title: Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005.
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.
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, India.
authors.forename: Prabuddha
authors.initials: P
authors.lastname: Dey
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, India.
authors.forename: Monami
authors.initials: M
authors.lastname: Chakraborty
authors.affiliation: Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India.
authors.forename: Maulik R
authors.initials: MR
authors.lastname: Kamdar
authors.affiliation: 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.
authors.forename: Mrinal K
authors.initials: MK
authors.lastname: Maiti
mesh_terms: D000595:Amino Acid Sequence; D041022:Candida tropicalis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D051048:Diacylglycerol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009584:Nitrogen / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013228:Stearates / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics; D014280:Triglycerides / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D013228:Stearates; D014280:Triglycerides; D051048:Diacylglycerol O-Acyltransferase; D009584:Nitrogen
references.citation: Phytochemistry. 2006 Dec;67(23):2541-9
references.pmid: 17084870
references.citation: N Biotechnol. 2009 Oct 1;26(1-2):11-6
references.pmid: 19501678
references.citation: J Mol Biol. 1982 May 5;157(1):105-32
references.pmid: 7108955
references.citation: J Microbiol Methods. 2004 Mar;56(3):331-8
references.pmid: 14967224
references.citation: Prog Lipid Res. 2012 Oct;51(4):350-77
references.pmid: 22705711
references.citation: BMC Genomics. 2011 Jun 02;12:286
references.pmid: 21635767
references.citation: Lipids. 2009 Oct;44(10):963-73
references.pmid: 19763656
references.citation: J Biol Chem. 2005 Apr 15;280(15):14755-64
references.pmid: 15671038
references.citation: J Biol Chem. 2011 Apr 15;286(15):13115-26
references.pmid: 21321129
references.citation: Plant Physiol. 2006 Aug;141(4):1533-43
references.pmid: 16798944
references.citation: Microbiology (Reading). 2013 Jan;159(Pt 1):155-166
references.pmid: 23103975
references.citation: J Biol Chem. 2006 Dec 29;281(52):40273-82
references.pmid: 17035227
references.citation: Plant Biotechnol J. 2010 Feb;8(2):184-95
references.pmid: 20078841
references.citation: J Biol Chem. 2003 Mar 7;278(10):8075-82
references.pmid: 12502715
references.citation: Yeast. 2012 Jan;29(1):25-38
references.pmid: 22189651
references.citation: J Appl Microbiol. 2013 May;114(5):1357-68
references.pmid: 23311514
references.citation: Plant Physiol. 2001 Jun;126(2):861-74
references.pmid: 11402213
references.citation: Plant J. 1999 Sep;19(6):645-53
references.pmid: 10571850
references.citation: PLoS One. 2013 Apr 11;8(4):e61363
references.pmid: 23593473
references.citation: Can J Biochem Physiol. 1959 Aug;37(8):911-7
references.pmid: 13671378
references.citation: Plant Biotechnol J. 2008 Oct;6(8):799-818
references.pmid: 18631243
references.citation: Bioresour Technol. 2010 Nov;101(22):8658-63
references.pmid: 20615689
references.citation: J Biol Chem. 2001 Oct 19;276(42):38862-9
references.pmid: 11481333
references.citation: J Biol Chem. 2004 Mar 19;279(12):11767-76
references.pmid: 14668353
references.citation: Bioinformation. 2009 Jun 28;3(9):394-8
references.pmid: 19759814
references.citation: Trends Biochem Sci. 2000 Mar;25(3):111-2
references.pmid: 10694878
references.citation: Biochem Soc Trans. 2002 Nov;30(Pt 6):1047-50
references.pmid: 12440969
references.citation: FEBS J. 2011 Oct;278(19):3651-66
references.pmid: 21812932
references.citation: Curr Opin Biotechnol. 2013 Apr;24(2):300-9
references.pmid: 22981869
references.citation: Plant Cell. 2006 Sep;18(9):2294-313
references.pmid: 16920778
references.citation: Biochem Biophys Res Commun. 2003 Oct 17;310(2):296-302
references.pmid: 14521909
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6487-92
references.pmid: 10829075
references.citation: J Biol Chem. 2003 Nov 21;278(47):47145-55
references.pmid: 12963726
references.citation: Plant Biotechnol J. 2010 Apr;8(3):277-87
references.pmid: 20051035
references.citation: J Lipid Res. 2011 Sep;52(9):1760-4
references.pmid: 21653930
references.citation: Plant Physiol Biochem. 2010 Jun;48(6):407-16
references.pmid: 20400321
references.citation: Appl Microbiol Biotechnol. 2011 May;90(4):1193-206
references.pmid: 21452033
references.citation: J Biol Chem. 2012 May 4;287(19):15811-25
references.pmid: 22403401
references.citation: Biochem Soc Trans. 2000 Dec;28(6):692-5
references.pmid: 11171173