title: Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation.
abstract: Malic enzyme (EC 1.1.1.40) converts L-malate to pyruvate and CO2 providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD(+) as the primary cofactor. Km values for NAD(+) and NADP(+) were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5-15 mM) increased NADP(+)- but not NAD(+)-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP(+)-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.
authors.affiliation: School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China, zhyuan004@126.com.
authors.forename: Huaiyuan
authors.initials: H
authors.lastname: Zhang
authors.forename: Luning
authors.initials: L
authors.lastname: Zhang
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.forename: Yong Q
authors.initials: YQ
authors.lastname: Chen
authors.forename: Colin
authors.initials: C
authors.lastname: Ratledge
authors.forename: Yuanda
authors.initials: Y
authors.lastname: Song
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D050356:Lipid Metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D019343:Citric Acid; D009249:NADP; D008291:Malate Dehydrogenase