Abstract

Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme in triacylglycerol biosynthesis in eukaryotic organisms. Triacylglycerols are important energy-storage oils in plants such as peanuts, soybeans and rape. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14’ using a homologous gene sequence method and rapid amplification of cDNA ends. To understand the role of AhDGAT2 in triacylglycerol biosynthesis, two AhDGAT2 nucleotide sequences that differed by three amino acids were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli Rosetta (DE3). Following IPTG induction, the isozymes (AhDGAT2a and AhDGAT2b) were expressed as 64.5 kDa GST fusion proteins. Both AhDGAT2a and AhDGAT2b occurred in the host cell cytoplasm and inclusion bodies, with larger amounts in the inclusion bodies. Overexpression of AhDGATs depressed the host cell growth rates relative to non-transformed cells, but cells harboring empty-vector, AhDGAT2a–GST, or AhDGAT2b–GST exhibited no obvious growth rate differences. Interestingly, induction of AhDGAT2a–GST and AhDGAT2b–GST proteins increased the sizes of the host cells by 2.4–2.5 times that of the controls (post-IPTG induction). The total fatty acid (FA) levels of the AhDGAT2a–GST and AhDGAT2a–GST transformants, as well as levels of C12:0, C14:0, C16:0, C16:1, C18:1n9c and C18:3n3 FAs, increased markedly, whereas C15:0 and C21:0 levels were lower than in non-transformed cells or those containing empty-vectors. In addition, the levels of some FAs differed between the two transformant strains, indicating that the two isozymes might have different functions in peanuts. This is the first time that a full-length recombinant peanut DGAT2 has been produced in a bacterial expression system and the first analysis of its effects on the content and composition of fatty acids in E. coli. Our results indicate that AhDGAT2 is a strong candidate gene for efficient FA production in E. coli.

Highlights

  • Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme of the Kennedy pathway for synthesizing triacylglycerols (TAGs) in eukaryotes

  • RACE, we identified two isozymes of AhDGAT2 from the peanut cultivar ‘Luhua 14’ and designated them AhDGAT2a and AhDGAT2b (GenBank accession numbers JF897614 and JF897615, respectively)

  • We constructed AhDGAT2a and AhDGAT2b plasmids to enable us to investigate their individual functions in E. coli

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Summary

Introduction

Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme of the Kennedy pathway for synthesizing triacylglycerols (TAGs) in eukaryotes. DGAT1 makes the major contribution to seed oil accumulation [1,4,7], whereas DGAT2 and PDAT both affect the specific accumulation of unusual fatty acids (FAs) in seed oil [2,8,9,10,11]. These enzymes have unique expression patterns in a variety of plant tissues [7] and may have other roles besides seed oil accumulation, such as FA mobilization [12] and leaf senescence [13]. The process by which such enzymes are regulated in developing seeds and other tissues remains poorly understood

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