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Document 2087471
2013 2nd International Conference on Environment, Energy and Biotechnology
IPCBEE vol.51 (2013) © (2013) IACSIT Press, Singapore
DOI: 10.7763/IPCBEE. 2013. V51. 29
Lauroyl-Acyl Carrier Protein Thioesterase: a Key Enzyme for
Regulation of Medium-Chain Fatty Acid Synthesis in E. Coli
Hsinju Hsieh 1, Chih-Chieh Huang 1, Liang-Jung Chien 2 and Jia-Hung Wang 1
1
Material and Chemical Research Laboratories, Industrial Technology Research Institute
Rm.403, Bldg.10, 321, Kuang Fu Rd., Sec. 2, Hsinchu 30011, Taiwan
2
Graduate School of Biochemaishical Engineering, Ming Chi University of Technology
84 Gungjuan Rd., Tan, Taipei 24301, Taiwan

Abstract. Long carbon chain nylon is a high-performance, high price of chemical material, due to its
unique thermal, physical, chemical and mechanical properties superior to other materials, for only specified
use in automotive fuel lines and brake system of high specification materials. Chemical process using high
active catalyst (Et2AlCl) water explosion as a major potential risk factors, the continued use of the risk is still
high. Therefore, this study will focus in the safety of long carbon-chain aliphatic dicarboxylic acids nylon
materials green process development, in order to develop a series of relatively long carbon chain aliphatic
dicarboxylic acids nylon materials in response to the majority of the market required.
Keywords: long carbon chain nylon, long carbon-chain aliphatic dicarboxylic acids, green process
development
1. Introduction
Goal of this study is to develop a biological method development of nylon materials, microbes as a cell
production factory; the key issue is to explore ways to enhance the content of in vivo ω-oxidation reaction
(ω-oxidation) substrate specificity (lauric acid), dodecanedioic acid for nylon materials - biological
metabolic paths to explore the long chain nylon raw materials-α, ω-dicarboxylic acid biosynthesis.
2. Material and Methods
2.1. Materials and cultivation condition
The E. coli strain was purchased from Yeastern Biotech Co. (Taiwan) and cultured with a rich medium
(LB) at 37°C. The standard of methyl caprate, methyl laurate, methyl myristate, Methyl pentadecanoate,
methyl palmitate, methyl palmitoleate, methyl stearate, methyl oleate and methyl linoleate were purchased
from Sigma Co. (USA).
2.2. Construction of lauroyl-ACP thioesterase genes
The E. coli–codon–optimized genes (BTE and FatB3) from Umbellularia californic and Cocos nucifera
were obtained through artificial oligonucleotide synthesis from GeneScript (USA) and transformed into E.
coli. The resulting chimeric genes were re-isolated as BamHI/SalI and EcoRI/HindIII fragments and cloned
into the pET24a vector. Kanamycin was used as the selection markers in E. coli. The β-oxidation mutants
was modified the acyl-CoA synthetase by double crossover of the lauroyl-ACP thioesterase genes to form a
new recombinant strain.

Corresponding author. Tel.: ++886-3-573-2873; fax: ++886-3-5743907.
E-mail address: [email protected]
153
2.3. Lipid extraction and gas chromatography analysis
The total lipids were estimated as fatty acid methyl esters (FAME) by the direct transesterification
method. Fatty acid methyl esters were analyzed by a gas chromatography (Agilent VARIAN 3900, USA)
equipping with a flame ionization detector (FID) and a Stabilwax column. Nitrogen (1.5 mLmin-1) was used
as the carrier gas. Temperature was programmed increasement from 130 °C to 180 °C with a 10 °C min-1 and
thereafter to 210 °C with a 15 °Cmin-1. Injector and detector were maintained at 220 °C and 250 °C,
respectively. FAME contents were determined from their corresponding peak areas using Methyl
pentadecanoate as the internal standard. The data presented are the average of three estimations.
3. Result and Discussions
3.1.
Effect of lauroyl-ACP thioesterase of BTE on redirecting of fatty acid composition
In this feasibility assessment study, lauryl acid production pathway regulation of E. coli by insertion an
artificial DNA fragment selected from Umbellularia californic (E. coli codon usage) to wild type and βoxidation mutants (*), shown in Fig. 1. To construct a transgenic E. coli of biological fatty acid metabolic
pathway control, regulation of lauryl acid production. The modified E. coli was cultured then analyzes the
fatty acid composition of the cell lysed, the results shown in Fig. 2. The data indicated that C16: 0 fatty acid
ratio presented reduced, and improved the C12: 0 fatty acid ratio of the modified E. coli.
Escherichia coli
KAS
Lauric acid (C12 FFA)
Dodecaneduoic acid
(C12 DCA)
Fig 1. The fatty acid biosynthesis pathway in E. coli
3.2.
Effect of lauroyl-ACP thioesterase of FatB3 on redirecting of fatty acid composition
In this feasibility assessment study, lauryl acid production pathway regulation of E. coli by insertion an
artificial DNA fragment selected from Cocos nucifera(E. coli codon usage) to wild type and β-oxidation
mutants (*), shown in Fig. 1. To construct a transgenic E. coli of biological fatty acid metabolic pathway
control, regulation of lauryl acid production. The modified E. coli was cultured then analyzes the fatty acid
composition of the cell lysed, the results shown in Fig. 3. The data indicated that C16: 0 fatty acid ratio
presented reduced, and improved the C12: 0 fatty acid ratio of the modified E. coli.
3.3.
Effect of lauroyl-ACP thioesterase of BTE and FatB3 on redirecting of fatty acid composition
In this feasibility assessment study, lauryl acid production pathway regulation of E. coli by insertion two
artificial DNA fragments selected from Cocos nucifera and Umbellularia californic (E. coli codon usage) to
wild type and β-oxidation mutants (*), shown in Fig. 1. To construct a transgenic E. coli of biological fatty
acid metabolic pathway control, regulation of lauryl acid production. The modified E. coli was cultured then
analyzes the fatty acid composition of the cell lysed, the results shown in Fig. 4. The data indicated that C16:
0 fatty acid ratio presented reduced, and improved the C12: 0 fatty acid ratio of the modified E. coli.
154
重大技術成果-1 (MCTE)
50
herichia coli )-ITRI’s lab
重大技術成果-1 (MCTE)
因轉殖,可將C16/C18轉換為提升
(*:Knockoutβ-oxidation)
erichia Control
coli )-ITRI’s
lab
-
Control+
轉殖,可將C16/C18轉換為提升
肪酸含量。
Control+ BTE
Control-
BTE
Lipid contentLipid
(%) content (%)
40
脂肪酸含量。
BTE
BTE*
50
BTE
BTE*
30
(*:Knockoutβ-oxidation)
40
20
30
10
20
0
C 10:0 C 12:0 C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2
10
50 acid composition of the modified E. coli of BTE insertion
Fig. 2 : The fatty
因轉殖,可將C16/C18轉換為提升
肪酸含量。
Control+ FatB3
FatB3
FatB3
FatB3*
30
40
(*:Knockoutβ-oxidation)
大腸桿菌(Escherichia coli )-ITRI’s lab
20
30
BTE與FatB3複合優化基因轉殖,可將C16/C18
轉換為提升46%的C12
脂肪酸含量。
10
20
Control-
Control+
0
10
C 10:0 C 12:0 C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2
0
C 10:0 C 12:0 C 14:0 C 16:0 C 16:1
FatB3
C 18:0 C 18:1 C 18:2
BTE
50
C 16:0
40
30
20
10
Fig. 3 : The fatty acid composition of the modified E. coli of FatB3 insertion
50
BTE+FatB3
BTE+FatB3*
40
(*:Knockoutβ-oxidation)
30
20
Lipid Content (%)
500
Lipid Content (%)
Control-
重大技術成果-2 (MCT
C 10:0 C 12:0 C 14:0 C 16:0 C 16:1(*:Knockoutβ-oxidation)
C 18:0 C 18:1 C 18:2
50
Lipid Content (%)
Control
Control
轉殖,可將C16/C18轉換為提升
肪酸含量。
+
0
40
Lipid Content
(%)
Lipid
Content (%)
-
FatB3
FatB3*
40
C 12:0
30
20
10
0
10
Host 1 2 3 4
Induction
0
C 10:0 C 12:0 C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2
Fig. 4: The fatty acid composition of the modified E. coli of BTE and FatB3 insertion
155
4. Acknowledgements
This work was under the auspices of Industrial Technology Research Institute and Ming Chi University
of Technology. Funding for this work was provided by (Department of Industrial Technology) Ministry of
Economics Affairs, Taiwan under Grant No.B301ARY4W1 and C301AR4Y10.
5. References
[1] F.Jing, D.C.Cantu, J.Tvaruzkova1, J. P.Chipman, B.J.Nikolau, M. D.Yandeau-Nelson1 and P. J.Reilly.
Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in
enzymatic specificity and active. BMC Biochem. 2011, 12 (44):1-16.
[2] N.M.D. Courchesne, A. Parisien, B. Wang, and C.Q. Lan. Enhancement of lipid production using biochemical,
genetic and transcription factor engineering approaches. J. Biotechnol. 2009, 141: 31–41.
[3] S. Zibek, S. Huf, W. Wagner, T. Hirth, S. Rupp. Fermentative production of α, ω-dicarboxylic 1,18
Octadecanedioic acid as the building block for biobased plastics. CHEM ENG TECHNOL. 2009, 81: 1797-1808.
[4] T.Liu, H.Vora, C.Khosla. Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab.Eng.
2010, 12: 378-386.
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