Complementary environmental analysis and functional characterization of lower glycolysis-gluconeogenesis in the diatom plastid.

Organic carbon fixed in chloroplasts through the Calvin-Benson-Bassham Cycle can be diverted towards different metabolic fates, including cyoplasmic and mitochondrial respiration, gluconeogenesis, and synthesis of diverse plastid metabolites via the pyruvate hub. In plants, pyruvate is principally produced via cytoplasmic glycolysis, although a plastid-targeted lower glycolytic pathway is known to exist in non-photosynthetic tissue. Here, we characterized a lower plastid glycolysis-gluconeogenesis pathway enabling the direct interconversion of glyceraldehyde-3-phosphate and phospho-enol-pyruvate in diatoms, ecologically important marine algae distantly related to plants. We show that two reversible enzymes required to complete diatom plastid glycolysis-gluconeogenesis, Enolase and bis-phospho-glycerate mutase (PGAM), originated through duplications of mitochondria-targeted respiratory isoforms. Through CRISPR-Cas9 mutagenesis, integrative 'omic analyses, and measured kinetics of expressed enzymes in the diatom Phaeodactylum tricornutum, we present evidence that this pathway diverts plastid glyceraldehyde-3-phosphate into the pyruvate hub, and may also function in the gluconeogenic direction. Considering experimental data, we show that this pathway has different roles dependent in particular on day length and environmental temperature, and show that the cpEnolase and cpPGAM genes are expressed at elevated levels in high latitude oceans where diatoms are abundant. Our data provide evolutionary, meta-genomic and functional insights into a poorly understood yet evolutionarily recurrent plastid metabolic pathway.

Prostaglandin production by the microalga with heterologous expression of cyclooxygenase.

Prostaglandins (PGs) are the physiologically active compounds synthesized from C20 polyunsaturated fatty acids (PUFAs) by cyclooxygenase (COX) and a series of PG synthases, and are utilized as pharmaceuticals. Currently, commercialized PGs are mainly produced by chemical synthesis under harsh conditions. By contrast, bioproduction of PGs can be an alternative, environmental-friendly, and inexpensive process with genetic engineering of model plants, although these conventional host organisms contain a limited quantity of PG precursors. In this study, we established an efficient PG production process using the genetically engineered microalga Fistulifera solaris which is rich in C20 PUFAs. A cox gene derived from the red alga Agarophyton vermiculophyllum was introduced into F. solaris. As a result, a transformant clone with high cox expression produced PGs (i.e., PGD2 , PGE2 , PGF2α , and 15-ketoPGF2α derived from arachidonic acid, and PGD3 , PGE3 , and PGF3α derived from eicosapentaenoic acid) as revealed by liquid chromatography/mass spectrometry. The total content of PGs was 1290.4 ng/g of dry cell weight, which was higher than that produced in the transgenic plant reported previously. The results obtained in this study indicate that the C20 PUFA-rich microalga functionally expressing COX is a promising host for PG bioproduction.

Metabolic Innovations Underpinning the Origin and Diversification of the Diatom Chloroplast.

: Of all the eukaryotic algal groups, diatoms make the most substantial contributions to photosynthesis in the contemporary ocean. Understanding the biological innovations that have occurred in the diatom chloroplast may provide us with explanations to the ecological success of this lineage and clues as to how best to exploit the biology of these organisms for biotechnology. In this paper, we use multi-species transcriptome datasets to compare chloroplast metabolism pathways in diatoms to other algal lineages. We identify possible diatom-specific innovations in chloroplast metabolism, including the completion of tocopherol synthesis via a chloroplast-targeted tocopherol cyclase, a complete chloroplast ornithine cycle, and chloroplast-targeted proteins involved in iron acquisition and CO2 concentration not shared between diatoms and their closest relatives in the stramenopiles. We additionally present a detailed investigation of the chloroplast metabolism of the oil-producing diatom Fistuliferasolaris, which is of industrial interest for biofuel production. These include modified amino acid and pyruvate hub metabolism that might enhance acetyl-coA production for chloroplast lipid biosynthesis and the presence of a chloroplast-localised squalene synthesis pathway unknown in other diatoms. Our data provides valuable insights into the biological adaptations underpinning an ecologically critical lineage, and how chloroplast metabolism can change even at a species level in extant algae.

Outdoor Cultivation of Marine Diatoms for Year-Round Production of Biofuels.

Biofuel production using microalgae is believed to have the advantage of continuous year-round production over crop plants, which have strong seasonality. However, actual year-round production of microalgal lipids using outdoor mass cultivation has rarely been demonstrated. In our previous study, it was demonstrated that the oleaginous diatom, Fistulifera solaris, was culturable in outdoor bioreactors from spring to autumn, whereas biomass and lipid production in winter failed because F. solaris did not grow below 15 °C. Therefore, another candidate strain that is culturable in winter is required. In this study, a cold-tolerant diatom, Mayamaea sp. JPCC CTDA0820, was selected as a promising candidate for biofuel production in winter. Laboratory-scale characterization revealed that this diatom was culturable at temperatures as low as 10 °C. Subsequently, F. solaris (April-October) and Mayamaea sp. JPCC CTDA0820 (November-March) were cultured in outdoor open-pond bioreactors, wherein year-round production of diatom lipids was successfully demonstrated. The maximal values of areal productivities of biomass and lipids reached to 9.79 and 1.80 g/(m² day) for F. solaris, and 8.62 and 0.92 g/(m² day) for Mayamaea sp. JPCC CTDA0820, respectively. With the combined use of these two diatom species, stable year-round production of microalgal lipids became possible.