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Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast Lipomyces starkeyi

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Abstract

The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5′ untranslated sequence and a 92-bp 3′ untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD+ or NADP+ as the coenzyme but preferred NAD+. The K m values for malic acid, NAD+ and NADP+ were 0.85 ± 0.05 mM, 0.34 ± 0.08 mM, and 7.4 ± 0.32 mM, respectively, at pH 7.3.

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Acknowledgment

This work was supported by the National High-Tech Research and Development Program of China (2007AA05Z403) and the National Basic Research Program of China (No. 2007CB707802).

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Authors and Affiliations

  1. Dalian Institute of Chemical Physics, CAS, Dalian, 116023, People’s Republic of China

    Wei Tang, Sufang Zhang, Haidong Tan & Zongbao K. Zhao

  2. Graduate School of the Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China

    Wei Tang

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Correspondence to Zongbao K. Zhao.

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Tang, W., Zhang, S., Tan, H. et al. Molecular Cloning and Characterization of a Malic Enzyme Gene from the Oleaginous Yeast Lipomyces starkeyi . Mol Biotechnol 45, 121–128 (2010). https://doi.org/10.1007/s12033-010-9255-8

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