Cloning of Acyl-ACP Thioesterase FatA fromArachis hypogaeaL. and Its Expression inEscherichia coli

Gao Chen,Yan Zhang,Qing-Fang He,Lan Li,Gui-Ying Tang,Ning Xuan,Lei Shan,Zhen-Ying Peng,Yu-Ping Bi

doi:10.1155/2012/652579

Abstract

In this study, a full-length cDNA of the acyl-ACP thioesterase, AhFatA, was cloned from developing seeds of Arachis hypogaea L. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of AhFatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli.

Highlights

In higher plants, fatty acid biosynthesis is catalyzed by the action of a type II fatty acid synthase, located in plastids [1,2,3,4]
Real-time quantitative Polymerase chain reaction RACE (PCR) analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging
Phylogenetic analysis (Supplementary Figure 1B) indicated that AhFatA has a higher similarity to FatAs, such as GarmFatA (Garcinia mangostana, 1930076) and RcFatA (Ricinus communis, 152206073), which have preference for 18:1-ACP and 16:1-ACP [4, 13]

Summary

Introduction

Fatty acid biosynthesis is catalyzed by the action of a type II fatty acid synthase, located in plastids [1,2,3,4]. Plant acyl-ACP thioesterases are plastid-targeted and nuclear-encoded proteins. Based on their sequence identity and substrate specificity, there are two gene families: FatA and FatB [9,10,11]. The FatA gene is one of the key genes involved in the plastidial fatty acid biosynthesis pathway and encodes thioesterase, with a higher specificity for 18:1-ACP and a lower activity for 18:0-ACP and 16:0-ACP [5, 12,13,14,15]. The FatB gene encodes thioesterases with a preference for saturated fatty acids with 8–18 carbons [4, 5, 7, 16]

Results

Discussion

Conclusion