Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005
Authors: Maulik R Kamdar
Mrinal K Maiti
Prabuddha Dey
Monami Chakraborty
Journal: PLoS ONE (2014)
Discipline: Multidisciplinary
Keywords: Diacylglycerol acyltransferase; DGAT; Candida tropicalis; triacylglycerol; gene homologues
Affiliations: Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, India., Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India., Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, India; Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India.

Abstract:  Diacylglycerol acyltransferase (DGAT) activity is an essential enzymatic step in the formation of neutral lipid i.e., triacylglycerol in all living cells capable of accumulating storage lipid. Previously, we characterized an oleaginous yeast Candida tropicalis SY005 that yields storage lipid up to 58% under a specific nitrogen-stress condition, when the DGAT-specific transcript is drastically up-regulated. Here we report the identification, differential expression and function of two DGAT2 gene homologues--CtDGAT2a and CtDGAT2b of this C. tropicalis. Two protein isoforms are unique with respect to the presence of five additional stretches of amino acids, besides possessing three highly conserved motifs known in other reported DGAT2 enzymes. Moreover, the CtDGAT2a and CtDGAT2b are characteristically different in amino acid sequences and predicted protein structures. The CtDGAT2b isozyme was found to be catalytically 12.5% more efficient than CtDGAT2a for triacylglycerol production in a heterologous yeast system i.e., Saccharomyces cerevisiae quadruple mutant strain H1246 that is inherently defective in neutral lipid biosynthesis. The CtDGAT2b activity rescued the growth of transformed S. cerevisiae mutant cells, which are usually non-viable in the medium containing free fatty acids by incorporating them into triacylglycerol, and displayed preferential specificity towards saturated acyl species as substrate. Furthermore, we document that the efficiency of triacylglycerol production by CtDGAT2b is differentially affected by deletion, insertion or replacement of amino acids in five regions exclusively present in two CtDGAT2 isozymes. Taken together, our study characterizes two structurally novel DGAT2 isozymes, which are accountable for the enhanced production of storage lipid enriched with saturated fatty acids inherently in C. tropicalis SY005 strain as well as in transformed S. cerevisiae neutral lipid-deficient mutant cells. These two genes certainly will be useful for further investigation on the novel structure-function relationship of DGAT repertoire, and also in metabolic engineering for the enhanced production of lipid feedstock in other organisms. 

Retracted: Yes

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 An email from Kaitlyn Keller, Publications Manager, PLOS ONE, indicates that this paper is set to be retracted: u00e2u0080u009cIn light of these concerns, and following an institutional investigation, we have decided to proceed with the retraction of pone.0111253 and pone.0094472. Additionally, we will be publishing a correction on the third article, pone.0150763.u00e2u0080u009d , 
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 Retraction, October 2017: https: journals.plos.org plosone article?id=10.1371 journal.pone.0187146  The authors and editors retract this publication [1] following an investigation into concerns around the images presented in several figures. After the publication of this article, readers raised concerns about potential image duplications in Figures 4A-C, 5A-B, 6C, 7A, and 7C. Specifically, * There are similarities between gel bands in Figure 4C and Figure 7B. * The backgrounds and first two lanes of Figures 4A and 7C appear to be identical. * Figure 4B: there are areas of overlap between figures in the WT, QM-CtDGAT2a and QM-CtDGAT2b panels. * Figure 5A: there are duplications between different yeast growth panels within the figure. * Figure 5B: there is a duplication between the left plates for C18:3 and C20:4. * Figure 6C: several yeast growth images appear to be derived from the same source image, involving panels u0394D2, u0394D4 and u0394D5. * Figure 7A: There appear to be areas of overlap between the right panels for u0394D1(1u201321) and u0394D5(481u2013571). The concerns were brought to the attention of the authors, who provided replicate images of the same experimental sets but were not able to provide the images underlying the figures included in the article. An institutional investigation conducted by The Indian Institute of Technology at Kharagpur confirmed the duplication of images and recommended the retraction of the article. The preparation of the figures falls below the standard of publication; in light of these concerns and in line with the institutional recommendation, the authors and the editors retract this publication. The authors sincerely apologize to the scientific community for the errors in the published article. I was Peer 1 above, and I reported these concerns to the journal in April 2014. ,