Baohua Zhu

Silencing UDP-glucose pyrophosphorylase gene in Phaeodactylum tricornutum affects carbon allocation

The effects of the suppression of UDP-glucose pyrophosphorylase (UGPase) on chrysolaminaran biosynthesis and carbon allocation were investigated in Phaeodactylum tricornutum. The 69% decrease in UGPase activity was accompanied by a 4.89 fold reduction in Ugp transcript abundance. Inactivation of UGPase in P. tricornutum led to a significant decrease in chrysolaminaran content and an increase in lipid synthesis. These findings suggest that UGPase is a rate-limiting enzyme and may play an important role in chrysolaminarin biosynthesis and carbon allocation. Our results support a theoretical deduction that Ugp is a good candidate for improving lipid synthesis in diatoms.

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE MARINE MICROALGA NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)1

Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae), a marine eukaryotic unicellular alga, is widely used in mariculture as live feed. It is considered to be of high nutritional value owing to its high content of proteins; polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA, C20:5n3); and diverse pigments. Previous studies of this microalga focused on its taxonomy, culture, and biochemistry, but little is known at the molecular level. Establishing a molecular base is vital to understand the biological processes of this alga. Therefore, we constructed a cDNA library using algal cells grown at exponential growth phase and carried out expressed sequence tag (EST) analysis. A total of 1,960 nonredundant sequences (NRSs) were generated for N. oculata clone CS‐179. Only 32.5% of NRSs showed significant similarity (E < 1e‐04) to proteins registered in the GenBank nonredundant protein database. The KOG (clusters of euKaryotic Orthologous Groups) profile database returned significant hits for 490 NRSs. Analysis revealed that a large proportion of NRSs could be unique to this microalga.

Large-scale biodiesel production using flue gas from coal-fired power plants with Nannochloropsis microalgal biomass in open raceway ponds

The potential use of microalgal biomass as a biofuel source has raised broad interest. Highly effective and economically feasible biomass generating techniques are essential to realize such potential. Flue gas from coal-fired power plants may serve as an inexpensive carbon source for microalgal culture, and it may also facilitate improvement of the environment once the gas is fixed in biomass. In this study, three strains of the genus Nannochloropsis (4-38, KA2 and 75B1) survived this type of culture and bloomed using flue gas from coal-fired power plants in 8000-L open raceway ponds. Lower temperatures and solar irradiation reduced the biomass yield and lipid productivities of these strains. Strain 4-38 performed better than the other two as it contained higher amounts of triacylglycerols and fatty acids, which are used for biodiesel production. Further optimization of the application of flue gas to microalgal culture should be undertaken.

Association of triacylglyceride content and transcript abundance of genes involving in lipid synthesis of nitrogen deficient Phaeodactylum tricornutum

Phaeodactylum tricornutum is a diatom that is rich in lipids. Recently, it has received much attention as a feedstock for biodiesel production. Nitrogen deficiency is widely known to increase the content of neutral lipids (mainly triacylglycerides, or TAGs) of microalgae, including P. tricornutum, but the mechanism is unclear. In this study, we deciphered the correlations between TAG content and nine key enzymatic genes involved in lipid synthesis in P. tricornutum. After being cultured under nitrogen-free conditions for 0, 4, 24, 48, 72, 120, and 168 h, the TAG contents of P. tricornutum cells were assayed and the transcript abundances of the target genes were monitored by quantitative real-time PCR. The results show that the abundances of four target gene transcripts (LACS3, G3PDH2, G3PDH3, and G3PDH5) were positively correlated with TAG content, indicating that these genes may be involved in TAG synthesis in P. tricornutum. The findings improve our understanding of the metabolic network and regulation of lipid synthesis and will guide the future genetic improvement of the TAG content of P. tricornutum.

Isolation and characterization of a long-chain acyl-coenzyme A synthetase encoding gene from the marine microalga Nannochloropsis oculata

Acyl-coenzyme A synthetases (ACSs) are associated with the anabolism and catabolism of fatty acids and play fundamental roles in various metabolic pathways. The cDNA of long-chain acyl-coenzyme A synthetase (LACS), one of the ACSs, was isolated from Nannochloropsis oculata and named as NOLACS. The predicted amino acid sequence was highly similar to LACSs of other species. NOLACS encodes a long-chain acyl-coenzyme A synthetase; it recovered the function of LACS in Saccharomyces cerevisiae YB525 (a LACS-deficient yeast strain). The substrate specificity of the enzyme was also assayed in yeast. It was found that NOLACS can activate saturated fatty acids (C12:0, C14:0, C16:0, and C18:0) and some unsaturated fatty acids (C18:2Δ9, 12 and C20:2Δ11, 14) with a preference for long-chain fatty acids. Our findings will provide a deep understanding of CoA-dependent fatty acid activation and also make some contribution to understanding the metabolic pathways of lipids in Nannochloropsis. These findings will also facilitate studies on the regulation of gene expression and genetic modification of fatty acid synthesis and storage of N. oculata.

Biomass and nutrient productivities of Tetraselmis chuii under mixotrophic culture conditions with various C:N ratios

Mass microalgal culture plays an irreplaceable role in aquaculture, but microalgal productivity is restricted by traditional autotrophic culture conditions. In the present study, a Tetraselmis chuii strain belonging to the phylum Chlorophyta was isolated from south Yellow Sea. The growth rate and biomass productivity of this strain was higher under mixotrophic conditions with different carbon:nitrogen (C:N) ratios than those under autotrophic conditions. When the C:N ratio was 16, the optical density and biomass productivity were 3.7- and 5-fold higher than their corresponding values under autotrophic culture conditions, respectively. Moreover, T. chuii synthesized more polysaccharides and total lipids under mixotrophic conditions. In addition, T. chuii cultured under mixotrophic conditions synthesized more types of fatty acids than autotrophic culture conditions. At a C:N ratio of 16, the percentage of C16:0 and C18:1 reached 30.08% and 24.65% of the total fatty acid (TFA) content, respectively. These findings may provide a basis for large-scale mixotrophic culture of T. chuii, as a potential bait-microalga.

Nuclear transition between the conjunction cells of Phaeodactylum tricornutum Bohlin (Bacillariophyta)

Phaeodactylum tricornutum is one of the important marine diatoms for oceanic primary production. Its reproduction has profound significance in the life cycle; however, the nuclear behavior during its sexual reproduction was not clear. In this study, we observed the nuclear transition and determined its correlation with cell conjunction. It was found that two cells jointed at their apices first and swung and aligned each other immediately, and nucleus from one cell was able to transfer into another one during cell conjugation. The cell pairs conjugated for nuclear transition were different from those formed in mitosis in hypovalve thickness and cellular arrangement. Our findings proved the existence of sexual reproduction in P. tricornutum.

Genetic transformation of Nannochloropsis oculata with a bacterial phleomycin resistance gene as dominant selective marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×107 cells mL−1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL−1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

Purification of a diatom and its identification to Cylindrotheca closterium

A diatom was purified with colony selection and continuous dilution methods. It was identified to Cylindrotheca closterium according to its morphological characteristics and rbcL and 18s rRNA gene sequences. The alga was not sensitive to ampicillin and neomycin, but sensitive to chloramphenicol which inhibited its growth at concentrations ranging from 50 to 150 μg mL−1. The purified alga was easy to culture and its specific growth rate was 0.207 ± 0.002 (d−1). It was resistant to pollution and could be harvested in an easy way. It was relatively high in lipid content (20.08% ± 0.67% of dry weight) and the combined amount of its 16:0 and 16:1 (n-7), the most suitable resource of biodiesel, was as high as 64% of the total fatty acids, while the amount of polyunsaturated fatty acids reached 19.96%–20% of the total fatty acids. Thus the purified C. closterium can be cultured as a biodiesel producer or a nutrition supplement producer.

The Role of Malic Enzyme on Promoting Total Lipid and Fatty Acid Production in Phaeodactylum tricornutum

To verify the function of malic enzyme (ME1), the ME1 gene was endogenously overexpressed in Phaeodactylum tricornutum. Overexpression of ME1 increased neutral and total lipid content and significantly increased saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs) in transformants, which varied between 23.19 and 25.32% in SFAs and between 49.02 and 54.04% in PUFAs, respectively. Additionally, increased ME1 activity was accompanied by elevated NADPH content in all three transformants, indicating that increased ME1 activity produced additional NADPH comparing with that of WT. These results indicated that ME1 activity is NADP-dependent and plays an important role in the NADPH levels required for lipid synthesis and fatty acid desaturation in P. tricornutum. Furthermore, our findings suggested that overexpression of endogenous ME1 represents a valid method for boosting neutral-lipid yield in diatom.