Production of rhamnolipids-producing enzymes of <Emphasis Type="Italic">Pseudomonas</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis> and structural characterization

Rhamnolipids are a class of biosurfactants that have a great potential to be used in industries. Five proteins/enzymes, namely RhlA, RhlB, RhlC, RhlG and RhlI, are critical for the production of rhamnolipids in Pseudomonas aeruginosa. Four of the 5 proteins except RhlC were successfully over-expressed in E. coli and three of them (RhlA, RhlB and RhlI) were purified and obtained in milligram quantities. The purified proteins were shown to be folded in solution. Homology models were built for RhlA, RhlB and RhlI. These results lay a basis for further structural and functional characterization of these proteins in vitro to favor the construction of super strains for rhamnolipids production.

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Expression of redesigned mussel silk-like protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Silks have been used widely for human beings due to their several extraordinary properties. Until now, the studies on silk proteins have mainly focused on spiders and silkworms. Because silk properties are organism-dependent, novel silk protein types can be found and developed through investigation of new silk-bearing organisms. We noticed that marine mussel has silk-like domains containing many repeats with abundance of glycine and alanine. In the present work, we redesigned mussel-derived silk-like gene sequence which contains alternating repeated and nonrepeated regions with optimized codons for Escherichia coli. For successful expression of recombinant mussel silk-like protein in E. coli cells, we employed several experimental strategies, including use of strong promoter, cold shock expression, and genetic fusions. We observed significant repression on cell growths by even low expression levels of soluble mussel silk-like proteins in cold shock- and glutathione s-transferase (GST) fusion-based systems. Thus, we finally used baculoviral polyhedrin protein as a fusion partner and successfully expressed insoluble mussel silk-like protein with relatively high expression level and without cell growth repression in E. coli.     

Biotransformation of medium-chain alkanes using recombinant P450 monooxygenase from <Emphasis Type="Italic">Alcanivorax borkumensis</Emphasis> SK2 expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A bioconversion system for medium-chain alkanes was constructed by using a recombinant Escherichia coli whole-cell biocatalyst expressing P450 monooxygenase genes, ferredoxin, and ferredoxin reductase cloned from Alcanivorax borkumensis as an operon. The recombinant E. coli harboring the P450 gene and two related expression component enzymes, ferredoxin and ferredoxin reductase, was constructed in a single vector pET21(a) and successfully expressed in E. coli BL21(DE3) as a soluble form, showing a molecular weight of 53 kDa on 10% SDS-PAGE. When the cell-free extract of E. coli BL21 expressing p450 monooxygenase was subjected to reduced CO difference spectral analysis, a soret band near 450 nm appeared indicating that the cloned P450 was expressed as a functionally active enzyme. The E. coli cells harboring the expressed P450 gene were able to convert n-octane and 1-decene, producing approximately 450 μg/ml of n-octanol and 290 μg/ml of 1,2-epoxydecane, respectively, at pH 7.0 and 30 °C. However, the recombinant E. coli cells were not able to convert the branched alkane, 2,6,10,14-tetramethylpentadecane (C19).     

Cloning of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles. The cloned gene was 1,480 nucleotides in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides. The open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide, with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases. However, this protein had a low homology with lipases of P. camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level. The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation. A distinct band with a M.W. of 33 kDa was detected on SDS-PAGE. Results of a Western blot analysis and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase. Its catalytic properties were not changed when compared with its natural counterpart. Zhikang Qian and Peihong Jiang are equal contributors as first author of this paper.     

Multiple-copy-gene integration on chromosome of <Emphasis Type="Italic">Escherichia coli</Emphasis> for beta-galactosidase production

Recombinant E. coli strains with 1–3 copies of lacZ genes on their chromosomes were constructed and their β-galactosidase (β-gal) expressions were examined. Serial dilution cultures were used to analyze the long-term genetic stability of the recombinant lacZ genes of the chromosomal or plasmid expression system. The strain with a 3-copy lacZ on the chromosome has a sustainable β-gal expression through 60 hours. However, the β-gal activity of the plasmid expression system lasted less than 36 hours under a no selection condition. Obviously, the genetic stability of the chromosomal expression system demonstrated in this study is better than that of the plasmid expression system under nonselective condition, such as a medium without antibiotics. The results demonstrated that the strains with a multiple-copy-gene on the chromosome are useful for protein production in industrial repeated fed-batch fermentation. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007.     

Constitutive overexpression of <Emphasis Type="Italic">Pseudoalteromonas carrageenovora</Emphasis> arylsulfatase in <Emphasis Type="Italic">E. coli</Emphasis> fed-batch culture

The arylsulfatase gene (astA) from Pseudoalteromonas carrageenovora genome was subcloned into pHCEIA vector, in which the hyper constitutive expression (HCE) promoter from the d-amino acid aminotransferase (DAAT) gene of Geobacillus toebii was employed. When the constructed pHCE-AST was introduced into E. coli, the transformant showed the hydrolyzing activity for 4-methylumbelliferyl-sulfate and p-nitrophenyl-sulfate. When the cell was cultured on fermentor containing MaxyBroth-HD medium with 1% glycerol, the enzyme activity reached 12.8 unit/mL. On MaxyBroth-HD medium with 2% glycerol, the cell showed 2.7-fold higher arylsulfatase expression than that with 1% glycerol. The fed-batch cultivation employing MaxyBroth-HD medium and additional feeding of glycerol gave about 143 unit/mL of arylsulfatase at 20 h, which corresponds to 4-fold higher enzyme activity than that of 2% glycerol batch culture. Most of arylsulfatase activity in fed-batch culture was produced in the extracellular medium, whereas the activity in the batch cultures was localized in the periplasmic cell space.     

Efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-1,2,4-butanetriol from <Emphasis Type="SmallCaps">d</Emphasis>-xylose by engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> whole-cell biocatalysts

We have developed a whole-cell bioconversion system for the production of d-1,2,4-butanetriol (BT) from renewable biomass. A plasmid pETduet-xylB-yjhG-T7-adhP-T7-mdlC was constructed and transformed to Escherichia coli BL21(DE3) to obtain the whole cells of E. coli BL21-XYMA capable of bioconversion d-xylose to BT. Then, the factors including carbon sources, nitrogen sources, metal ions, and culture conditions (pH, temperature, IPTG) were identified, and their effects on the whole-cell activity for BT production were investigated. To obtain the highest whole-cell activity, the optimal cultivation parameters are: 15 g·L^–1 yeast extract, 5 g·L^–1 sucrose, 3 g·L^–1 KH₂PO₄, 5 g·L^–1 NaCl, 3 g·L^–1 NH₄Cl, 0.25 g·L^–1 MgSO₄·7H₂O and 1 mL·L^–1 the mixture of trace elements. With the optimized whole cells of E. coli BL21-XYMA, 60 g·L^–1 of xylose was converted to 28 g·L^–1 BT with a molar yield of 66 %, which is higher than those reported in the biotechnological system.

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Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> strains bind bovine milk gangliosides in a ceramide-dependent process

Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is the main infectious disease of newborn calves. The first step of infection involves bacterial attachment to the intestinal mucosa. This adhesion is mediated by fimbriae that recognize some glycoconjugates on the host cell surface, in particular, several gangliosides. Because milk also contains gangliosides, these have been suggested to serve as ligands for bacterial fimbriae and thus prevent the bacterial attachment to mucosa. The most relevant ETEC strains in calves, including those with K99 and F41 fimbriae, were assayed to determine whether they are able to bind gangliosides isolated from several stages of bovine lactation. Both GM3 and GD3, the main gangliosides of milk, were recognized by ETEC strains, although the different fimbriae showed diverse levels of affinity. Unexpectedly, the adhesion to colostral gangliosides was considerably weaker than that to gangliosides from the other stages of lactation. Because the carbohydrate moiety did not change and because differences in the percentages of unsaturated FA and sphingosine between colostrum and other stages were observed, we conclude that the differences in adhesion could be due to a different composition of the ganglioside caramide.     

2-Methoxylated FA Display Unusual Antibacterial Activity Towards Clinical Isolates of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (CIMRSA) and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The naturally occurring (6Z)-(±)-2-methoxy-6-hexadecenoic acid (1) and (6Z)-(±)-2-methoxy-6-octadecenoic acid (2) were synthesized in 7–8 steps with 38 and 13% overall yields, respectively, by using an acetylide coupling approach, which made it possible to obtain a 100% cis-stereochemistry for the double bonds. In a similar fashion, the acetylenic analogs (±)-2-methoxy-6-hexadecynoic acid (3) and (±)-2-methoxy-6-octadecynoic acid (4) were also synthesized in 6–7 steps with 48 and 16% overall yields, respectively. The antibacterial activity of acids 1–4 was determined against clinical isolates of methicillin-resistant Staphylococcus aureus (ClMRSA) and Escherichia coli. Among the series of compounds, acid 4 was the most active bactericide towards CIMRSA displaying IC_50s (half maximal inhibitory concentrations) between 17 and 37 μg/mL, in sharp contrast to the 6-octadecynoic acid, which was not bactericidal at all. On the other hand, acids 1 and 3 were the only acids that displayed antibacterial activity towards E. coli, but 1 stood out as the best candidate with an IC₅₀ of 21 μg/mL. The critical micelle concentrations (CMCs) of acids 1–4 were also determined. The C18 acids 2 and 4 displayed a five-fold lower CMC (15–20 μg/mL) than the C16 analogs 1 and 3 (70–100 μg/mL), indicating that 4 exerts its antibacterial activity in a micellar state. None of the studied acids were inhibitory towards S. aureus DNA gyrase discounting this type of enzyme inhibition as a possible antibacterial mechanism. It was concluded that the combination of α-methoxylation and C-6 unsaturation increases the bactericidal activity of the C16 and C18 FA towards the studied bacterial strains. Acids 1 and 4 stand out as viable candidates to be used against E. coli and CIMRSA, respectively.     

Construction of methanol sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by the introduction of novel chimeric MxcQZ/OmpR two-component system from <Emphasis Type="Italic">Methylobacterium organophilum</Emphasis> XX

Methylobacterium organophilum XX is a type II facultative methylotroph that can grow on methanol. In M. organophilum XX, the MxcQ/MxcE two-component system (TCS) is involved in methanol metabolism. EnvZ/OmpR in E. coli TCS was exploited to develop a methanol biosensor by engaging the MxcQ/MxcE TCS system. The MxcQZ/OmpR methanol sensing chimeric TCS was constructed by integrating the sensing domain of M. organophilum MxcQ with the transmitter domain of E. coli EnvZ. The response regulator of the chimeric TCS system is OmpR, which regulates the expression of the ompC and gfp. The expression of ompC was monitored by real-time quantitative PCR analysis. The expression of gfp also confirmed the expression of the ompC. The maximum expression of ompC and gfp occurred with 0.05% of methanol, and the expression started to decline with further increases in methanol concentration. This system delivers rapid detection of methanol in the environment.     

<Emphasis Type="Italic">Santalum </Emphasis><Emphasis Type="Italic">insulare</Emphasis> Acetylenic Fatty Acid Seed Oils: Comparison within the <Emphasis Type="Italic">Santalum</Emphasis> Genus

The sandalwood kernels of Santalum insulare (Santalaceae) collected in French Polynesia give seed oils containing significant amounts of ximenynic acid, E-11-octadecen-9-oic acid (64–86%). Fatty acid (FA) identifications were performed by gas chromatography/mass spectrometry (GC/MS) of FA methyl esters. Among the other main eight identified fatty acids, oleic acid was found at a 7–28% level. The content in stearolic acid, octadec-9-ynoic acid, was low (0.7–3.0%). An inverse relationship was demonstrated between ximenynic acid and oleic acid using 20 seed oils. Results obtained have been compared to other previously published data on species belonging to the Santalum genus, using multivariate statistical analysis. The relative FA S. insulare composition, rich in ximenynic acid is in the same order of those given for S. album or S. obtusifolium. The other compared species (S. acuminatum, S. lanceolatum, S. spicatum and S. murrayanum) are richer in oleic acid (40–59%) with some little differences in linolenic content.     

Optimal culture condition for the production of phenyalanine ammonia lyase from <Emphasis Type="Italic">E. coli</Emphasis>

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal composition of the batch medium was 2% glucose, 1% yeast extract, 0.7% K₂HPO₄, 0.8% KH₂PO₄, 0.5% (NH₄)₂SO₄, 0.1% MgSO₄·7H₂O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37°C to 42°C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD₆₀₀ about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.     

Extraction of microbial transglutaminase from <Emphasis Type="Italic">Amycolatopsis</Emphasis> sp. fermentation broth using aqueous two-phase system

Partitioning of microbial transglutaminase (MTG) from Amycolatopsis sp. in the polyethylene glycol (PEG)/salt-based ATPS was investigated for the first time. The key parameters such as the molecular weight of PEG (PEG 600-6000), the type and concentration of phase-forming salt (ammonium sulfate or phosphates), the pH of system (pH 5.0-8.5), and the concentration of neutral salt (0-6% NaCl, w/w) were determined. The partition coefficient of the enzyme was not linear with PEG molecular weight; PEG1000 gave better yield than others. The concentration of PEG1000, ammonium sulfate and NaCl, and the system pH showed effects with different extents on specific activity (SA) and yield of the enzyme. In the ATPS of 26% w/w PEG 1000 and 19% w/w ammonium sulfate in the presence of 5% w/w NaCl and at pH 6.0, MTG was partitioned into the PEG-rich phase with a maximum yield of 86.51% and SA was increased to 0.83. The results of SDS-PAGE showed the MTG produced by the test strain differed from the enzymes reported before. Thus, this study proves that ATPS can be used as a preliminary step for partial purification of MTG from Amycolatopsis sp. fermentation broth.     

Effects of carbon source and metabolic engineering on butyrate production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Butyrate was produced in recombinant Escherichia coli strains by applying metabolic engineering strategies. The genes for producing butyrate were cloned from Clostridium acetobutylicum and then expressed in E. coli. To study important factors for improving the productivity of butyrate, we deleted pta and ptsG genes in E. coli and compared the effects of these gene deletions in E. coli B and K strains. The effect of carbon sources, glucose and glycerol, was also compared. A significant improvement of butyrate production was made when glycerol was used as a carbon source, resulting in 0.56 g/l of butyrate in LB medium with 1% (v/v) glycerol.     

(1<Emphasis Type="Italic">S</Emphasis>,3<Emphasis Type="Italic">R</Emphasis>)-<Emphasis Type="Italic">cis</Emphasis>-Chrysanthemyl Tiglate: Sex Pheromone of the Striped Mealybug, <Emphasis Type="Italic">Ferrisia virgata</Emphasis>

Derivatives of 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylic acid (chrysanthemic acid) are classic natural pyrethroids discovered in pyrethrum plants and show insecticidal activity. Chrysanthemic acid, with two asymmetric carbons, has four possible stereoisomers, and most natural pyrethroids have the (1R,3R)-trans configuration. Interestingly, chrysanthemic acid–related structures are also found in insect sex pheromones; carboxylic esters of (1R,3R)-trans-(2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropyl)methanol (chrysanthemyl alcohol) have been reported from two mealybug species. In the present study, another ester of chrysanthemyl alcohol was discovered from the striped mealybug, Ferrisia virgata (Cockerell), as its pheromone. By means of gas chromatography–mass spectrometry, nuclear magnetic resonance spectrometry, and high-performance liquid chromatography analyses using a chiral stationary phase column and authentic standards, the pheromone was identified as (1S,3R)-(?)-cis-chrysanthemyl tiglate. The (1S,3R)-enantiomer strongly attracted adult males in a greenhouse trapping bioassay, whereas the other enantiomers showed only weak activity. The cis configuration of the chrysanthemic acid–related structure appears to be relatively scarce in nature, and this is the first example reported from arthropods.     

Comparison of Postprandial Oleic Acid, 9<Emphasis Type="Italic">c,</Emphasis>11<Emphasis Type="Italic">t</Emphasis> CLA and 10<Emphasis Type="Italic">t,</Emphasis>12<Emphasis Type="Italic">c</Emphasis> CLA Oxidation in Healthy Moderately Overweight Subjects

Few studies report the individual effect of 9c,11t- and 10t,12c-CLA on human energy metabolism. We compared the postprandial oxidative metabolism of 9c,11t- and 10t,12c-CLA and oleic acid (9c-18:1) in 22 healthy moderately overweight volunteers. After 24 weeks supplementation with 9c,11t-, 10t,12c-CLA or 9c-18:1 (3 g/day), subjects consumed a single oral bolus of the appropriate [1-¹³C]-labeled fatty acid. 8 h post-dose, cumulative oxidation was similar for 9c-18:1 and 10t,12c (P = 0.66), but significantly higher for 9c,11t (P < 0.01).     

Roquefortine/Oxaline Biosynthesis Pathway Metabolites in <Emphasis Type="Italic">Penicillium</Emphasis> ser. <Emphasis Type="Italic">Corymbifera</Emphasis>: <Emphasis Type="Italic">In Planta</Emphasis> Production and Implications for Competitive Fitness

Three strains of each of the seven taxa comprising the Penicillium series Corymbifera were surveyed by direct injection mass spectrometry (MS) and liquid chromatography–MS for the production of terrestric acid and roquefortine/oxaline biosynthesis pathway metabolites when cultured upon macerated tissue agars prepared from Allium cepa, Zingiber officinale, and Tulipa gesneriana, and on the defined medium Czapek yeast autolysate agar (CYA). A novel solid-phase extraction methodology was applied for the rapid purification of roquefortine metabolites from a complex matrix. Penicillium hordei and P. venetum produced roquefortine D and C, whereas P. hirsutum produced roquefortine D and C and glandicolines A and B. P. albocoremium, P. allii, and P. radicicola carried the pathway through to meleagrin, producing roquefortine D and C, glandicolines A and B, and meleagrin. P. tulipae produced all previously mentioned metabolites yet carried the pathway through to an end product recognized as epi-neoxaline, prompting the proposal of a roquefortine/epi-neoxaline biogenesis pathway. Terrestric acid production was stimulated by all Corymbifera strains on plant-derived media compared to CYA controls. In planta, production of terrestric acid, roquefortine C, glandicolines A and B, meleagrin, epi-neoxaline, and several other species-related secondary metabolites were confirmed from A. cepa bulbs infected with Corymbifera strains. The deposition of roquefortine/oxaline pathway metabolites as an extracellular nitrogen reserve for uptake and metabolism into growing mycelia and the synergistic role of terrestric acid and other Corymbifera secondary metabolites in enhancing the competitive fitness of Corymbifera species in planta are proposed.     

Oxidation kinetics for <Emphasis Type="Italic">cis</Emphasis>-9,<Emphasis Type="Italic">trans</Emphasis>-11 and <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12 isomers of CLA

The autoxidation processes of the cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) isomers of CLA were separately observed at ca. 0% RH and different temperatures. The t10,c12 CLA oxidized faster than the c9,t11 isomer at all tested temperatures. The first half of the oxidation process of t10,c12 CLA obeyed an autocatalytic-type rate expression, but the latter half followed first-order kinetics. On the other hand, the entire oxidation process of c9,t11 CLA could be expressed by the autocatalytic-type rate expression. The apparent activation energies and frequency factors for the autoxidation of the isomers were estimated from the rate constants obtained at various temperatures based on the Arrhenius equation. The apparent activation energies for the CLA isomers were greater than those for the nonconjugated n−6 and n−3 PUFA or their esters. However, the enthalpyentropy compensation held during the autoxidation of both the CLA and PUFA. This suggested that the autoxidation mechanisms for the CLA and PUFA were essentially the same.     

Synthesis of <Emphasis Type="Italic">p</Emphasis><Emphasis Type="Bold">-</Emphasis> and <Emphasis Type="Italic">n</Emphasis>-type Gels Doped with Ionic Charge Carriers

In this study, we synthesized the new kinds of semiconducting polymeric gels having negative (n-type) and positive (p-type) counter ions as charge carriers. The polyacrylamide gel was doped with pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt), having \textSO₃ ^- {\text{SO}}_{3}^{ - } ions as side groups and Na⁺ as counter ions, so-called p-type semiconducting gel. The doping process was performed during the polymerization where the pyranine binds to the polymer strands over OH group chemically via radical addition. In a similar way, N-isopropylacrylamide (NIPA) gel was doped with methacrylamidopropyltrimethyl ammonium chloride (MAPTAC), having Cl⁻ as counter ions, so-called n-type semiconducting gel. Here MAPTAC was embedded by copolymerization within the polymer network (NIPA). These semiconducting gels can show different electrical properties by changing the concentration of the doping agents, swelling ratio etc. We have shown that the pn junction, formed by combining p-type and n-type gels together in close contact, rectifies the current similar to the conventional Si and Ge diodes.     

Biosynthesis of (3<Emphasis Type="Italic">Z</Emphasis>,6<Emphasis Type="Italic">Z</Emphasis>,9<Emphasis Type="Italic">Z</Emphasis>)-3,6,9-Octadecatriene: The Main Component of the Pheromone Blend of <Emphasis Type="Italic">Erannis bajaria</Emphasis>

The hydrocarbons (3Z,6Z,9Z)-3,6,9-octadecatriene (3Z,6Z,9Z-18:H) and (3Z,6Z,9Z)-3,6,9-nonadecatriene (3Z,6Z,9Z-19:H) constitute the pheromone of the winter moth, Erannis bajaria. These compounds belong to a large group of lepidopteran pheromones which consist of unsaturated hydrocarbons and their corresponding oxygenated derivatives. The biosynthesis of such hydrocarbons with an odd number of carbons in the chain is well understood. In contrast, knowledge about the biosynthesis of even numbered derivatives is lacking. We investigated the biosynthesis of 3Z,6Z,9Z-18:H by applying deuterium-labeled precursors to females of E. bajaria followed by gas chromatography–mass spectrometry analysis of extracts of the pheromone gland. A mixture of deuterium-labeled [17,17,18,18-²H₄]-3Z,6Z,9Z-18:H and the unlabeled 3Z,6Z,9Z-18:H was obtained after topical application and injection of (10Z,13Z,16Z)-[2,2,3,3-²H₄]-10,13,16-nonadecatrienoic acid ([2,2,3,3-²H₄]-10Z,13Z,16Z-19:acid) or (11Z,14Z,17Z)-[3,3,4,4-²H₄]-11,14,17-icosatrienoic acid ([3,3,4,4-²H₄]-11Z,14Z,17Z-20:acid). These results are consistent with a biosynthetic pathway that starts with α-linolenic acid (9Z,12Z,15Z-18:acid). Chain elongation leads to 11Z,14Z,17Z-20:acid, which is shortened by α-oxidation as the key step to yield 10Z,13Z,16Z-19:acid. This acid can be finally reduced to an aldehyde and decarbonylated or decarboxylated to furnish the pheromone component 3Z,6Z,9Z-18:H. A similar transformation of 11Z,14Z,17Z-20:acid yields the second pheromone component, 3Z,6Z,9Z-19:H.