Silencing of a Pseudo‐nitzschia arenysensis lipoxygenase transcript leads to reduced oxylipin production and impaired growth

Abstract

Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists.Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo-and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15.PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms.The presence in the genome of a single LOX gene, whose downregulation impairs both 12and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.

Structural and Functional Characterization of Lipoxygenases from Diatoms by Bioinformatics and Modelling Studies

“…The difference in terms of LOX products is attributable to the different composition of the oxygen channel that allows the oxidation in the deepest portion of the substrate (C12). The result obtained is also in agreement with the literature data, which report a possible role of LOX from P. arenysensis as LOX12 or 15, due to the discovery of oxylipins produced by this diatom by stereo-and regio-selective oxidation of EPA at C12 and C15 [47]. An example of this is also given by the hypothetical LOX from P. tricornutum, for which, as explained in the result section, the active site presents many differences that can be related to the absence of interaction with EPA and the possible binding with the 9,12-hexa-decadienoic acid.…”

Empirical evidence for metabolic drift in plant and algal lipid biosynthesis pathways

“…This would suggest enzyme replacement, although the full demonstration by biochemical characterization of such a bifunctional LOX is still missing. For the moment, only a few LOX homologous sequences have been reported in diatoms, and although one of them is very likely involved in fatty acid peroxidation ( Sabatino et al., 2022 ), there is no report of a fusion protein bearing a catalase-like domain coupled to a LOX domain. In conclusion, similar final products of the oxylipin biosynthesis pathway have been identified in different organisms, but in diatoms, the conversion of the epoxidized substrate is not performed by a cytochrome P450, but by a catalase-like hemoprotein, thus, suggesting the occurrence of an enzyme replacement.…”

“…This would suggest enzyme replacement, although the full demonstration by biochemical characterization of such a bifunctional LOX is still missing. For the moment, only a few LOX homologous sequences have been reported in diatoms, and although one of them is very likely involved in fatty acid peroxidation (Sabatino et al, 2022), there is no report of a fusion protein bearing a catalase-like domain coupled Enzymatic replacement in the oxylipin biosynthesis pathway. Comparison of metabolic pathways for oxylipin biosynthesis.…”

De novo transcriptome assembly of a lipoxygenase knock-down strain in the diatom Pseudo-nitzschia arenysensis

“…To investigate the ecological roles of these secondary metabolites, studies that examine their structure and biosynthesis should be paralleled with functional studies on the enzyme-coding genes involved in their synthesis. In recent work, we explored the role of LOX in P. arenysensis (PaLOX) 26 , a diatom known to synthesize oxylipins throughout both 12-and 15S-LOX pathways 14,16,24 . Taking advantage of sequence information from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) 7 we discovered that a unique LOX transcript is present in this species and generated mutants in which this gene was silenced 26 .…”

“…In recent work, we explored the role of LOX in P. arenysensis (PaLOX) 26 , a diatom known to synthesize oxylipins throughout both 12-and 15S-LOX pathways 14,16,24 . Taking advantage of sequence information from the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) 7 we discovered that a unique LOX transcript is present in this species and generated mutants in which this gene was silenced 26 .…”