Morakot Tanticharoen

Production and partial purification of γ-linolenic acid and some pigments fromSpirulina platensis

The polyunsaturated fatty acid γ-linolenic acid (GLA, 18:3ω6) is of potential pharmaceutical value. The cyanobacteriumSpirulina platensis could become an excellent source for this fatty acid, provided that GLA content could be increased and a GLA concentrate could be obtained at a low cost. Increasing the cell concentration inSpirulina platensis enhanced the fatty acid content and thus the GLA content. This effect was used to further enhance the GLA content of GLA-overproducing strains. Separation of the galactolipids and their purification via urea complexes formation, resulted in a GLA concentrate of over 90% purity.

Optimization of γ-linolenic acid (GLA) production inSpirulina platensis

The cyanobacteriumSpirulina platensis is one of the most promising sources of the polyunsaturated fatty acid γ-linolenic acid (GLA). The GLA content ofSpirulina can be enhanced by cultivation under light-dark cycles in the laboratory or outdoors. Thus, in strain BP, the GLA content increased from 1.2 to 1.6% when cultivated under light-dark cycles. Moreover, in the derived mutant Z19, the GLA content reached 2.4% when cultivated outdoors. To the best of our knowledge, this is the highest GLA content ever reported for any alga.

A combined stress response analysis of Spirulina platensis in terms of global differentially expressed proteins, and mRNA levels and stability of fatty acid biosynthesis genes.

Changes in gene expression play a critical role in enhancing the ability of cyanobacteria to survive under cold conditions. In the present study, Spirulina platensis cultures were grown at the optimal growth temperature, in the light, before being transferred to dark conditions at 22 degrees C. Two dimensional-differential gel electrophoresis was then performed to separate differentially expressed proteins that were subsequently identified by MS. Among all differentiated proteins identified, a protein involved in fatty acid biosynthesis, (3R)-hydroxymyristoyl-[acyl-carrier-protein]-dehydratase encoded by fabZ, was the most up-regulated protein. However, the fatty-acid desaturation proteins were not significantly differentiated. This raised the question of how the unsaturated fatty acid, especially gamma-linolenic acid, content in the cells in the cold-dark shift remained stable compared with that of the cold shift. Thus, a study at the transcriptional level of these desaturase genes, desC, desA and desD, and also of the fabZ gene was conducted. The results indicated that in the dark, where energy is limited, mRNA stability was enhanced by exposure to low temperatures. The data demonstrate that when the cells encounter cold stress with energy limitation, they can maintain their homeoviscous adaptation ability via mRNA stability.

Differential responses of three acyl-lipid desaturases to immediate temperature reduction occurring in two lipid membranes of Spirulina platensis strain C1

In cyanobacteria, the elevation of unsaturated fatty acid levels in membrane lipids has been shown to play a major role in the response to temperature change. The cyanobacterium Spirulina platensis strain C1 contains three desaturases--delta9, delta12 and delta6 desaturases--which are encoded by desC, desA and desD, respectively. In light of the above, a study was conducted of the regulation of desaturase gene expression in response to temperature change in S. platensis strain C1. The two lipid membranes, thylakoid and plasma, were separated, while the expressions of the desaturase genes to the downward shift of growth temperature were studied in the translation level by Western blot analysis. The results revealed that the expression of delta9 desaturase is independent of temperature. In the case of delta12 desaturase, two forms of the enzyme were found, at 45 and 40 kDa. In terms of correlation with the results in the transcription level, it is more likely that the 45 kDa-delta12 desaturase and the 40 kDa-delta12 desaturase are translated from 1.7 kb and 1.5 kb mRNA, respectively. Taken together, the results indicate that the expression of the 40 kDa-delta12 desaturase is temperature independent, whereas, the 45 kDa protein form demonstrates a response to the immediate temperature reduction. Furthermore, the activity of delta6 desaturase in the two lipid membranes is possibly regulated by temperature reduction. However, alteration in the level of gamma-linolenic acid, the product synthesized by delta6 desaturase, was observed in the plasma membrane prior to the thylakoid membrane.

Herbicide-resistant lines of microalgae: Growth and fatty acid composition

Cell lines of Spirulina platensis and Porphyridium cruentum resistant to growth inhibition by the herbicide SAN 9785 had a significantly higher growth rate than their respective wild-type strains. These lines were also shown to overproduce y-linolenic acid (GLA) and eicosapentaenoic acid (EPA), respectively, in the presence and absence of the inhibitor, as compared with wild-type cultures under similar conditions. The effect was most conspicuous in polar lipids. Thus, the proportion of GLA in the galactolipid (GL) fraction of the SAN 9785-resistant strain of S. platensis, SRS-1, increased in the absence of the inhibitor from 33.3% in the wild-type to 39.0%. Similarly, the proportion of EPA in the GL fraction of the resistant strain of P. cruentum, SRP, increased in the presence of the inhibitor from 29.1 to 45.4%.

Productivity of the cyanobacterium Spirulina platensis in cultures using sunlight

The growth kinetics of a vacuolated Spirulina platensis strain BP and a non-vacuolated Spirulina platensis strain 12D in open pond cultures were studied. The changes in the dissolved oxygen concentration of the cultures were parallel to the sunlight intensity profiles during the day. The specific growth rate remained unchanged over a large part of the day, although the growth rate in the early morning varied as a function of cell concentration. The variation in the dark respiration rate during the day was about 30%. The photosynthesis of the cultures was not photoinhibited in the early morning. The degree of photoinhibition increased in the morning to reach a value of 15–24%, depending on the cell concentration and light intensity at noon, and then decreased gradually in the afternoon. The study suggests that the rate limiting step in the growth of Spirulina cultures using sunlight was not at the light harvesting step (photosynthesis), but the rate of uptake of a key substrate, or the activity of a key enzyme in the biochemical pathways.

Effect of two intermediate electron donors, NADPH and FADH2, on Spirulina Δ6-desaturase co-expressed with two different immediate electron donors, cytochrome b5 and ferredoxin, in Escherichia coli

When the gene desD encoding Spirulina Δ6-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b5 domain from Mucor rouxii, the results showed the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ6-desaturase. The results revealed that in E. coli cells, where cytochrome b5 is absent and ferredoxin, a natural electron donor of Δ6-desaturase, is present at a very low level, the cytochrome b5 domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Δ6-desaturase in E. coli. In comparison to the co-expression of cytochrome b5 with the Δ6-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Δ6-desaturase co-expressed with cytochrome b5 and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH2 (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.