title: Co-option of the sphingolipid metabolism for the production of nitroalkene defensive chemicals in termite soldiers.
abstract: The aliphatic nitroalkene (E)-1-nitropentadec-1-ene (NPD), reported in early seventies in soldiers of the termite genus Prorhinotermes, was the first documented nitro compound produced by insects. Yet, its biosynthetic origin has long remained unknown. Here, we investigated in detail the biosynthesis of NPD in P. simplex soldiers. First, we track the dynamics in major metabolic pathways during soldier ontogeny, with emphasis on likely NPD precursors and intermediates. Second, we propose a hypothesis of NPD formation and verify its individual steps using in vivo incubations of putative precursors and intermediates. Third, we use a de novo assembled RNA-Seq profiles of workers and soldiers to identify putative enzymes underlying NPD formation. And fourth, we describe the caste- and age-specific expression dynamics of candidate initial genes of the proposed biosynthetic pathway. Our observations provide a strong support to the following biosynthetic scenario of NPD formation, representing an analogy of the sphingolipid pathway starting with the condensation of tetradecanoic acid with l-serine and leading to the formation of a C16sphinganine. The C16sphinganine is then oxidized at the terminal carbon to give rise to 2-amino-3-hydroxyhexadecanoic acid, further oxidized to 2-amino-3-oxohexadecanoic acid. Subsequent decarboxylation yields 1-aminopentadecan-2-one, which then proceeds through six-electron oxidation of the amino moiety to give rise to 1-nitropentadecan-2-one. Keto group reduction and hydroxyl moiety elimination lead to NPD. The proposed biosynthetic sequence has been constructed from age-related quantitative dynamics of individual intermediates and confirmed by the detection of labeled products downstream of the administered labeled intermediates. Comparative RNA-Seq analyses followed by qRT-PCR validation identified orthologs of serine palmitoyltransferase and 3-ketodihydrosphingosine reductase genes as highly expressed in the NPD production site, i.e. the frontal gland of soldiers. A dramatic onset of expression of the two genes in the first days of soldier's life coincides with the start of NPD biosynthesis, giving further support to the proposed biosynthetic hypothesis.
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia.
authors.forename: Anna
authors.initials: A
authors.lastname: Jirošová
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia.
authors.forename: Andrej
authors.initials: A
authors.lastname: Jančařík
authors.affiliation: Max-Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany.
authors.forename: Riya C
authors.initials: RC
authors.lastname: Menezes
authors.affiliation: Biology Centre CAS, Branišovská 31, CZ-37005 České Budějovice, Czechia.
authors.forename: Olga
authors.initials: O
authors.lastname: Bazalová
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia; Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague, Czechia.
authors.forename: Klára
authors.initials: K
authors.lastname: Dolejšová
authors.affiliation: Max-Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Vogel
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Jedlička
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia.
authors.forename: Aleš
authors.initials: A
authors.lastname: Buček
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia.
authors.forename: Jana
authors.initials: J
authors.lastname: Brabcová
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia.
authors.forename: Pavel
authors.initials: P
authors.lastname: Majer
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia. Electronic address: robert@uochb.cas.cz.
authors.forename: Robert
authors.initials: R
authors.lastname: Hanus
authors.affiliation: The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo n. 2, 166 10 Prague, Czechia; Max-Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany. Electronic address: svatos@ice.mpg.de.
authors.forename: Aleš
authors.initials: A
authors.lastname: Svatoš
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D005227:Fatty Acids / Q000378:metabolism; D019476:Insect Proteins / Q000378:metabolism; D020049:Isoptera / Q000378:metabolism*; D007659:Ketones / Q000378:metabolism*; D055442:Metabolome; D009595:Nitroparaffins / Q000378:metabolism*; D051102:Serine C-Palmitoyltransferase / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005227:Fatty Acids; D019476:Insect Proteins; D007659:Ketones; D009595:Nitroparaffins; D013107:Sphingolipids; D000429:Alcohol Oxidoreductases; D051102:Serine C-Palmitoyltransferase
keywords: Biosynthesis; Chemical defense; Labeled probes; Metabolomics; Prorhinotermes simplex; Transcriptomics