The isocitrate dehydrogenase gene of oleaginous yeast Lipomyces starkeyi is linked to lipid accumulation.

The oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids to over 60% of its cell dry mass under a nitrogen-limited condition. We showed that extracellular and intracellular citrate concentrations of L. starkeyi AS 2.1560 increased and the nicotinamide adenine dinucleotide - isocitrate dehydrogenase (NAD+-IDH) activity decreased at the beginning of the lipid accumulation, suggesting that the attenuation of the NAD+-IDH activity might initiate lipid storage. We next cloned the IDH gene by the methods of degenerate PCR and rapid amplification of cDNA ends. Phylogenetic analyses of the evolutionary relationships among LsIDH1, LsIDH2, and other yeast NAD+-IDHs revealed that the L. starkeyi IDH had a closer relationship with the IDHs of Yarrowia lipolytica. Further real-time PCR analysis showed that the expression levels of both LsIDH1 and LsIDH2 decreased concurrently with the evolution of cellular lipids. Our data should be valuable for understanding the biology of oleaginous yeasts and for further strain engineering of L. starkeyi.

[1]  L. D. Barnes,et al.  Yeast diphosphopyridine nucleotide specific isocitrate dehydrogenase. Regulation of activity and unidirectional catalysis. , 1972, Biochemistry.

[2]  C. Ratledge,et al.  Fatty Acid Distribution in Triglycerides of Yeasts Grown on Glucose or n-Alkanes , 1972 .

[3]  Colin Ratledge,et al.  ATP: Citrate Lyase - The Regulatory Enzyme for Lipid Biosynthesis in Lipomyces starkeyi? , 1981 .

[4]  Colin Ratledge,et al.  Correlation of Lipid Accumulation in Yeasts with Possession of ATP: Citrate Lyase , 1981 .

[5]  C. Ratledge,et al.  Use of Transition Studies in Continuous Cultures of Lipomyces starkeyi, an oleaginous yeast, to Investigate the Physiology of Lipid Accumulation , 1983 .

[6]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[7]  R. Fall,et al.  Bioconversion of Xylan to Triglycerides by Oil-Rich Yeasts , 1984, Applied and environmental microbiology.

[8]  E. Hammond,et al.  Biomodification of fats and oils: Trials withCandida lipolytica , 1984 .

[9]  C. T. Evans,et al.  Possible regulatory roles of ATP:citrate lyase, malic enzyme, and AMP deaminase in lipid accumulation by Rhodosporidium toruloides CBS 14 , 1985 .

[10]  C. T. Evans,et al.  The role of the mitochondrial NAD+: isocitrate dehydrogenase in lipid accumulation by the oleaginous yeast Rhodosporidium toruloides CBS 14 , 1985 .

[11]  L. McAlister-Henn,et al.  Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae , 1990, Journal of bacteriology.

[12]  C. Ratledge.  Microorganisms for lipids , 1991 .

[13]  H. Weiss,et al.  The role of the proton-pumping and alternative respiratory chain NADH:ubiquinone oxidoreductases in overflow catabolism of Aspergillus niger. , 1993, European journal of biochemistry.

[14]  L. McAlister-Henn,et al.  Kinetic analysis of NAD(+)-isocitrate dehydrogenase with altered isocitrate binding sites: contribution of IDH1 and IDH2 subunits to regulation and catalysis. , 1993, Biochemistry.

[15]  M. McCammon,et al.  Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase. , 2001, Biochemistry.

[16]  C Ratledge,et al.  Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. , 2001, Microbiology.

[17]  C. Ratledge,et al.  Regulation of lipid accumulation in oleaginous micro-organisms. , 2002, Biochemical Society transactions.

[18]  C. Ratledge,et al.  The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. , 2002, Advances in applied microbiology.

[19]  L. McAlister-Henn,et al.  Isocitrate Binding at Two Functionally Distinct Sites in Yeast NAD+-specific Isocitrate Dehydrogenase* , 2002, The Journal of Biological Chemistry.

[20]  L. McAlister-Henn,et al.  Homologous Binding Sites in Yeast Isocitrate Dehydrogenase for Cofactor (NAD+) and Allosteric Activator (AMP)* , 2003, The Journal of Biological Chemistry.

[21]  Xiwen Chen,et al.  Cloning and activity analysis ofin vitro expression of plant NAD-IDH genes , 2004 .

[22]  S. Papanikolaou,et al.  Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats , 2001, Antonie van Leeuwenhoek.

[23]  C. Ratledge,et al.  Cryptococcus terricolus, an oleaginous yeast re-appraised , 1984, Applied Microbiology and Biotechnology.

[24]  Z. Zong-bao.  Toward Cheaper Microbial Oil for Biodiesel Oil , 2005 .

[25]  Weiwen Zhang,et al.  Optimization of RNA isolation from the archaebacterium Methanosarcina barkeri and validation for oligonucleotide microarray analysis. , 2006, Journal of microbiological methods.

[26]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[27]  Fengwu Bai,et al.  High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture , 2007 .

[28]  S. Papanikolaou,et al.  Organic nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accumulation in Cunninghamella echinulata , 2008, Journal of applied microbiology.

[29]  Q. Wang,et al.  Determination of organic acids in the presence of inorganic anions by ion chromatography with suppressed conductivity detection. , 2008, Journal of chromatography. A.

[30]  Xin Zhao,et al.  Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi , 2008 .

[31]  M Mittelbach,et al.  Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production. , 2008, Bioresource technology.