Overexpression of peanut diacylglycerol acyltransferase 2 in Escherichia coli
Authors: Lianqun Yang
Zhenying Peng
Gao Chen
Yuping Bi
Bin Zhang
Lan Li
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: Diacylglycerol acyltransferase; Triacylglycerol biosynthesis; Arachis hypogaea; Peanut cultivar; AhDGAT2
Affiliations: High-Tech Research Center, Shandong Academy of Agricultural Science, Jinan, China.

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. 

Retracted: Yes

PubPeer comments:
null, 
 Retraction, June 4, 2021: https: journals.plos.org plosone article?id=10.1371 journal.pone.0252974 It reads:  Article Metrics Comments Media Coverage Reference Reader Comments (0) Following the publication of this article [1], concerns were raised regarding results presented in Figs 1 and 7. Specifically, The background signal directly surrounding the bands presented in Fig 5B appears to be cut off at straight horizontal and vertical edges and appears to be inconsistent with the overall background of the blot. Fig 7 panels B, F, and H, demonstrate square and rectangular areas where the background colour and pattern does not appear to match the overall background signal of the rest of the panel. In addition, the background signal surrounding the cells in the Fig 7 A, C, D, E, and G panels appears to be cut off at straight horizontal and vertical edges and does not appear to match the overall background of these panels. The first author indicated that the corresponding author is retired and that the raw data underlying the results presented in this study are no longer available. In light of the concerns affecting multiple figure panels that question the integrity of these data, the PLOS ONE Editors retract this article. The authors either did not respond to the editorial decision to retract this article or could not be reached. I was Peer 1 and I reported this paper to the journal in October 2015. The PubPeer post in #1 was posted on 10 25 2014. ,