We investigated the lipid synthesis using the green alga Myrmecia incisa as a model system. This alga is known as an arachidonic acid (ArA) producer, where ArA is mainly stored in the form of triacylglycerols (TAGs), a class of neutral lipids. We conducted the transcriptome analysis of M. incisa using 454 pyrosequencing, and a total of 754,208 high-quality reads were obtained. Through the homology search against the database, we cloned several cDNAs encoding putative diacylglycerol acyltransferases (DGATs), a group of key enzymes responsible for TAG synthesis in eukaryotic organisms. The complementation assay was carried out in the mutant strain of Saccharomyces cerevisiae to verify the function of MiDGATs. According to the results of thin-layer chromatography and BODIPY staining, both MiDGAT1 and MiDGAT2s were able to restore TAG synthesis and lipid body formation. The substrate preference was evaluated by GC-MS analysis. Quantitative RT-PCR results showed that the transcript level of MiDGAT2A was regulated by nitrogen starvation, which was consistent with TAG accumulation in M. incisa.

Fig. 1 Fluorescent staining of yeast cells with BODIPY. Lipid bodies where neutral lipids accumulate were visualized in the yeast cells with BODIPY fluorescence. The wild type strain Scy62 was used as positive control; the mutant H1246 and the mutant harboring the empty vector (pYES2) were used as negative controls. The mutant expressing MiDGAT1, MiDGAT2A, MiDGAT2B or MiDGAT1-ΔPH was analyzed. All bars represent the length of 5 μm.

 

 

To explore microalgae as the feedstock for biodiesel, the first step, strain selection, is of fundamental importance. It is expected that an ideal algal strain for biodiesel production should grow fast with high cell density, accumulate a large quantity of oils and perform well in downstream processes. To this end, we have established an algal strain bank with over 1,000 strains collected from different environments. Systematic screening and characterization was conducted, and subsequently some high-performance strains were further evaluated in large-scale outdoor systems. For example, the green alga Chlorella protothecoides was demonstrated to have high biomass and lipid productivities in outdoor panel photobioreactors, where up to 1.25 and 0.59 g L-1 day-1, or 44. 1 and 16.1 g m-2 day-1 were achieved, respectively.

Fig. 2 Outdoor facilities for large-scale algal cultivation. Left: open ponds; Middle: tubular PBRs; Right: flat plate PBRs.

  

The high productivity and photosynthetic efficiency of Saccharina japonica suggest it may possess the carbon concentrating mechanism (CCM) to use HCO-3 in the seawater as an exogenous (inorganic carbon) Ci source. In the reported CCMs, carbonic anhydrases (CAs) play important roles in transition and transportation of Ci to supply enough CO2 around RuBisCo. Based on the high-throughput sequencing data of S. japonica and combining with our preparatory work, we determined that this kelp has twelve CA genes that are separately coded as α, β, and γ. Functions of CAs are usually associated with their locations. Among S. japonica CAs, one α-CA has been found in the chloroplast and thylakoid membrane of S. japonica gametocyte under immunogold electron microscopy. To explore each CA’s roles in S. japonica CCM, the subcellular localization of other CA will be examined soon. The completion of this study is conducive to the CCM modeling of sporophyte and gametophyte in this kelp.

Fig. 3 Transmission electron micrograph showing the immunogold labeling in the gametophyte cells of Saccharina japonica probed with the SjCA antibody. a) Ultrastructure of a whole cell; b–d) Enlarged images corresponding to the marked areas 1, 2, and 3 showing the immunogold labeling distribution; e) Immunogold labeling distribution especially in a pyrenoid. Gold particles were localized on the envelopes and thylakoid membranes of chloroplasts as indicated by white arrows. Ch chloroplast, Nu nucleus, Py pyrenoid.

 

 

Macroscopic monoecious sporophytes (2n) of Saccharina japonica can produce meiospores which are able to germinate microscopic female and male gametophytes (n). The 1:1 ratio of female to male gametophytes and all the female offsprings from the parthenogenetic sporophytes predict that the kelp has a XY-like sex determination. Three molecular markers related to the sex of kelp gametophytes were developed by our research group to identify their gender because the chromosomes are too small that a karyogram cannot be easily obtained. By use of fluorescence in situ hybridization, their locations on the chromosomes of gametophytes were determined. This research will pave the way for sex differentiation, sex chromosome and sex determination of this kelp.

 

 Fig. 4 FISH of the FRML-494 marker(red) on Saccharina japonica chromosomes counterstained with DAPI (blue).

(A) Metaphase chromosomes of the female gametophytes (n = 31); (B) hybridization signal of the FRML-494 marker (red) as indicated by the arrow on the same spread as image A; the inset is the enlarged chromosome where this marker was located; (C) an interphase nucleus of the female gametophytes with hybridization signal (red) as indicated by the arrow; (D) metaphase chromosomes of the male gametophytes (n = 31); (E) an interphase nucleus of the male gametophytes without a hybridization signal; (F) an interphase nucleus of the sporophytes with a hybridization signal (red) as indicated by the arrow; (G) metaphase chromosomes of the sporophytes (2n = 62); (H) hybridization signal (red) as indicated by the arrow present on only one metaphase chromosome of the same spread as image G; the inset is the enlarged chromosome where this marker was located; and the arrowheads in images D and G show the constriction of the chromosomes.