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Thioesterase from Haematococcus pluvialis

Acyl-[acyl-carrier-protein] thioesterase (FATA)

Usage and Biology

This part contains a promoter BBa_J23119, the RBS* BBa_K2924009, a coding region for the acyl-acyl carrier protein thioesterase (FATA) of Haematococcus pluvialis BBa_K2924002 and a double terminator BBa_B0015.

Background

Fatty acids are long aliphatic chained carboxylic acids, which can be saturated or unsaturated. They have mostly an even number of carbon atoms from 4 to 28.

Fig. 1: Structural formula of hexadecanoic acid. Gray spheres represent a carbon atom, red spheres represent oxygen atom and the white spheres represent hydrogen atoms.

Fig. 2: Structural formula of octadecanoic acid. Gray spheres represent a carbon atom, red spheres represent oxygen atom and the white spheres represent hydrogen atoms.

Hexadecanoic acid (Fig. 1) is a saturated long-chain fatty acid, which contains 16 carbon-atoms and is also called palmitic acid. It has a white or colorless crystalline form and a slightly characteristic odor ¹. It has a molecular weight of 256,42 g/mol.

The octadecanoic acid (Fig. 2) is a saturated long-chain fatty acid, which contains 18 carbon-atoms and is also called stearic acid. It is a white or slightly yellow, wax-like solid with mild odor ². It has a molecular weight of 284,48 g/mol.

Biosynthesis

Fatty acids are synthesized and elongated by fatty acid synthases, which is a complex containing multiple enzymes. The determination of its length is controlled by thioesterases, a subgroup of hydrolases. They hydrolyze acetyl-CoA esters or acyl carrier protein esters to the corresponding free fatty acid and the Coenzyme A or the acyl carrier protein.

The thioesterase of the microalgae Haematococcus pluvialis (FATA), one of the key rate-limiting genes of the fatty acid synthesis, uses acyl-ACP as substrates and determines the chain-length of fatty acids producing palmitic and stearic acid ³. The microalgae shows high potential in producing C16:0 and C18:0 lipids and furthermore FATA could become a potential candidate for genetically engineered production for fatty acids and biofuel ³.

Usage of palmitic and stearic acid

The long-chain fatty acid palmitic acid is found naturally in human milk, in cow's milk fat for triglycerides or in vegetable oils and it is commonly used in infant formulas ⁴. It is the most abundant fatty acid in cow’s milk ⁵.

Palmitic acid is an important chemical, which is used to make soaps or cosmetics agents, lubricating oils, waterproofing materials, food additives and other chemicals ¹.

Stearic acid is also found naturally in animal milk and vegetable oils ² and used in manufacturing of different pharmaceuticals, cosmetics, soaps, candles, food packaging, modeling compounds and other chemicals. It is found sometimes in pesticides ².

Moreover, they play an important role for the intracellular biological functions. They mainly serve as sources of metabolic energy or as substrates for the cell membrane biosynthesis. Another function is serving as precursor, such as PGs, leukotrienes and more, for different signaling pathways ⁶. It has been shown that saturated fatty acids, such as palmitic and stearic acid, induce apoptosis of human granulosa cells, which could be used for further medical investigations ⁶. Both fatty acids are used for special dietary to increase the milk fat yield and as an energy source for milk production ⁵. Sequence and Features

Characterization

In the following, the thioesterase FATA is labeled as TeHP (thioesterase of Haematococcus pluvialis.

For the fatty acid analysis in Synechocystis sp. and E.coli BL21, the E. coli transformants as well as the Synechocystis conjugants and the wild type as control were grown under the same grow conditions for each strain. 4 optical density units of the cells, usually, an equivalent of 4 ml cells at OD₆₀₀ = 1, were isolated and used for extraction and derivatization of fatty acids for comparison. The extract was used for gas chromatography-mass spectrometry (GC-MS) to identify different fatty acids compositions in Synechocystis and E. coli (Fig. 3).

Fig. 3: Intracellular levels of fatty acids tested via GC-MS. E. coli transformants (red) and Synechocystis conjugants (orange) with the TeHP were tested and the fatty acids profiles were compared to the E. coli (blue) and Synechocystis (green) controls.

In the following, the thioesterase FATA is labeled as TeHP (thioesterase of Haematococcus pluvialis).

For the fatty acid analysis in Synechocystis and Escherichia coli BL21, the E. coli transformants as well as the Synechocystis conjugants and the wild type as control were grown under the same grow conditions for each strain. 4 optical density units of the cells, usually, an equivalent of 4 ml cells at OD₆₀₀ = 1, were isolated and used for extraction and derivatization of fatty acids for comparison. The extract was used for gas chromatography-mass spectrometry (GC-MS) to identify different fatty acids compositions in Synechocystis and E. coli (Fig. 3).

The results show, that both organisms exhibit a differentiatial free fatty acid profile (Fig. 3). E.coli transformants compared to the control show a slightly higher yield in C18:0 and C18:1 fatty acids as the control. C16:1, C18:3 and C20:1 fatty acids show a slightly lower yield as the control. As for Synechocystis sp., there is a clearly higher intracellular level compared to the control for the C20:1 fatty acid. This results may be due to the heterologous expressed thioesterase of Haematococcus pluvialis.

Interestingly, the thioesterase of Haematococcus pluvialis has a different impact on the different organisms. While in Synechocystis the changes are not that huge, and C18 fatty acids are targeted, in E. coli TeHP shows an enormous increase in free C20:1 fatty acid, making it a suitable host for its production.

References

[1]: National Center for Biotechnology Information. PubChem Database. Palmitic acid, CID=985, https://pubchem.ncbi.nlm.nih.gov/compound/Palmitic-acid (accessed on Oct. 5, 2019)

[2]: National Center for Biotechnology Information. PubChem Database. Stearic acid, CID=5281, https://pubchem.ncbi.nlm.nih.gov/compound/Stearic-acid (accessed on Oct. 5, 2019)

[3]: Saha, Sushanta Kumar, et al. "Effect of various stress-regulatory factors on biomass and lipid production in microalgaHaematococcus pluvialis." Bioresource technology 128 (2013): 118-124.

[4]: Carnielli, Virgilio P., et al. "Structural position and amount of palmitic acid in infant formulas: effects on fat, fatty acid, and mineral balance." Journal of pediatric gastroenterology and nutrition 23.5 (1996): 553-560.

[5]: Mu, Yi-Ming, et al. "Saturated FFAs, palmitic acid and stearic acid, induce apoptosis in human granulosa cells." Endocrinology 142.8 (2001): 3590-3597.

[6]:Loften, J. R., et al. "Invited review: Palmitic and stearic acid metabolism in lactating dairy cows." Journal of dairy science 97.8 (2014): 4661-4674.