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.

Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation-induced DNA damage.

The nucleoids of radiation-resistant Deinococcus species show a high degree of compaction maintained after ionizing irradiation. We identified proteins recruited after irradiation in nucleoids of Deinococcus radiodurans and Deinococcus deserti by means of comparative proteomics. Proteins in nucleoid-enriched fractions from unirradiated and irradiated Deinococcus were identified and semiquantified by shotgun proteomics. The ssDNA-binding protein SSB, DNA gyrase subunits GyrA and GyrB, DNA topoisomerase I, RecA recombinase, UvrA excinuclease, RecQ helicase, DdrA, DdrB, and DdrD proteins were found in significantly higher amounts in irradiated nucleoids of both Deinococcus species. We observed, by immunofluorescence microscopy, the subcellular localization of these proteins in D. radiodurans, showing for the first time the recruitment of the DdrD protein into the D. radiodurans nucleoid. We specifically followed the kinetics of recruitment of RecA, DdrA, and DdrD to the nucleoid after irradiation. Remarkably, RecA proteins formed irregular filament-like structures 1 h after irradiation, before being redistributed throughout the cells by 3 h post-irradiation. Comparable dynamics of DdrD localization were observed, suggesting a possible functional interaction between RecA and DdrD. Several proteins involved in nucleotide synthesis were also seen in higher quantities in the nucleoids of irradiated cells, indicative of the existence of a mechanism for orchestrating the presence of proteins involved in DNA metabolism in nucleoids in response to massive DNA damage. All MS data have been deposited in the ProteomeXchange with identifier PXD00196 (http://proteomecentral.proteomexchange.org/dataset/PXD000196).

Development of a forward genetic screen to isolate oil mutants in the green microalga Chlamydomonas reinhardtii.

BACKGROUND: Oils produced by microalgae are precursors to biodiesel. To achieve a profitable production of biodiesel from microalgae, identification of factors governing oil synthesis and turnover is desirable. The green microalga Chlamydomonas reinhardtii is amenable to genetic analyses and has recently emerged as a model to study oil metabolism. However, a detailed method to isolate various types of oil mutants that is adapted to Chlamydomonas has not been reported. RESULTS: We describe here a forward genetic approach to isolate mutants altered in oil synthesis and turnover from C. reinhardtii. It consists of a three-step screening procedure: a primary screen by flow cytometry of Nile red stained transformants grown in 96-deep-well plates under three sequential conditions (presence of nitrogen, then absence of nitrogen, followed by oil remobilization); a confirmation step using Nile red stained biological triplicates; and a validation step consisting of the quantification by thin layer chromatography of oil content of selected strains. Thirty-one mutants were isolated by screening 1,800 transformants generated by random insertional mutagenesis (1.7%). Five showed increased oil accumulation under the nitrogen-replete condition and 13 had altered oil content under nitrogen-depletion. All mutants were affected in oil remobilization. CONCLUSION: This study demonstrates that various types of oil mutants can be isolated in Chlamydomonas based on the method set-up here, including mutants accumulating oil under optimal biomass growth. The strategy conceived and the protocol set-up should be applicable to other microalgal species such as Nannochloropsis and Chlorella, thus serving as a useful tool in Chlamydomonas oil research and algal biotechnology.

A comparative proteomic approach to better define Deinococcus nucleoid specificities.

Compared to radiation-sensitive bacteria, the nucleoids of radiation-resistant Deinococcus species show a higher degree of compaction. Such a condensed nucleoid may contribute to the extreme radiation resistance of Deinococcus by limiting dispersion of radiation-induced DNA fragments. Architectural proteins may play a role in this high degree of nucleoid compaction, but comparative genomics revealed only a limited number of Deinococcus homologs of known nucleoid-associated proteins (NAPs) from other species such as Escherichia coli. A comparative proteomic approach was used to identify potentially novel proteins from isolated nucleoids of Deinococcus radiodurans and Deinococcus deserti. Proteins in nucleoid enriched fractions were identified and semi-quantified by shotgun proteomics. Based on normalized spectral counts, the histone-like DNA-binding protein HU appeared to be the most abundant among candidate NAPs from both micro-organisms. By immunofluorescence microscopy, D. radiodurans HU and both DNA gyrase subunits were shown to be distributed throughout the nucleoid structure and absent from the cytoplasm. Taken together, our results suggest that D. radiodurans and D. deserti bacteria contain a very low diversity of NAPs, with HU and DNA gyrase being the main proteins involved in the organization of the Deinococcus nucleoids.