Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway.

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway.

Role of HoxE subunit in Synechocystis PCC6803 hydrogenase.

Cyanobacterial NAD(P)(+)-reducing reversible hydrogenases comprise five subunits. Four of them (HoxF, HoxU, HoxY, and HoxH) are also found in the well-described related enzyme from Ralstonia eutropha. The fifth one (HoxE) is not encoded in the R. eutropha genome, but shares homology with the N-terminal part of R. eutropha HoxF. However, in cyanobacteria, HoxE contains a 2Fe-2S cluster-binding motif that is not found in the related R. eutropha sequence. In order to obtain some insights into the role of HoxE in cyanobacteria, we deleted this subunit in Synechocystis PCC6803. Three types of interaction of the cyanobacterial hydrogenase with pyridine nucleotides were tested: (a) reductive activation of the NiFe site, for which NADPH was found to be more efficient than NADH; (b) H(2) production, for which NADH appeared to be a more efficient electron donor than NADPH; and (c) H(2) oxidation, for which NAD(+) was a much better electron acceptor than NADP(+). Upon hoxE deletion, the Synechocystis hydrogenase active site remained functional with artificial electron donors or acceptors, but the enzyme became unable to catalyze H(2) production or uptake with NADH/NAD(+). However, activation of the electron transfer-independent H/D exchange reaction by NADPH was still observed in the absence of HoxE, whereas activation of this reaction by NADH was lost. These data suggest different mechanisms for diaphorase-mediated electron donation and catalytic site activation in cyanobacterial hydrogenase.

The chlorophyll-binding protein IsiA is inducible by high light and protects the cyanobacterium Synechocystis PCC6803 from photooxidative stress.

The products of the isiAB operon are a chlorophyll antenna protein (IsiA) and flavodoxin (IsiB), which accumulate in cyanobacteria grown under iron starvation conditions. Here we show that strong light triggers de-repression of isiAB transcription and leads to IsiA and flavodoxin accumulation under iron replete conditions. Genetic deletion of isiAB resulted in a photosensitive phenotype, with accumulation of reactive oxygen species and cell bleaching in high light, while the flavodoxin-deficient isiB null mutant expressing isiA was phototolerant. We conclude that IsiA protects cyanobacteria from photooxidative stress. IsiA is the first example of a chlorophyll antenna protein outside the extended LHC family that is induced transiently by high light and that fulfills a photoprotective role.

Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex.

The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and carbohydrate-mediated reduction of the plastoquinone pool.

Elimination of high-light-inducible polypeptides related to eukaryotic chlorophyll a/b-binding proteins results in aberrant photoacclimation in Synechocystis PCC6803.

The hli genes, present in cyanobacteria, algae and vascular plants, encode small proteins [high-light-inducible polypeptides (HLIPs)] with a single membrane-spanning alpha-helix related to the first and third helices of eukaryotic chlorophyll a/b-binding proteins. The HLIPs are present in low amounts in low light and they accumulate transiently at high light intensities. We are investigating the function of those polypeptides in a Synechocystis PCC6803 mutant lacking four of the five hli genes. Growth of the quadruple hli mutant was adversely affected by high light intensities. The most striking effect of the quadruple hli mutation was an alteration of cell pigmentation. Pigment changes associated with cell acclimation to increasing light intensity [i.e. decrease in light-harvesting pigments, accumulation of the carotenoid myxoxanthophyll and decrease in photosystem I (PSI)-associated chlorophylls] were strongly exacerbated in the quadruple hli mutant, resulting in yellowish cultures that bleached in high light and died as light intensities exceeded (>500 micromol photon m(-2) s(-1)). However, these pigment changes were not associated with an inhibition of photosynthesis, as probed by in vivo chlorophyll fluorescence, photoacoustic and O(2)-evolution measurements. On the contrary, the HLIP deficiency was accompanied by a stimulation of the photochemical activity, especially in high-light-grown cells. Western blot analyses revealed that the PSI reaction center level (PsaA/B) was noticeably reduced in the quadruple hli mutant relative to the wild type, whereas the abundance of the PSII reaction center protein D1 was comparatively little affected. The hli mutations did not enhance photoinhibition and photooxidation when cells were exposed over a short term to a very high light intensity. Together, the results of this study indicate that HLIPs are critical in the adaptation of the cyanobacterium to variations in light intensity. The data are consistent with the idea that HLIPs are involved, through a direct or indirect means, in nonphotochemical dissipation of absorbed light energy.