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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.

SUBMITTER: Dey P

PROVIDER: S-EPMC3986092 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005.

Dey Prabuddha P Chakraborty Monami M Kamdar Maulik R MR Maiti Mrinal K MK

PloS one 20140414 4

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 ...[more]

PMID: 24732323

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Project description:Background:Without sufficient alternatives to crude oil, as demand continues to rise, the global economy will undergo a drastic decline as oil prices explode. Dependence on crude oil and growing environmental impairment must eventually be overcome by creating a sustainable and profitable alternative based on renewable and accessible feedstock. One of the promising solutions for the current and near-future is the substitution of fossil fuels with sustainable liquid feedstock for biofuel production. Among the different renewable liquid feedstock's studied, wastewater is the least explored one for biodiesel production. Sago wastewater is the byproduct of the cassava processing industry and has starch content ranging from 4 to 7%. The present investigation was aimed to produce microbial lipids from oleaginous yeast, Candida tropicalis ASY2 for use as biodiesel feedstock and simultaneously decontaminate the sago processing wastewater for reuse. Initial screening of oleaginous yeast to find an efficient amylolytic with maximum lipid productivity resulted in a potent oleaginous yeast strain, C. tropicalis ASY2, that utilizes SWW as a substrate. Shake flask experiments are conducted over a fermentation time of 240 h to determine a suitable fatty acid composition using GC-FID for biodiesel production with simultaneous removal of SWW pollutants using ASY2. Results:The maximum biomass of 0.021 g L-1 h-1 and lipid productivity of 0.010 g L-1 h-1 was recorded in SWW with lipid content of 49%. The yeast strain degraded cyanide in SWW (79%) and also removed chemical oxygen demand (COD), biological oxygen demand (BOD), nitrate (NO3), ammoniacal (NH4), and phosphate (PO4) ions (84%, 92%, 100%, 98%, and 85%, respectively). GC-FID analysis of fatty acid methyl esters (FAME) revealed high oleic acid content (41.33%), which is one of the primary fatty acids for biodiesel production. Conclusions:It is evident that the present study provides an innovative and ecologically sustainable technology that generates valuable fuel, biodiesel using SWW as a substrate and decontaminates for reuse.

Dataset Information

Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005.

Publications

Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005.

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