Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli.
Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS-PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1Delta9) and cis-vaccenate (18:1Delta11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1Delta9) was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase.
Cao
Yujin
Y
Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, 266101 Qingdao, China.
Xian
Mo
M
Yang
Jianming
J
Xu
Xin
X
Liu
Wei
W
Li
Liangzhi
L
D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D029681:Arabidopsis Proteins; D005227:Fatty Acids; D011993:Recombinant Fusion Proteins; D044943:Fatty Acid Desaturases; C433487:stearoyl ACP desaturase, Arabidopsis