2012 International Congress on Informatics, Environment, Energy and Applications-IEEA 2012
IPCSIT vol.38 (2012) © (2012) IACSIT Press, Singapore
Construction of A Platform for Fatty Acid Secretion from Chlorella
Chih-Yao Changa, Szu-Ting Lina, and Chien-Chia Wang1*
a
Department of Life Sciences, National Central University, No.300, Jung-da Rd., Jung-li City 32001, Taiwan
(ROC)
Abstract. Concerns about petroleum supplies, high energy costs, and environmental pollutions are drawing
considerable attention to find renewable biofuels. Biodiesel is produced from renewable biomass by
transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with
short-chain alcohols. Production of biodiesel by the transesterification process using alkali catalysts has been
widely accepted for its high conversion and reaction rates. However, such a process has also raised serious
concerns about its impact on environment. In contrast, enzymatic transesterification has recently become the
method of choice for biodiesel production as it produces high-purity product and enables easy separation of
glycerol from the byproducts. Hence, more attention has been focused on the application of genetically
engineered microalgae in the production of biodiesel. However, there is an upper limit in the lipid content of
the microalgae (~50%). In addition, an energy-consuming step is required to break down the microalgae to
extract the intracellular lipids. Up to date, the price of biodiesel is still much higher than that of petroleumbased diesel fuel. Therefore, a new strategy for producing low-cost biodiesel is imperative. To this end, we
constructed a highly efficient fatty acid (FA) secretion system in chlorella, a genus of single-celled green
algae. This strategy can not only reduce the cost of biodiesel by skipping the breaking-down step, but also aid
in the development of a consolidated bioprocess.
Keywords: chlorella, biodiesel, fatty acid secretion.
1. Introduction
1.1. Chlorella
The name Chlorella is derived from the Greek word chloros, meaning “green” and the Latin diminutive
suffix ella, meaning "small". They are typically small (2 to 10 μm in diameter), unicellular, coccoid, and
nonmotile. Chlorella harbors the green photosynthetic pigments in its chloroplast. It multiplies rapidly and
requires only carbon dioxide, water, sunlight, and a small amount of minerals to reproduce.
1.2. Biodiesel
Biofuels are a variety of fuels derived from biomass. Biodiesels are fatty acid methyl esters (FAMEs)
made from transesterification of plant oils or fats. It is a liquid, similar in composition to fossil/mineral diesel
[1]. In addition, biodiesel is the most common biofuel because it can be used in any diesel engine in any ratio.
There are several important steps in order to make biodiesel from algae, including culture, fatty acid
extraction, and transesterification [2]. Transesterification, a frequently used organic chemistry conversion
technology, is the process of exchanging the organic group R" of an ester with the organic group R' of an
alcohol. In other words, it is a chemical process that triglycerides are reacted with methanol to yield glycerol
and fatty acid methyl esters (FAME) [3].
1.3. Fatty acid secretion platform
There are many undergoing studies on increasing the biomass and oil content [4, 5]. However, fatty acid
extraction is still tough and needs to be improved. Regardless of using pressure or ultrasonic to extract fatty
* Corresponding author. Tel.: +886-3-422-7151-65061; fax: +886-3-422-8482.
E-mail address: dukewang@cc.ncu.edu.tw.
acids, they consume a great deal of energy. So, a new way is needed to solve this problem. There is little
information on how the fatty acids are secreted from Chlorella. In contrast, there are many studies on how
plants release fatty acids to extracellular matrix. We summarized here four mechanisms by which fatty acids
can be secreted from plants or microorganisms (Fig. 1). The first is a carrier protein, which can bind
hydrophobic substance and move to or through cell membrane [6]. The second is a channel protein. This type
of protein contains at least one trans-membrane domain and is embedded in the cell membrane, becoming a
channel. Substrates can pass through the membrane from inside to outside by the channel [7]. The third is a
pump protein, which is typically a protein complex embedded in the cell membrane. Only some specific
substrates can bind to the pump protein and pass through the cell membrane [8]. The last is an as-yetundefined system, which releases fatty acid not by carrier protein, channel protein, or pump protein. For
example, knock-down of fatty acyl-CoA synthetase in S. cerevisiae increases oil content with fatty acids
secreted in culture medium [9].
1.4. Summary
Little is known about fatty acid secretion in Chlorella. Our primary goal is to create a highly efficient
fatty acid secretion platform in Chlorella using one of the four systems mentioned above. The target genes of
interest were cloned in appropriate plasmids (Table 1), and the resulting constructs were transformed into E.
coli or chlorella for functional assays. The intracellular lipids were assayed by nile red fluorescent staining
(Fig. 2), and the ratio of the fatty acids between intracellular and extracellular matrixes was further analyzed
by GC (gas chromatography) (Fig. 3). The data showed that a carrier protein from Synechocystis sp. 6803
and A. thaliana significantly increases fatty acid secretion from E. coli.
Fig 1. Mechanisms of fatty acid secretion. FABP (Fatty Acid Binding Protein) and Lipid transfer protein (LTP) are
carrier proteins that accelerate the transfer of fatty acids and lipids to cell membrane or outside. CER5 is a channel
protein that ships fatty acids or lipids form inside to outside [10].
Table 1. The target genes of interest.
Carrier protein
A. thaliana
FABP-A
A. thaliana
FABP-B
Synechocystis sp 6803
FABP-C
Synechocystis sp 6803
FABP-D
Other
A. thaliana
Acyl-ACP thioesterase A (FatA)
A. thaliana
Acyl-ACP thioesterase B (FatB)
E. coli
Lysophopholipase (tesA)
Fig 2. Neil red fluorescent staining. Plasmids that carry target genes of interest were transformed into E. coli . The
transformants were stained with nile red and visualized under a fluorescence microscope.
AtFABP-A AtFABP-B
AtCPX
SspFABP-D
SspCPB
Fig 3. Lipid distribution analysis by gas chromatography. E. coli was transformed with various genes from
Synechocystis sp 6803 and A. thaliana, and the lipid distribution in the transformants was examined by gas
chromatography.
2. Acknowledgements
Here, we would like to thank the support of the National Science Council and Industrial Technology
Research Institute .
3. References
[1] Sheehan, J., Camobreco, V., Duffiled, J., Graboski, M., Shapouri, H. National Renewable Energy Laboratory, US.
Department of Agriculture and U.S. Department of Energy, Prepared under Task No. BF886002; 1998.
[2] Scott SA, Davey MP, Dennis JS, Horst I, Howe CJ, Lea-Smith DJ, Smith AG. Biodiesel from algae: challenges
and prospects. Curr Opin Biotechnol 2010;3:277-86.
[3] Ranganathan SV, Narasimhan SL, Muthukumar K. An overview of enzymatic production of biodiesel Bioresour
Technol. 2008;99:3975-81.
[4] Liu X, Brune D, Vermaas W, Curtiss R. Production and secretion of fatty acids in genetically engineered
cyanobacteria Proc Natl Acad Sci 2010;10:1073.
[5] Liu J, Huang J, Sun Z, Zhong Y, Jiang Y, Chen F. Differential lipid and fatty acid profiles of photoautotrophic and
heterotrophic Chlorella zofingiensis: assessment of algal oils for biodiesel production Bioresour Technol
2011;102:106-10.
[6] Guerbette F, Grosbois M, Jolliot-Croquin A, Kader J. C., Zachowski A. Lipid-transfer proteins from plants:
structure and binding properties Mol Cell Biochem 1999;192: 157-61.
[7] David A. B. The role of ABC transporters in cuticular lipid secretion Plant Science 2008;174:563–9.
[8] Tal N., Schuldiner S. A coordinated network of transporters with overlapping specificities provides a robust
survival strategy Proc Natl Acad Sci 2009;106:9051-6.
[9] Michinaka Y, Shimauchi T, Aki T, Nakajima T, Kawamoto S, Shigeta S, Suzuki O, Ono K. Extracellular secretion
of free fatty acids by disruption of a fatty acyl-CoA synthetase gene in Saccharomyces cerevisiae J Biosci Bioeng
2003;5,435-40.
[10] Thoma S, Kaneko Y, Somerville C. A non-specific lipid transfer protein from Arabidopsis is a cell wall protein
Plant J 1993;3:427-36.