Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

Consequences of Mixotrophy on Cell Energetic Metabolism in Microchloropsis gaditana Revealed by Genetic Engineering and Metabolic Approaches.

Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via ß-oxidation in this oleaginous alga.

In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases.

BACKGROUND: P450 fatty acid decarboxylases represented by the unusual CYP152 peroxygenase family member OleTJE have been receiving great attention recently since these P450 enzymes are able to catalyze the simple and direct production of 1-alkenes for potential applications in biofuels and biomaterials. To gain more mechanistic insights, broader substrate spectra, and improved decarboxylative activities, it is demanded to discover and investigate more P450 fatty acid decarboxylases. RESULTS: Here, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46Δ29, that are capable of decarboxylating straight-chain saturated fatty acids. Both enzymes were found to catalyze the decarboxylation and hydroxylation of a broad range of free fatty acids (C10-C20) with overlapping substrate specificity, yet distinct chemoselectivity. CYP-Sm46Δ29 works primarily as a fatty (lauric) acid decarboxylase (66.1 ± 3.9% 1-undecene production) while CYP-Aa162 more as a fatty (lauric) acid hydroxylase (72.2 ± 0.9% hydroxy lauric acid production). Notably, the optical spectroscopic analysis of functional CYP-Sm46Δ29 revealed no characteristic P450 band, suggesting a unique heme coordination environment. Active-site mutagenesis analysis showed that substitution with the proposed key decarboxylation-modulating residues, His85 and Ile170, enhanced the decarboxylation activity of CYP-Aa162 and P450BSß, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46Δ29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent. CONCLUSIONS: Our data identify CYP-Sm46Δ29 as an efficient OleTJE-like fatty acid decarboxylase. Oxidative decarboxylation chemoselectivity of the CYP152 decarboxylases is largely dependent upon the carbon chain length of fatty acid substrates and their precise positioning in the enzyme active site. Finally, the kinetic mode analysis of the enzymes could provide important guidance for future process design.