Characterisation of Acetyl‐CoA Thiolase: The First Enzyme in the Biosynthesis of Terpenic Sex Pheromone Components in the Labial Gland of Bombus terrestris

Buff‐tailed bumblebees, Bombus terrestris, use a male sex pheromone for premating communication. Its main component is a sesquiterpene, 2,3‐dihydrofarnesol. This paper reports the isolation of a thiolase (acetyl‐CoA thiolase, AACT_BT), the first enzyme involved in the biosynthetic pathway leading to formation of isoprenoids in the B. terrestris male sex pheromone. Characterisation of AACT_BT might contribute to a better understanding of pheromonogenesis in the labial gland of B. terrestris males. The protein was purified to apparent homogeneity by column chromatography with subsequent stepwise treatment. AACT_BT showed optimum acetyltransferase activity at pH 7.1 and was strongly inhibited by iodoacetamide. The enzyme migrated as a band with an apparent mass of 42.9 kDa on SDS‐PAGE. MS analysis of an AACT_BT tryptic digest revealed high homology to representatives of the thiolase family. AACT_BT has 96 % amino acid sequence identity with the previously reported Bombus impatiens thiolase.     

Chemistry of the Cephalic Labial Gland Secretions of Male Bombus morrisoni and B. rufocinctus, Two North American Bumblebee Males with Perching Behavior

The labial gland secretions from males of the North American bumblebees Bombus morrisoni Cresson and B. rufocinctus Cresson were analyzed by gas chromatography/mass spectrometry. In both species, 3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraenyl acetate was found as the major compound of a complex mixture of alkenols, acetates, hydrocarbons, and wax-type esters. In addition to a mixture of saturated and mono-unsaturated straight chain hydrocarbons, the labial gland of both species contained the isoprenoid hydrocarbons (6E, 10E)-3,7,15-trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene [β-springene], three isomers of 3,7,11,15-tetramethyl-hexadeca-1,3,6,10,14-pentaene [α-springene], and two further unidentified cyclic diterpenes. In B. morrisoni, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol and 3,7,11,15-tetramethyl-6,10,14-hexadecatrien-1-ol were detected as characteristic alcohols, as well as small amounts of 9-hexadecenol and hexadecanol. Furthermore, a large peak of hexadecyl dodecanoate and minor amounts of 9-hexadecenyl, 9-octadecenyl, and eicosenyl 9-tetradecenoate were found as typical esters in this species. In B. rufocinctus, 9-hexadecenol, hexadecanol, and 9-octadecenol were present in considerable amounts, with their acetates and 9-tetradecenoic, tetradecanoic, hexadecanoic, and 11-octadecenoic acids. The chemical composition of cephalic labial glands in male bumblebees with perching behavior is discussed.     

Biosynthesis of the spiroacetal suite in Bactrocera tryoni

In pursuit of a more environmentally benign method of controlling the highly pestiferous Queensland fruit fly, Bactrocera tryoni, the biosynthesis of the minor components in the suite of spiroacetals released by females has been investigated. This follows on the biosynthetic definition of the pathway to the major component, (E,E)-1. The origins of the C(12) and C(13) spiroacetals (E,E)-2 and (E,E)-3, respectively, have been investigated by the administration of over 30 deuterated potential precursors. Analysis of the relative incorporation levels and identification of some of the exceptionally minor spiroacetals that were biosynthesised established that B. tryoni processes fatty acids to 2,6-dioxygenated precursors by a modified β-oxidation pathway, with a suite of putative cytochromes P450 employed in the crucial oxidative steps, prior to cyclisation of the proposed ketodiol.     

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Hydroxycinnamic acid esters (HCEs) are widely‐distributed phenylpropanoid‐derived plant natural products. Rosmarinic acid (RA), the most well‐known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, microbial production of HCEs could be a sustainable, controlled means of production. Through the overexpression of a six‐enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof‐of‐concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non‐natural HCEs.     

The impact of fatty acid profile on the physicochemical properties of commercial margarines in Brazil

This study aimed to evaluate how lipid profiles affect the physicochemical properties, fatty acid profiles, and nutritional qualities of Brazilian margarines. We analyzed the texture profiles of 13 margarine samples and characterized their fatty acid composition, solid fat content, crystallization kinetics by NMR and thermal behavior by differential scanning calorimetry. The samples had total fat content ranging from 20% to 82% and low trans fatty acid (TFA) levels, except for two samples (5–7% elaidic acid). The fatty acid compositions of all samples showed a predominance of linoleic (23%–46%), oleic (20%–46%), and palmitic acids (7%–14%), indicating that they were formulated with soybean and palm oils. Saturated fat content ranged from 23% to 31%. Compared to the other evaluated samples, those with higher content of lipid and saturated fatty acids (SFAs) exhibited increased hardness and stickiness but reduced spreadability and adhesiveness. The presence of TFAs resulted in increased plasticity of the samples. Reformulation resulted in products with greater SFA levels, which had a negative impact as it increased the atherogenic index (AI: 0.22–0.48). The HF55 sample contained canola oil-based fat and presented the best nutritional and physical properties. This study is the first to report a complete evaluation of representative margarines, with essential information in reformulating to achieve lower SFA.     

Biosynthesis of Long Chain Polyunsaturated Fatty Acids in the Marine Ichthyosporean Sphaeroforma arctica

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the ω3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80 % of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a Δ8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate Δ8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a Δ4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies.     

Unusual Fatty Acids in the Fat Body of the Early Nesting Bumblebee, <Emphasis Type="Italic">Bombus pratorum</Emphasis>

Unusual fatty acids with 24, 26, and 28 carbon atoms were found in triacylglycerols (TAGs) isolated from fat body tissue of bumblebee Bombus pratorum. The most abundant one was (Z,Z)-9,19-hexacosadienoic acid. Its structure was determined by mass spectrometry after derivatization with dimethyl disulfide and by infrared spectroscopy. ECL (equivalent chain length) values of its methyl ester were determined on both DB-1 and DB-WAX capillary columns. (Z,Z)-9,19-Hexacosadienoic acid is quite rare in nature. So far it has been identified only in marine sponges, and this work is the first evidence of its occurrence in a terrestrial organism. HPLC/MS analysis of the bumblebee TAGs showed that (Z,Z)-9,19-hexacosadienoic acid is present in one third of all TAG molecular species. As it was found in all sn-TAG positions, it is likely that (Z,Z)-9,19-hexacosadienoic acid is transported to tissues. Interestingly, labial gland secretion of B. pratorum was found to contain (Z,Z)-7,17-pentacosadiene, a hydrocarbon with markedly similar double bond positions and geometry. Possible biosynthetic relationships between these two compounds are discussed.     

Expression of Genes Controlling Unsaturated Fatty Acids Biosynthesis and Oil Deposition in Developing Seeds of Sacha Inchi (Plukenetia volubilis L.)

Sacha inchi (Plukenetia volubilis L., Euphorbiaceae) seed oil is rich in α-linolenic acid, a kind of n-3 fatty acids with many health benefits. To discover the mechanism underlying α-linolenic acid accumulation in sacha inchi seeds, preliminary research on sacha inchi seed development was carried out from one week after fertilization until maturity, focusing on phenology, oil content, and lipid profiles. The results suggested that the development of sacha inchi seeds from pollination to mature seed could be divided into three periods. In addition, investigations on the effect of temperature on sacha inchi seeds showed that total oil content decreased in the cool season, while unsaturated fatty acid and linolenic acid concentrations increased. In parallel, expression profiles of 17 unsaturated fatty acid related genes were characterized during seed development and the relationships between gene expression and lipid/unsaturated fatty acid accumulation were discussed.     

Genomic Regions Governing the Biosynthesis of Unsaturated Fatty Acids in Recombinant Inbred Lines of Soybean Raised across Multiple Growing Years

The ratio of oleic acid to the combined value of linoleic and α-linolenic acids determines the oxidative stability, and the ratio of linoleic acid to α-linolenic acid is the key to the nutritional value of soybean oil. The present study was conducted to identify genomic regions associated with oleic, linoleic, and α-linolenic acids in recombinant inbred lines (RIL), developed from LSb1 × NRC7, across 5 cropping years. These RIL were genotyped using 105 polymorphic SSR markers across soybean genome and analyzed for fatty acid composition. SSR markers, namely, Satt245 (LGp M), Satt556 (LGpB2), Sat_042 (LGp C1), Staga002 (LGp D1b), Satt684 (LGp A1), and AI856415 (LGpD1b) showed significant (P < 0.05) association with oleic acid for all the 5 years, though this association was weak in the years when the growing temperature during active seed formation stage was high. Quantitative trait loci (QTL) linked to Satt684 (LGp A1), Satt556 (LGp B2), Sat_042 (LGp C1), and AI856415 (LGp D1b) showed pleiotropic influence on the levels of unsaturated fatty acids. Complementation of favorable QTL from LSb1 and NRC7 generated 60% oleic acid and less than 4% α-linolenic acid RIL, stable across 5 cropping years. New SSR markers, namely, Satt245, AI856415, and Staga002 identified to be associated with different unsaturated fatty acids may be useful in improving the efficiency of marker-assisted breeding for enhancing the monounsaturated to polyunsaturated fatty acids ratio of soybean oil.     

Double Bond Position Plays an Important Role in Delta-9 Desaturation and Lipogenic Properties of Trans 18:1 Isomers in Mouse Adipocytes

The objective of this research was to study the delta‐9 desaturation of individual trans (t) fatty acids that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on lipogenic gene expression in adipocytes. It was hypothesized that delta‐9 desaturation and lipogenic properties of t‐18:1 isomers depend on the position of double bond. Differentiated 3T3‐L1 adipocytes were treated with 200 µM of t6–18:1, t9–18:1, t11–18:1, t13–18:1 or t16–18:1, cis (c)‐9 18:1 or bovine serum albumin (BSA) vehicle control for 48 h. Cells were then harvested for fatty acid and gene expression analyses using gas chromatography and quantitative PCR respectively. Among t‐18:1 isomers, t13–18:1 and t11–8:1 had the greatest percent delta‐9 desaturation (44 and 41 % respectively) followed by t16–18:1 and t6–18:1 (32 and 17 % respectively), while c9–18:1 and t9–18:1 did not undergo delta‐9 desaturation. Trans9–18:1 up‐regulated (P < 0.05) the expression of lipogenic genes including fatty acid synthase and stearoyl‐CoA desaturase‐1 (P < 0.05), whereas the expression of these genes were not affected with other t‐18:1 isomers (P > 0.05). Consistent with gene expression results, t9–18:1 increased the de novo lipogenic index (16:0/18:2n‐6) compared with control cells and increased delta‐9 desaturation index (c9–16:1/18:0) compared to other t‐18:1 isomers (P < 0.05). The current study provides further evidence that the predominant trans fatty acid in PHVO (t9–18:1) has isomer specific lipogenic properties.     

Comparison of Age-dependent Quantitative Changes in the Male Labial Gland Secretion of <Emphasis Type="Italic">Bombus Terrestris</Emphasis> and <Emphasis Type="Italic">Bombus Lucorum</Emphasis>

Age-related changes of antennal-active components of male labial gland extracts were studied in two closely related bumblebee species, Bombus terrestris and B. lucorum. In B. terrestris, compounds eliciting electroantennogram (EAG) responses of virgin queens were ethyl dodecanoate, 2,3-dihydrofarnesal, 2,3-dihydrofarnesol, hexadecan-1-ol, octadeca-9,12,15-trien-1-ol, and geranylcitronellol. Compounds that elicited EAG responses from queens of B. lucorum were ethyl dodecanoate, ethyl tetradec-7-enoate, ethyl tetradec-9-enoate, ethyl hexadec-9-enoate, hexadecan-1-ol, hexadec-7-enal, octadeca-9,12-dien-1-ol, octadeca-9,12,15-trien-1-ol, and octadecan-1-ol. Quantities of these compounds in the labial glands changed significantly over the lifetime of the respective males of the two species. In both species, concentrations of the respective compounds reached their maximum within seven days after eclosion. Subsequently, a rapid decrease in the amount of EAG-active compounds occurred in B. terrestris, whereas in B. lucorum the amount of active compounds stayed approximately constant or decreased at a slow rate. Microscopy showed that in B. terrestris secretory cells of the labial glands undergo apoptosis from the fifth to the tenth day of life, whilst in B. lucorum labial gland cells remain unchanged throughout the life of the males.     

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a d ‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l ‐aculose or l ‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an l ‐aculose moiety to 3‐OH of the angucycline core.     

Geographic Variation and Tissue Distribution of Endogenous Terpenoids in the Northeastern Pacific Ocean Dorid Nudibranch Cadlina luteomarginata: Implications for the Regulation of De Novo Biosynthesis

Gas chromatography (GC) analyses of whole animal skin extracts and individual tissue extracts obtained from specimens of Cadlina luteomarginata collected in British Columbia and southern California were used to determine if concentrations of the nudibranch's biosynthetic products—albicanyl acetate (1), cadlinaldehyde (2), and luteone (3)—vary significantly between two populations, among individuals of a population, and among body tissues of individual specimens. The major biosynthetic product, albicanyl acetate (1), has the same concentration in both British Columbia and California populations, while the British Columbia population contains greater total amounts of 2 and 3 than the California population. Within individuals from one population, the largest proportion of endogenous metabolites is in the dorsum, specifically in the mantle dermal formations and margins. The GC analyses show that across geographically separated populations and within geographically localized populations the concentration of endogenous metabolites is inversely correlated with availability of structurally similar compounds from dietary sources. This suggests that the de novo biosynthesis of defensive compounds might be regulated according to need.