communications. While many intraspecies signaling factors have been found and characterized, discovery of factors for interspecies communication is lagging behind. To facilitate the discovery of such factors, we explored the potential of a mixed microbial culture (MMC) derived from wheatgrass, in which heterogeneity of this microbial community might elicit signaling factors for interspecies communication. The stability of Wheatgrass MMC in terms of community structure and metabolic output was first characterized by 16S ribosomal RNA amplicon sequencing and liquid chromatography/mass spectrometry (LC/MS), respectively. In addition, detailed MS analyses led to the identification of 12-hydroxystearic acid (12-HSA) as one of the major metabolites produced by Wheatgrass MMC. Stereochemical analysis revealed that Wheatgrass MMC produces mostly the (R)-isomer, although a small amount of the (S)-isomer was also observed. Furthermore, 12-HSA was found to modulate planktonic growth and biofilm formation of various marine bacterial strains. The current study suggests that naturally derived MMCs could serve as a simple and reproducible platform to discover potential signaling factors for interspecies communication. In addition, the study indicates that hydroxylated long-chain fatty acids, such as 12-HSA, may constitute a new class of interspecies signaling factors.

Novel mannosylerythritol lipid biosurfactant structures from castor oil revealed by advanced structure analysis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by fungi of the Ustilaginaceae family in the presence of hydrophobic carbon sources like plant oils. In the present study, we investigated the structural composition of MELs produced from castor oil using seven different microorganisms and compared them to MEL structures resulting from other plant oils. Castor oil is an industrially relevant plant oil that presents as an alternative to currently employed edible plant oils like rapeseed or soybean oil. The main fatty acid in castor oil is the mono-hydroxylated ricinoleic acid, providing the possibility to produce novel MEL structures with interesting features. Analysis of the produced MELs from castor oil by different chromatographic and mass spectrometry techniques revealed that all seven microorganisms were generally able to integrate hydroxylated fatty acids into the MEL molecule, although at varying degrees. These novel MELs containing a hydroxy fatty acid (4-O-[2'-O-alka(e)noyl-3'-O-hydroxyalka(e)noyl-4'/6'-O-acetyl-ß-D-mannopyranosyl]-erythritol) were more hydrophilic than conventional MEL and therefore showed a different elution behavior in chromatography. Large shares of novel hydroxy MELs (around 50% of total MELs) were found for the two MEL-B/C producing species Ustilago siamensis and Ustilago shanxiensis, but also for the MEL-A/B/C producer Moesziomyces aphidis (around 25%). In addition, tri-acylated hydroxylated MELs with a third long-chain fatty acid esterified to the free hydroxyl group of the hydroxy fatty acid were identified for some species. Overall, production of MEL from castor oil with the investigated organisms provided a complex mixture of various novel MEL structures that can be exploited for further research.

Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

Catalytic residues, substrate specificity, and role in carbon starvation of the 2-hydroxy FA dioxygenase Mpo1 in yeast.

The yeast protein Mpo1 belongs to a protein family that is widely conserved in bacteria, fungi, protozoa, and plants, and is the only protein of this family whose function has so far been elucidated. Mpo1 is an Fe2+-dependent dioxygenase that catalyzes the α-oxidation reaction of 2-hydroxy (2-OH) long-chain FAs (LCFAs) produced in the degradation pathway of the long-chain base phytosphingosine. However, several biochemical characteristics of Mpo1, such as its catalytic residues, membrane topology, and substrate specificity, remain unclear. Here, we report that yeast Mpo1 contains two transmembrane domains and that both its N- and C-terminal regions are exposed to the cytosol. Mutational analyses revealed that three histidine residues conserved in the Mpo1 family are especially important for Mpo1 activity, suggesting that they may be responsible for the formation of coordinate bonds with Fe2+ We found that, in addition to activity toward 2-OH LCFAs, Mpo1 also exhibits activity toward 2-OH very-long-chain FAs derived from the FA moiety of sphingolipids. These results indicate that Mpo1 is involved in the metabolism of long-chain to very-long-chain 2-OH FAs produced in different pathways. We noted that the growth of mpo1Δ cells is delayed upon carbon deprivation, suggesting that the Mpo1-mediated conversion of 2-OH FAs to nonhydroxy FAs is important for utilizing 2-OH FAs as a carbon source under carbon starvation. Our findings help to elucidate the as yet unknown functions and activities of other Mpo1 family members.

Oleate hydratase from Staphylococcus aureus protects against palmitoleic acid, the major antimicrobial fatty acid produced by mammalian skin.

Oleate hydratases (OhyAs) belong to a large family of bacterial proteins catalyzing the hydration or isomerization of double bonds in unsaturated fatty acids. A Staphylococcus aureus gene (Sa0102) is predicted to encode an OhyA. Here, we recombinantly expressed and purified SaOhyA and found that it forms a homodimer that requires FAD for activity. SaOhyA hydrates only unsaturated fatty acids containing cis-9 double bonds, but not fatty acids with trans-9 double bonds or cis double bonds at other positions. SaOhyA products were not detected in S. aureus phospholipids and were released into the growth medium. S. aureus does not synthesize unsaturated fatty acids, and the SaOhyA substrates are derived from infection sites. Palmitoleate (16:1(9Z)) is a major mammalian skin-produced antimicrobial fatty acid that protects against S. aureus infection, and we observed that it is an SaOhyA substrate and that its hydroxylated derivative is not antimicrobial. Treatment of S. aureus with 24 µm 16:1(9Z) immediately arrested growth, followed by growth resumption after a lag period of 2 h. The ΔohyA mutant strain did not recover from the 16:1(9Z) challenge, and increasing SaOhyA expression using a plasmid system prevented the initial growth arrest. Challenging S. aureus with sapienic acid (16:1(6Z)), an antimicrobial fatty acid produced only by human skin, arrested growth without recovery in WT, ΔohyA, and SaOhyA-overexpressing strains. We conclude that SaOhyA protects S. aureus from palmitoleic acid, the antimicrobial unsaturated fatty acid produced by most mammals, and that sapienic acid, uniquely produced by humans, counters the OhyA-dependent bacterial defense mechanism.

Castor patatin-like phospholipase A IIIß facilitates removal of hydroxy fatty acids from phosphatidylcholine in transgenic Arabidopsis seeds.

KEY MESSAGE: Castor patatin-like phospholipase A IIIß facilitates the exclusion of hydroxy fatty acids from phosphatidylcholine in developing transgenic Arabidopsis seeds. Hydroxy fatty acids (HFAs) are industrial useful, but their major natural source castor contains toxic components. Although expressing a castor OLEATE 12-HYDROXYLASE in Arabidopsis thaliana leads to the synthesis of HFAs in seeds, a high proportion of the HFAs are retained in phosphatidylcholine (PC). Thus, the liberation of HFA from PC seems to be critical for obtaining HFA-enriched seed oils. Plant phospholipase A (PLA) catalyzes the hydrolysis of PC to release fatty acyl chains that can be subsequently channeled into triacylglycerol (TAG) synthesis or other metabolic pathways. To further our knowledge regarding the function of PLAs from HFA-producing plant species, two class III patatin-like PLA cDNAs (pPLAIIIß or pPLAIIIδ) from castor or Physaria fendleri were overexpressed in a transgenic line of A. thaliana producing C18-HFA, respectively. Only the overexpression of RcpPLAIIIß resulted in a significant reduction in seed HFA content with concomitant changes in fatty acid composition. Reductions in HFA content occurred in both PC and TAG indicating that HFAs released from PC were not incorporated into TAG. These results suggest that RcpPLAIIIß may catalyze the removal of HFAs from PC in the developing seeds synthesizing these unusual fatty acids.

Production of long-chain hydroxy fatty acids by Starmerella bombicola.

To decrease our dependency for the diminishing source of fossils resources, bio-based alternatives are being explored for the synthesis of commodity and high-value molecules. One example in this ecological initiative is the microbial production of the biosurfactant sophorolipids by the yeast Starmerella bombicola. Sophorolipids are surface-active molecules mainly used as household and laundry detergents. Because S. bombicola is able to produce high titers of sophorolipids, the yeast is also used to increase the portfolio of lipophilic compounds through strain engineering. Here, the one-step microbial production of hydroxy fatty acids by S. bombicola was accomplished by the selective blockage of three catabolic pathways through metabolic engineering. Successful production of 17.39 g/l (ω-1) linked hydroxy fatty acids was obtained by the successive blockage of the sophorolipid biosynthesis, the ß-oxidation and the ω-oxidation pathways. Minor contamination of dicarboxylic acids and fatty aldehydes were successfully removed using flash chromatography. This way, S. bombicola was further expanded into a flexible production platform of economical relevant compounds in the chemical, food and cosmetic industries.

Lipidomic Profiling of the Olive (Olea europaea L.) Fruit towards Its Valorisation as a Functional Food: In-Depth Identification of Triacylglycerols and Polar Lipids in Portuguese Olives.

Olives (Olea europaea L.) are classic ingredients in the Mediterranean diet with well-known health benefits, but their lipid composition has not been fully addressed. In this work, we characterised triacylglycerol (TAG) and polar lipid profiles of the olive pulp while using a complementary methodological approach that was based on solid-phase extraction to recover the neutral lipid (NL) and the polar lipid-rich fractions. The TAG profile was analysed in the NL-fraction by C30 reversed-phase liquid chromatography (LC) and the polar lipid profile by normal-phase hydrophilic interaction liquid chromatography (HILIC), with both being coupled to electrospray ionization-mass spectrometry (ESI-MS) and ESI-MS/MS. This approach identified 71 TAG ions that were attributed to more than 350 molecular species, with fatty acyl chain lengths from C11:0 to C26:0, including different polyunsaturated acyl chains. The polar lipids included 107 molecular species that belonged to 11 lipid classes that comprised phospholipids, glyceroglycolipids, glycosphingolipids, and betaine lipids. In addition to polyunsaturated fatty acids, some of the phospholipids, glycolipids, and glycosphingolipids that were identified in the olive pulp have been described as biologically active molecules. Lipidomic phenotyping of the olive pulp has led to the discovery of compounds that will allow for a better assessment of its nutritional value and new applications of bioactive lipid components in this functional food.

Overexpression of Seipin1 Increases Oil in Hydroxy Fatty Acid-Accumulating Seeds.

While plant oils are an important source of food, plants also produce oils containing specialized fatty acids with chemical and physical properties valued in industry. Ricinoleic acid, a hydroxy fatty acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized fatty acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.

Personal exposure of dairy workers to dust, endotoxin, muramic acid, ergosterol, and ammonia on large-scale dairies in the high plains Western United States.

Dairy workers experience a high degree of bioaerosol exposure, composed of an array of biological and chemical constituents, which have been tied to adverse health effects. A better understanding of the variation in the magnitude and composition of exposures by task is needed to inform worker protection strategies. To characterize the levels and types of exposures, 115 dairy workers grouped into three task categories on nine farms in the high plains Western United States underwent personal monitoring for inhalable dust, endotoxin, 3-hydroxy fatty acids (3-OHFA), muramic acid, ergosterol, and ammonia through one work shift. Eighty-nine percent of dairy workers were exposed to endotoxin at concentrations exceeding the recommended exposure guidelines (adjusted for a long work shift). The proportion of workers with exposures exceeding recommended guidelines was lower for inhalable dust (12%), and ammonia (1%). Ergosterol exposures were only measurable on 28% of samples, primarily among medical workers and feed handlers. Milking parlor workers were exposed to significantly higher inhalable dust, endotoxin, 3-OHFA, ammonia, and muramic acid concentrations compared to workers performing other tasks. Development of large modern dairies has successfully made progress in reducing worker exposures and lung disease prevalence. However, exposure to endotoxin, dust, and ammonia continues to present a significant risk to worker health on North American dairies, especially for workers in milking parlors. This study was among the first to concurrently evaluate occupational exposure to assayable endotoxin (lipid A), 3-hydroxy fatty acids or 3-OHFA (a chemical measure of cell bound and noncell-bound endotoxins), muramic acid, ergosterol, and ammonia among workers on Western U.S. dairies. There remains a need for cost-effective, culturally acceptable intervention strategies integrated in OHS Risk Management and production systems to further optimize worker health and farm productivity.